Mise à jour le : 14-07-2014
Les derniers abstracts de la revue JVI Current Issue : |
Date de mise en ligne : Mardi 01 juillet 2014
Editorial Board [Masthead]
Date de mise en ligne : Mardi 01 juillet 2014
Articles of Significant Interest Selected from This Issue by the Editors [Spotlight]
Date de mise en ligne : Mardi 01 juillet 2014 Mammalian cells have the ability to recognize virus infection and mount a powerful antiviral transcriptional response that provides an initial barrier to replication and impacts both innate and adaptive immune responses. Retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) proteins mediate intracellular virus recognition and are activated by viral RNA ligands to induce antiviral signal transduction. While the mechanisms of RIG-I regulation are already well understood, less is known about the more enigmatic melanoma differentiation-associated 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2). Emerging evidence suggests that these two RLRs are intimately associated as both accomplices and antagonists of antiviral signal transduction.
Rodriguez, K. R., Bruns, A. M., Horvath, C. M.
MDA5 and LGP2: Accomplices and Antagonists of Antiviral Signal Transduction [Minireviews]
Date de mise en ligne : Mardi 01 juillet 2014 The genus beta human papillomaviruses (beta HPVs) cause cutaneous lesions and are thought to be involved in the initiation of some nonmelanoma skin cancers (NMSCs), particularly in patients with the genetic disorder epidermodysplasia verruciformis (EV). We have previously reported that at least two of the genus beta HPV E6 proteins bind to and/or increase the steady-state levels of p53 in squamous epithelial cells. This is in contrast to a well-characterized ability of the E6 proteins of cancer-associated HPVs of genus alpha HPV, which inactivate p53 by targeting its ubiquitin-mediated proteolysis. In this study, we have investigated the ability of genus beta E6 proteins from eight different HPV types to block the transactivation of p53 target genes following DNA damage. We find that the E6 proteins from diverse beta HPV species and types vary in their capacity to block the induction of MDM2, p21, and proapoptotic genes after genotoxic stress. We conclude that some genus beta HPV E6 proteins inhibit at least some p53 target genes, although perhaps not by the same mechanism or to the same degree as the high-risk genus alpha HPV E6 proteins. IMPORTANCE This study addresses the ability of various human papillomavirus E6 proteins to block the activation of p53-responsive cellular genes following DNA damage in human keratinocytes, the normal host cell for HPVs. The E6 proteins encoded by the high-risk, cancer-associated HPV types of genus alpha HPV have a well-established activity to target p53 degradation and thereby inhibit the response to DNA damage. In this study, we have investigated the ability of genus beta HPV E6 proteins from eight different HPV types to block the ability of p53 to transactivate downstream genes following DNA damage. We find that some, but not all, genus beta HPV E6 proteins can block the transactivation of some p53 target genes. This differential response to DNA damage furthers the understanding of cutaneous HPV biology and may help to explain the potential connection between some beta HPVs and cancer.
White, E. A., Walther, J., Javanbakht, H., Howley, P. M.
Genus Beta Human Papillomavirus E6 Proteins Vary in Their Effects on the Transactivation of p53 Target Genes [Virus-Cell Interactions]
Date de mise en ligne : Mardi 01 juillet 2014 Chikungunya virus (CHIKV) is a reemerging mosquito-transmitted alphavirus that causes epidemics of debilitating polyarthritis in humans. A prior study identified two anti-CHIKV monoclonal antibodies ([MAbs] CHK-152 and CHK-166) against the E2 and E1 structural proteins, which had therapeutic efficacy in immunocompetent and immunocompromised mice. Combination MAb therapy was required as administration of a single MAb resulted in the rapid selection of neutralization escape variants and treatment failure in mice. Here, we initially evaluated the efficacy of combination MAb therapy in a nonhuman primate model of CHIKV infection. Treatment of rhesus macaques with CHK-152 and CHK-166 reduced viral spread and infection in distant tissue sites and also neutralized reservoirs of infectious virus. Escape viruses were not detected in the residual viral RNA present in tissues and organs of rhesus macaques. To evaluate the possible significance of MAb resistance, we engineered neutralization escape variant viruses (E1-K61T, E2-D59N, and the double mutant E1-K61T E2-D59N) that conferred resistance to CHK-152 and CHK-166 and tested them for fitness in mosquito cells, mammalian cells, mice, and Aedes albopictus mosquitoes. In both cell culture and mosquitoes, the mutant viruses grew equivalently and did not revert to wild-type (WT) sequence. All escape variants showed evidence of mild clinical attenuation, with decreased musculoskeletal disease at early times after infection in WT mice and a prolonged survival time in immunocompromised Ifnar1–/– mice. Unexpectedly, this was not associated with decreased infectivity, and consensus sequencing from tissues revealed no evidence of reversion or compensatory mutations. Competition studies with CHIKV WT also revealed no fitness compromise of the double mutant (E1-K61T E2-D59N) neutralization escape variant in WT mice. Collectively, our study suggests that neutralization escape viruses selected during combination MAb therapy with CHK-152 plus CHK-166 retain fitness, cause less severe clinical disease, and likely would not be purified during the enzootic cycle. IMPORTANCE Chikungunya virus (CHIKV) causes explosive epidemics of acute and chronic arthritis in humans in Africa, the Indian subcontinent, and Southeast Asia and recently has spread to the New World. As there are no approved vaccines or therapies for human use, the possibility of CHIKV-induced debilitating disease is high in many parts of the world. To this end, our laboratory recently generated a combination monoclonal antibody therapy that aborted lethal and arthritogenic disease in wild-type and immunocompromised mice when administered as a single dose several days after infection. In this study, we show the efficacy of the antibody combination in nonhuman primates and also evaluate the significance of possible neutralization escape mutations in mosquito and mammalian cells, mice, and Aedes albopictus vector mosquitoes. Our experiments show that escape viruses from combination antibody therapy cause less severe CHIKV clinical disease, retain fitness, and likely would not be purified by mosquito vectors.
Pal, P., Fox, J. M., Hawman, D. W., Huang, Y.-J. S., Messaoudi, I., Kreklywich, C., Denton, M., Legasse, A. W., Smith, P. P., Johnson, S., Axthelm, M. K., Vanlandingham, D. L., Streblow, D. N., Higgs, S., Morrison, T. E., Diamond, M. S.
Chikungunya Viruses That Escape Monoclonal Antibody Therapy Are Clinically Attenuated, Stable, and Not Purified in Mosquitoes [Pathogenesis and Immunity]
Date de mise en ligne : Mardi 01 juillet 2014 Adeno-associated virus serotype 2 (AAV2) can efficiently replicate in cells that have been infected with helper viruses, such as adenovirus or herpesvirus. However, in the absence of helper virus infection, AAV2 establishes latency by integrating its genome site specifically into PPP1R12C, a gene located on chromosome 19. This integration target site falls into one of the most gene-dense regions of the human genome, thus inviting the question as to whether the virus has evolved mechanisms to control this complex transcriptional environment in order to facilitate integration, maintain an apparently innocuous latency, and/or establish conditions that are conducive to the rescue of the integrated viral genome. The viral replication (Rep) proteins control and direct every known aspect of the viral life cycle and have been shown to tightly control all AAV2 promoters. In addition, a number of heterologous promoters are repressed by the AAV2 Rep proteins. Here, we demonstrate that Rep proteins efficiently repress expression from the target site PPP1R12C promoter. We find evidence that this repression employs mechanisms similar to those described for Rep-mediated AAV2 p5 promoter regulation. Furthermore, we show that the repression of the cellular target site promoter is based on two distinct mechanisms, one relying on the presence of a functional Rep binding motif within the 5' untranslated region (UTR) of PPP1R12C, whereas the second pathway requires only an intact nucleoside triphosphate (NTP) binding site within the Rep proteins, indicating the possible reliance of this pathway on interactions of the Rep proteins with cellular proteins that mediate or regulate cellular transcription. IMPORTANCE The observation that repression of transcription from the adeno-associated virus serotype 2 (AAV2) p5 and integration target site promoters is mediated by shared mechanisms highlights the possible coevolution of virus and host and could lead to the identification of host factors that the virus exploits to navigate its life cycle.
Dutheil, N., Smith, S. C., Agundez, L., Vincent-Mistiaen, Z. I., Escalante, C. R., Linden, R. M., Henckaerts, E.
Adeno-Associated Virus Rep Represses the Human Integration Site Promoter by Two Pathways That Are Similar to Those Required for the Regulation of the Viral p5 Promoter [Virus-Cell Interactions]
Date de mise en ligne : Mardi 01 juillet 2014 The RV144 HIV-1 vaccine trial demonstrated partial efficacy of 31% against HIV-1 infection. Studies into possible correlates of protection found that antibodies specific to the V1 and V2 (V1/V2) region of envelope correlated inversely with infection risk and that viruses isolated from trial participants contained genetic signatures of vaccine-induced pressure in the V1/V2 region. We explored the hypothesis that the genetic signatures in V1 and V2 could be partly attributed to selection by vaccine-primed T cells. We performed a T-cell-based sieve analysis of breakthrough viruses in the RV144 trial and found evidence of predicted HLA binding escape that was greater in vaccine versus placebo recipients. The predicted escape depended on class I HLA A*02- and A*11-restricted epitopes in the MN strain rgp120 vaccine immunogen. Though we hypothesized that this was indicative of postacquisition selection pressure, we also found that vaccine efficacy (VE) was greater in A*02-positive (A*02+) participants than in A*02– participants (VE = 54% versus 3%, P = 0.05). Vaccine efficacy against viruses with a lysine residue at site 169, important to antibody binding and implicated in vaccine-induced immune pressure, was also greater in A*02+ participants (VE = 74% versus 15%, P = 0.02). Additionally, a reanalysis of vaccine-induced immune responses that focused on those that were shown to correlate with infection risk suggested that the humoral responses may have differed in A*02+ participants. These exploratory and hypothesis-generating analyses indicate there may be an association between a class I HLA allele and vaccine efficacy, highlighting the importance of considering HLA alleles and host immune genetics in HIV vaccine trials. IMPORTANCE The RV144 trial was the first to show efficacy against HIV-1 infection. Subsequently, much effort has been directed toward understanding the mechanisms of protection. Here, we conducted a T-cell-based sieve analysis, which compared the genetic sequences of viruses isolated from infected vaccine and placebo recipients. Though we hypothesized that the observed sieve effect indicated postacquisition T-cell selection, we also found that vaccine efficacy was greater for participants who expressed HLA A*02, an allele implicated in the sieve analysis. Though HLA alleles have been associated with disease progression and viral load in HIV-1 infection, these data are the first to suggest the association of a class I HLA allele and vaccine efficacy. While these statistical analyses do not provide mechanistic evidence of protection in RV144, they generate testable hypotheses for the HIV vaccine community and they highlight the importance of assessing the impact of host immune genetics in vaccine-induced immunity and protection. (This study has been registered at ClinicalTrials.gov under registration no. NCT00223080.)
Gartland, A. J., Li, S., McNevin, J., Tomaras, G. D., Gottardo, R., Janes, H., Fong, Y., Morris, D., Geraghty, D. E., Kijak, G. H., Edlefsen, P. T., Frahm, N., Larsen, B. B., Tovanabutra, S., Sanders-Buell, E., deCamp, A. C., Magaret, C. A., Ahmed, H., Goodridge, J. P., Chen, L., Konopa, P., Nariya, S., Stoddard, J. N., Wong, K., Zhao, H., Deng, W., Maust, B. S., Bose, M., Howell, S., Bates, A., Lazzaro, M., O'Sullivan, A., Lei, E., Bradfield, A., Ibitamuno, G., Assawadarachai, V., O'Connell, R. J., deSouza, M. S., Nitayaphan, S., Rerks-Ngarm, S., Robb, M. L., Sidney, J., Sette, A., Zolla-Pazner, S., Montefiori, D., McElrath, M. J., Mullins, J. I., Kim, J. H., Gilbert, P. B., Hertz, T.
Analysis of HLA A*02 Association with Vaccine Efficacy in the RV144 HIV-1 Vaccine Trial [Vaccines and Antiviral Agents]
Date de mise en ligne : Mardi 01 juillet 2014 We report that the human cytomegalovirus (HCMV) high-molecular-weight tegument protein (HMWP, pUL48; 253 kDa) and the HMWP-binding protein (hmwBP, pUL47; 110 kDa) can be recovered as a complex from virions disrupted by treatment with 50 mM Tris (pH 7.5), 0.5 M NaCl, 0.5% NP-40, and 10 mM dithiothreitol [DTT]. The subunit ratio of the complex approximates 1:1, with a shape and structure consistent with an elongated heterodimer. The HMWP/hmwBP complex was corroborated by reciprocal coimmunoprecipitation experiments using antipeptide antibodies and lysates from both infected cells and disrupted virus particles. An interaction of the amino end of pUL48 (amino acids [aa] 322 to 754) with the carboxyl end of pUL47 (aa 693 to 982) was identified by fragment coimmunoprecipitation experiments, and a head-to-tail self-interaction of hmwBP was also observed. The deubiquitylating activity of pUL48 is retained in the isolated complex, which cleaves K11, K48, and K63 ubiquitin isopeptide linkages. IMPORTANCE Human cytomegalovirus (HCMV, or human herpesvirus 5 [HHV-5]) is a large DNA-containing virus that belongs to the betaherpesvirus subfamily and is a clinically important pathogen. Defining the constituent elements of its mature form, their organization within the particle, and the assembly process by which it is produced are fundamental to understanding the mechanisms of herpesvirus infection and developing drugs and vaccines against them. In this study, we report isolating a complex of two large proteins encoded by HCMV open reading frames (ORFs) UL47 and UL48 and identifying the binding domains responsible for their interaction with each other and of pUL47 with itself. Our calculations indicate that the complex is a rod-shaped heterodimer. Additionally, we determined that the ubiquitin-specific protease activity of the ORF UL48 protein was functional in the complex, cleaving K11-, K48-, and K63-linked ubiquitin dimers. This information builds on and extends our understanding of the HCMV tegument protein network that is required to interface the HCMV envelope and capsid.
Tullman, J. A., Harmon, M.-E., Delannoy, M., Gibson, W.
Recovery of an HMWP/hmwBP (pUL48/pUL47) Complex from Virions of Human Cytomegalovirus: Subunit Interactions, Oligomer Composition, and Deubiquitylase Activity [Structure and Assembly]
Date de mise en ligne : Mardi 01 juillet 2014 The Paramyxoviridae form an increasingly diverse viral family, infecting a wide variety of different hosts. In recent years, they have been linked to disease emergence in many different animal populations and in humans. Bats and rodents have been identified as major animal populations capable of harboring paramyxoviruses, and host shifting between these animals is likely to be an important driving factor in the underlying evolutionary processes that eventually lead to disease emergence. Here, we have studied paramyxovirus circulation within populations of endemic and introduced wild small mammals of the southwestern Indian Ocean region and belonging to four taxonomic orders: Rodentia, Afrosoricida, Soricomorpha, and Chiroptera. We report elevated infection levels as well as widespread paramyxovirus dispersal and frequent host exchange of a newly emerging genus of the Paramyxoviridae, currently referred to as the unclassified morbillivirus-related viruses (UMRVs). In contrast to other genera of the Paramyxoviridae, where bats have been shown to be a key host species, we show that rodents (and, in particular, Rattus rattus) are significant spreaders of UMRVs. We predict that the ecological particularities of the southwestern Indian Ocean, where small mammal species often live in densely packed, multispecies communities, in combination with the increasing invasion of R. rattus and perturbations of endemic animal communities by active anthropological development, will have a major influence on the dynamics of UMRV infection. IMPORTANCE Identification of the infectious agents that circulate within wild animal reservoirs is essential for several reasons: (i) infectious disease outbreaks often originate from wild fauna; (ii) anthropological expansion increases the risk of contact between human and animal populations and, as a result, the risk of disease emergence; (iii) evaluation of pathogen reservoirs helps in elaborating preventive measures to limit the risk of disease emergence. Many paramyxoviruses for which bats and rodents serve as major reservoirs have demonstrated their potential to cause disease in humans and animals. In the context of the biodiversity hot spot of southwestern Indian Ocean islands and their rich endemic fauna, we show that highly diverse UMRVs exchange between various endemic animal species, and their dissemination likely is facilitated by the introduced Rattus rattus. Hence, many members of the Paramyxoviridae appear well adapted for the study of the viral phylodynamics that may be associated with disease emergence.
Wilkinson, D. A., Melade, J., Dietrich, M., Ramasindrazana, B., Soarimalala, V., Lagadec, E., le Minter, G., Tortosa, P., Heraud, J.-M., de Lamballerie, X., Goodman, S. M., Dellagi, K., Pascalis, H.
Highly Diverse Morbillivirus-Related Paramyxoviruses in Wild Fauna of the Southwestern Indian Ocean Islands: Evidence of Exchange between Introduced and Endemic Small Mammals [Genetic Diversity and Evolution]
Date de mise en ligne : Mardi 01 juillet 2014 Influenza virus neuraminidase (NA) is an interesting target of small-molecule antiviral drugs. We isolated a set of H5N1 NA-specific single-domain antibodies (N1-VHHm) and evaluated their in vitro and in vivo antiviral potential. Two of them inhibited the NA activity and in vitro replication of clade 1 and 2 H5N1 viruses. We then generated bivalent derivatives of N1-VHHm by two methods. First, we made N1-VHHb by genetically joining two N1-VHHm moieties with a flexible linker. Second, bivalent N1-VHH-Fc proteins were obtained by genetic fusion of the N1-VHHm moiety with the crystallizable region of mouse IgG2a (Fc). The in vitro antiviral potency against H5N1 of both bivalent N1-VHHb formats was 30- to 240-fold higher than that of their monovalent counterparts, with 50% inhibitory concentrations in the low nanomolar range. Moreover, single-dose prophylactic treatment with bivalent N1-VHHb or N1-VHH-Fc protected BALB/c mice against a lethal challenge with H5N1 virus, including an oseltamivir-resistant H5N1 variant. Surprisingly, an N1-VHH-Fc fusion without in vitro NA-inhibitory or antiviral activity also protected mice against an H5N1 challenge. Virus escape selection experiments indicated that one amino acid residue close to the catalytic site is required for N1-VHHm binding. We conclude that single-domain antibodies directed against influenza virus NA protect against H5N1 virus infection, and when engineered with a conventional Fc domain, they can do so in the absence of detectable NA-inhibitory activity. IMPORTANCE Highly pathogenic H5N1 viruses are a zoonotic threat. Outbreaks of avian influenza caused by these viruses occur in many parts of the world and are associated with tremendous economic loss, and these viruses can cause very severe disease in humans. In such cases, small-molecule inhibitors of the viral NA are among the few treatment options for patients. However, treatment with such drugs often results in the emergence of resistant viruses. Here we show that single-domain antibody fragments that are specific for NA can bind and inhibit H5N1 viruses in vitro and can protect laboratory mice against a challenge with an H5N1 virus, including an oseltamivir-resistant virus. In addition, plant-produced VHH fused to a conventional Fc domain can protect in vivo even in the absence of NA-inhibitory activity. Thus, NA of influenza virus can be effectively targeted by single-domain antibody fragments, which are amenable to further engineering.
Cardoso, F. M., Ibanez, L. I., Van den Hoecke, S., De Baets, S., Smet, A., Roose, K., Schepens, B., Descamps, F. J., Fiers, W., Muyldermans, S., Depicker, A., Saelens, X.
Single-Domain Antibodies Targeting Neuraminidase Protect against an H5N1 Influenza Virus Challenge [Vaccines and Antiviral Agents]
Date de mise en ligne : Mardi 01 juillet 2014 On 30 March 2013, a novel avian influenza A H7N9 virus causing severe human respiratory infections was identified in China. Preliminary sequence analyses have shown that the virus is a reassortant of H7N9 and H9N2 avian influenza viruses. In this study, we conducted enhanced surveillance for H7N9 virus in Guangdong, China, from April to August 2013. We isolated two H7N9 viral strains from environmental samples associated with poultry markets and one from a clinical patient. Sequence analyses showed that the Guangdong H7N9 virus isolated from April to May shared high sequence similarity with other strains from eastern China. The A/Guangdong/1/2013 (H7N9) virus isolated from the Guangdong patient on 10 August 2013 was divergent from previously sequenced H7N9 viruses and more closely related to local circulating H9N2 viruses in the NS and NP genes. Phylogenetic analyses revealed that four internal genes of the A/Guangdong/1/2013 (H7N9) virus—the NS, NP, PB1, and PB2 genes—were in clusters different from those for H7N9 viruses identified previously in other provinces of China. The discovery presented here suggests that continuing reassortment led to the emergence of the A/Guangdong/1/2013 (H7N9) virus as a novel H7N9 virus in Guangdong, China, and that viral adaptation to avian and human hosts must be assessed. IMPORTANCE In this study, we isolated and characterized the avian influenza A H7N9 virus in Guangdong, China, from April to August 2013. We show that the viruses isolated from Guangdong environmental samples and chickens from April to May 2013 were highly similar to other H7N9 strains found in eastern China. The H7N9 virus isolated from the clinical patient in Guangdong in August 2013 was divergent from previously identified H7N9 viruses, with the NS and NP genes originating from recent H9N2 viruses circulating in the province. This study provides direct evidence that continuing reassortment occurred and led to the emergence of a novel H7N9 influenza virus in Guangdong, China. These results also shed light on how the H7N9 virus evolved, which is critically important for future monitoring and tracing of viral transmission.
Lu, J., Wu, J., Zeng, X., Guan, D., Zou, L., Yi, L., Liang, L., Ni, H., Kang, M., Zhang, X., Zhong, H., He, X., Monagin, C., Lin, J., Ke, C.
Continuing Reassortment Leads to the Genetic Diversity of Influenza Virus H7N9 in Guangdong, China [Genetic Diversity and Evolution]
Date de mise en ligne : Mardi 01 juillet 2014 Monoclonal-antibody (MAb)-resistant mutants were used to map antigenic sites on foot-and-mouth disease virus (FMDV), which resulted in the identification of neutralizing epitopes in the flexible βG-βH loop in VP1. For FMDV SAT2 viruses, studies have shown that at least two antigenic sites exist. By use of an infectious SAT2 cDNA clone, 10 structurally exposed and highly variable loops were identified as putative antigenic sites on the VP1, VP2, and VP3 capsid proteins of SAT2/Zimbabwe (ZIM)/7/83 (topotype II) and replaced with the corresponding regions of SAT2/Kruger National Park (KNP)/19/89 (topotype I). Virus neutralization assays using convalescent-phase antisera raised against the parental virus, SAT2/ZIM/7/83, indicated that the mutant virus containing the TQQS-to-ETPV mutation in the N-terminal part of the βG-βH loop of VP1 showed not only a significant increase in the neutralization titer but also an increase in the index of avidity to the convalescent-phase antisera. Furthermore, antigenic profiling of the epitope-replaced and parental viruses with nonneutralizing SAT2-specific MAbs led to the identification of two nonneutralizing antigenic regions. Both regions were mapped to incorporate residues 71 to 72 of VP2 as the major contact point. The binding footprint of one of the antigenic regions encompasses residues 71 to 72 and 133 to 134 of VP2 and residues 48 to 50 of VP1, and the second antigenic region encompasses residues 71 to 72 and 133 to 134 of VP2 and residues 84 to 86 and 109 to 11 of VP1. This is the first time that antigenic regions encompassing residues 71 to 72 of VP2 have been identified on the capsid of a SAT2 FMDV. IMPORTANCE Monoclonal-antibody-resistant mutants have traditionally been used to map antigenic sites on foot-and-mouth disease virus (FMDV). However, for SAT2-type viruses, which are responsible for most of the FMD outbreaks in Africa and are the most varied of all seven serotypes, only two antigenic sites have been identified. We have followed a unique approach using an infectious SAT2 cDNA genome-length clone. Ten structurally surface-exposed, highly varied loops were identified as putative antigenic sites on the VP1, VP2, and VP3 capsid proteins of the SAT2/ZIM/7/83 virus. These regions were replaced with the corresponding regions of an antigenically disparate virus, SAT2/KNP/19/89. Antigenic profiling of the epitope-replaced and parental viruses with SAT2-specific MAbs led to the identification of two unique antibody-binding footprints on the SAT2 capsid. In this report, evidence for the structural engineering of antigenic sites of a SAT2 capsid to broaden cross-reactivity with antisera is provided.
Opperman, P. A., Rotherham, L. S., Esterhuysen, J., Charleston, B., Juleff, N., Capozzo, A. V., Theron, J., Maree, F. F.
Determining the Epitope Dominance on the Capsid of a Serotype SAT2 Foot-and-Mouth Disease Virus by Mutational Analyses [Pathogenesis and Immunity]
Date de mise en ligne : Mardi 01 juillet 2014 Hantavirus cardiopulmonary syndrome (HCPS) is a rodent-borne disease with a high case-fatality rate that is caused by several New World hantaviruses. Each pathogenic hantavirus is naturally hosted by a principal rodent species without conspicuous disease and infection is persistent, perhaps for life. Deer mice (Peromyscus maniculatus) are the natural reservoirs of Sin Nombre virus (SNV), the etiologic agent of most HCPS cases in North America. Deer mice remain infected despite a helper T cell response that leads to high-titer neutralizing antibodies. Deer mice are also susceptible to Andes hantavirus (ANDV), which causes most HCPS cases in South America; however, deer mice clear ANDV. We infected deer mice with SNV or ANDV to identify differences in host responses that might account for this differential outcome. SNV RNA levels were higher in the lungs but not different in the heart, spleen, or kidneys. Most ANDV-infected deer mice had seroconverted 14 days after inoculation, but none of the SNV-infected deer mice had. Examination of lymph node cell antigen recall responses identified elevated immune gene expression in deer mice infected with ANDV and suggested maturation toward a Th2 or T follicular helper phenotype in some ANDV-infected deer mice, including activation of the interleukin 4 (IL-4) pathway in T cells and B cells. These data suggest that the rate of maturation of the immune response is substantially higher and of greater magnitude during ANDV infection, and these differences may account for clearance of ANDV and persistence of SNV. IMPORTANCE Hantaviruses persistently infect their reservoir rodent hosts without pathology. It is unknown how these viruses evade sterilizing immune responses in the reservoirs. We have determined that infection of the deer mouse with its homologous hantavirus, Sin Nombre virus, results in low levels of immune gene expression in antigen-stimulated lymph node cells and a poor antibody response. However, infection of deer mice with a heterologous hantavirus, Andes virus, results in a robust lymph node cell response, signatures of T and B cell maturation, and production of antibodies. These findings suggest that an early and aggressive immune response to hantaviruses may lead to clearance in a reservoir host and suggest that a modest immune response may be a component of hantavirus ecology.
Schountz, T., Quackenbush, S., Rovnak, J., Haddock, E., Black, W. C., Feldmann, H., Prescott, J.
Differential Lymphocyte and Antibody Responses in Deer Mice Infected with Sin Nombre Hantavirus or Andes Hantavirus [Pathogenesis and Immunity]
Date de mise en ligne : Mardi 01 juillet 2014 VSV-FH is a hybrid vesicular stomatitis virus (VSV) with a deletion of its G glycoprotein and encoding the measles virus (MV) fusion (F) and hemagglutinin (H) envelope glycoproteins. VSV-FH infects cells expressing MV receptors and is fusogenic and effective against myeloma xenografts in mice. We evaluated the fusogenic activities of MV and VSV-FH in relationship to the density of receptor on the target cell surface and the kinetics of F and H expression in infected cells. Using a panel of cells expressing increasing numbers of the MV receptor CD46, we evaluated syncytium size in MV- or VSV-FH-infected cells. VSV-FH is not fusogenic at low CD46 density but requires less CD46 for syncytium formation than MV. The size of each syncytium is larger in VSV-FH-infected cells at a specific CD46 density. While syncytium size reached a plateau and did not increase further in MV-infected CHO cells expressing ≥4,620 CD46 copies/cell, there was a corresponding increase in syncytium size with increases in CD46 levels in VSV-FH-infected CD46-expressing CHO (CHO-CD46) cells. Further analysis in VSV-FH-infected cell lines shows earlier and higher expression of F and H mRNAs and protein. However, VSV-FH cytotoxic activity was reduced by pretreatment of the cells with type I interferon. In contrast, the cytopathic effects are not affected in MV-infected cells. In summary, VSV-FH has significant advantages over MV as an oncolytic virus due to its higher viral yield, faster replication kinetics, and larger fusogenic capabilities but should be used in cancer types with defective interferon signaling pathways. IMPORTANCE We studied the cytotoxic activity of a vesicular stomatitis/measles hybrid virus (VSV-FH), which is superior to that of measles virus (MV), in different cancer cell lines. We determined that viral RNA and protein were produced faster and in higher quantities in VSV-FH-infected cells. This resulted in the formation of larger syncytia, higher production of infectious particles, and a more potent cytopathic effect in permissive cells. Importantly, VSV-FH, similar to MV, can discriminate between low- and high-expressing CD46 cells, a phenotype important for cancer therapy as the virus will be able to preferentially infect cancer cells that overexpress CD46 over low-CD46-expressing normal cells.
Ayala-Breton, C., Russell, L. O. J., Russell, S. J., Peng, K.-W.
Faster Replication and Higher Expression Levels of Viral Glycoproteins Give the Vesicular Stomatitis Virus/Measles Virus Hybrid VSV-FH a Growth Advantage over Measles Virus [Gene Delivery]
Date de mise en ligne : Mardi 01 juillet 2014 A key characteristic of arenaviruses is their ability to establish persistent infection in their natural host. Different factors like host age, viral dose strain, and route of infection may contribute to the establishment of persistence. However, the molecular mechanisms governing persistence are not fully understood. Here, we describe gain-of-function mutations of lymphocytic choriomeningitis virus (LCMV) expressing Lassa virus (LASV) GP, which can prolong viremia in mice depending on the sequences in the GP-2 cytoplasmic tail. The initial mutant variant (rLCMV/LASV mut GP) carried a point mutation in the cytosolic tail of the LASV glycoprotein GP corresponding to a K461G substitution. Unlike what occurred with the original rLCMV/LASV wild-type (wt) GP, infection of C57BL/6 mice with the mutated recombinant virus led to a detectable viremia of 2 weeks' duration. Further replacement of the entire sequence of the cytosolic tail from LASV to LCMV GP resulted in increased viral titers and delayed clearance of the viruses. Biosynthesis and cell surface localization of LASV wt and mut GPs were comparable. IMPORTANCE Starting from an emerging virus in a wild-type mouse, we engineered a panel of chimeric Lassa/lymphocytic choriomeningitis viruses. Mutants carrying a viral envelope with the cytosolic tail from the closely related mouse-adapted LCMV were able to achieve a productive viral infection lasting up to 27 days in wild-type mice. Biochemical assays showed a comparable biosynthesis and cell surface localization of LASV wt and mut GPs. These recombinant chimeric viruses could allow the study of immune responses and antivirals targeting the LASV GP.
Sommerstein, R., Ramos da Palma, J., Olschlager, S., Bergthaler, A., Barba, L., Lee, B. P.- L., Pasquato, A., Flatz, L.
Evolution of Recombinant Lymphocytic Choriomeningitis Virus/Lassa Virus In Vivo Highlights the Importance of the GPC Cytosolic Tail in Viral Fitness [Genetic Diversity and Evolution]
Date de mise en ligne : Mardi 01 juillet 2014 Increasing data suggest that NK cells can mediate antiviral activity in HIV-1-infected humans, and as such, novel approaches harnessing the anti-HIV-1 function of both T cells and NK cells represent attractive options to improve future HIV-1 immunotherapies. Chronic progressive HIV-1 infection has been associated with a loss of CD4+ T helper cell function and with the accumulation of anergic NK cells. As several studies have suggested that cytokines produced by CD4+ T cells are required to enhance NK cell function in various infection models, we hypothesized that reconstitution of HIV-1-specific CD4+ T-cell responses by therapeutic immunization would restore NK cell activity in infected individuals. Using flow cytometry, we examined the function of CD4+ T cells and NK cells in response to HIV-1 in subjects with treated chronic HIV-1 infection before and after immunization with an adjuvanted HIV-1 Gp120/NefTat subunit protein vaccine candidate provided by GlaxoSmithKline. Vaccination induced an increased expression of interleukin-2 (IL-2) by Gp120-specific CD4+ T cells in response to HIV-1 peptides ex vivo, which was associated with enhanced production of gamma interferon (IFN-) by NK cells. Our data show that reconstitution of HIV-1-specific CD4+ T-cell function by therapeutic immunization can enhance NK cell activity in HIV-1-infected individuals. IMPORTANCE NK cells are effector cells of the innate immune system and are important in the control of viral infection. Recent studies have demonstrated the crucial role played by NK cells in controlling and/or limiting acquisition of HIV-1 infection. However, NK cell function is impaired during progressive HIV-1 infection. We recently showed that therapeutic immunization of treated HIV-1-infected individuals reconstituted strong T-cell responses, measured notably by their production of IL-2, a cytokine that can activate NK cells. The current study suggests that reconstitution of T-cell function by therapeutic vaccination can enhance NK cell activity in individuals with chronic HIV-1 infection. Our findings provide new insights into the interplay between adaptive and innate immune mechanisms involved in HIV-1 immunity and unveil opportunities to harness NK cell function in future therapeutic vaccine strategies to target HIV-1.
Jost, S., Tomezsko, P. J., Rands, K., Toth, I., Lichterfeld, M., Gandhi, R. T., Altfeld, M.
CD4+ T-Cell Help Enhances NK Cell Function following Therapeutic HIV-1 Vaccination [Vaccines and Antiviral Agents]
Date de mise en ligne : Mardi 01 juillet 2014 Viruses are dependent on their host cells for replication and thus have evolved in intimate association with them. The identification of host factors required for viral infection has led to advances in both viral and cellular biology. Vesicular stomatitis virus (VSV), a negative-sense RNA virus, replicates in all eukaryotic cells in culture, suggesting that the host requirements for its replication are ubiquitous. In this study, we performed a genome-wide small interfering RNA screen of human cells in culture and identified multiple cellular genes that influence the entry and replication of VSV. From a list of >300 genes, we selected the most promising candidates to perform further analysis to assign their functions to either the entry or intracellular replication step of infection. We implicate 3 new factors in VSV entry and 20 new factors in viral gene expression. These proteins have diverse cellular roles, including S-adenosylmethionine synthesis, respiration, and host translation machinery, underscoring the intimate relationship between VSV and the host cell. Together, these results provide a curated list of genes required for VSV replication. IMPORTANCE Replication of vesicular stomatitis virus (VSV) has long served as a model for understanding host-virus interactions and neuropathogenesis. We performed a genome-wide analysis of host factors and revealed genes critical for viral replication, including some involved in vesicular trafficking, cell cycling, and protein modification. Our results provide an enriched list of host factors that are required for specific stages of VSV entry and gene expression. This study may also potentially expand the repertoire of targets for antiviral therapy against negative-strand RNA viruses.
Lee, A. S.-Y., Burdeinick-Kerr, R., Whelan, S. P. J.
A Genome-Wide Small Interfering RNA Screen Identifies Host Factors Required for Vesicular Stomatitis Virus Infection [Virus-Cell Interactions]
Date de mise en ligne : Mardi 01 juillet 2014 Hepatitis C virus (HCV) causes not only severe liver problems but also extrahepatic manifestations, such as insulin resistance (IR). Wild-type peroxisome proliferator-activated receptor gamma coactivator 1 alpha (WT-PGC-1α) is essential in hepatic gluconeogenesis and has recently been demonstrated to link HCV infection to hepatic insulin resistance (IR). A recent study has characterized a novel human liver-specific PGC-1α (L-PGC-1α) transcript, which is proposed to reflect human adaption to more complex pathways. However, the effect of HCV infection on L-PGC-1α expression and the mechanism by which HCV modulates WT-PGC-1α/L-PGC-1α remain unclear. In this study, we showed that HCV infection upregulated both WT-PGC-1α and L-PGC-1α, which further promoted HCV production. The upregulation of both PGC-1α isoforms depended on HCV RNA replication. By using promoter-luciferase reporters, kinase inhibitors, and dominant negative mutants, we further observed that the HCV-induced upregulation of WT-PGC-1α was mediated by the phosphorylation of cyclic AMP (cAMP)-responsive element-binding protein (CREB), whereas that of L-PGC-1α was mediated by CREB phosphorylation and forkhead box O1 dephosphorylation. Moreover, HCV infection induced endoplasmic reticulum (ER) stress, and pharmacological induction of ER stress upregulated WT-PGC-1α/L-PGC-1α and phosphorylated CREB. In contrast, pharmacological inhibition of HCV-induced ER stress impaired WT-PGC-1α/L-PGC-1α upregulation along with decreased phosphorylated CREB. The correlation of hepatic mPGC-1α with ER stress was further confirmed in mice. Overall, HCV infection upregulates both WT-PGC-1α and L-PGC-1α through an ER stress-mediated, phosphorylated CREB-dependent pathway, and both PGC-1α isoforms promote HCV production in turn. IMPORTANCE HCV causes not only severe liver problems but also extrahepatic manifestations, such as insulin resistance (IR). As a key regulator in energy metabolism, wild-type PGC-1α (WT-PGC-1α), has recently been demonstrated to link HCV infection to hepatic IR. A recent study has characterized a novel human liver-specific PGC-1α (L-PGC-1α), which reflects human adaption to more complex pathways. However, the effect of HCV infection on L-PGC-1α expression and the mechanism by which HCV regulates WT-PGC-1α/L-PGC-1α remain unclear. In this study, we showed that HCV infection upregulated both WT-PGC-1α and L-PGC-1α, which further promoted HCV production. WT-PGC-1α upregulation was mediated by CREB phosphorylation, whereas L-PGC-1α upregulation was mediated by CREB phosphorylation and FoxO1 dephosphorylation. HCV-induced ER stress mediated WT-PGC-1α/L-PGC-1α upregulation and CREB phosphorylation. Overall, this study provides new insights into the mechanism by which HCV upregulates WT-PGC-1α/L-PGC-1α and highlights the novel intervention of HCV-ER stress-PGC-1α signaling for HCV therapy and HCV-induced IR therapy.
Yao, W., Cai, H., Li, X., Li, T., Hu, L., Peng, T.
Endoplasmic Reticulum Stress Links Hepatitis C Virus RNA Replication to Wild-Type PGC-1{alpha}/Liver-Specific PGC-1{alpha} Upregulation [Virus-Cell Interactions]
Date de mise en ligne : Mardi 01 juillet 2014 Although alteration in host cellular translation machinery occurs in virus-infected cells, the role of such alteration and the precise pathogenic processes are not well understood. Influenza A virus (IAV) infection shuts off host cell gene expression at transcriptional and translational levels. Here, we found that the protein level of eukaryotic translation initiation factor 4B (eIF4B), an integral component of the translation initiation apparatus, was dramatically reduced in A549 cells as well as in the lung, spleen, and thymus of mice infected with IAV. The decrease in eIF4B level was attributed to lysosomal degradation of eIF4B, which was induced by viral NS1 protein. Silencing eIF4B expression in A549 cells significantly promoted IAV replication, and conversely, overexpression of eIF4B markedly inhibited the viral replication. Importantly, we observed that eIF4B knockdown transgenic mice were more susceptible to IAV infection, exhibiting faster weight loss, shorter survival time, and more-severe organ damage. Furthermore, we demonstrated that eIF4B regulated the expression of interferon-induced transmembrane protein 3 (IFITM3), a critical protein involved in immune defense against a variety of RNA viruses, including influenza virus. Taken together, our findings reveal that eIF4B plays an important role in host defense against IAV infection at least by regulating the expression of IFITM3, which restricts viral entry and thereby blocks early stages of viral production. These data also indicate that influenza virus has evolved a strategy to overcome host innate immunity by downregulating eIF4B protein. IMPORTANCE Influenza A virus (IAV) infection stimulates the host innate immune system, in part, by inducing interferons (IFNs). Secreted IFNs activate the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, leading to elevated transcription of a large group of IFN-stimulated genes that have antiviral function. To circumvent the host innate immune response, influenza virus has evolved multiple strategies for suppressing the production of IFNs. Here, we show that IAV infection induces lysosomal degradation of eIF4B protein; and eIF4B inhibits IAV replication by upregulating expression of interferon-induced transmembrane protein 3 (IFITM3), a key protein that protects the host from virus infection. Our finding illustrates a critical role of eIF4B in the host innate immune response and provides novel insights into the complex mechanisms by which influenza virus interacts with its host.
Wang, S., Chi, X., Wei, H., Chen, Y., Chen, Z., Huang, S., Chen, J.-L.
Influenza A Virus-Induced Degradation of Eukaryotic Translation Initiation Factor 4B Contributes to Viral Replication by Suppressing IFITM3 Protein Expression [Virus-Cell Interactions]
Date de mise en ligne : Mardi 01 juillet 2014 Interaction between E and prM proteins in flavivirus-infected cells is a major factor for virus-like particle (VLP) production. The prM helical (prM-H) domain is topologically close to and may interact with domain II of the E protein (EDII). In this study, we investigated prM-H domain amino acid residues facing Japanese encephalitis virus EDII using site-directed mutagenesis to determine their roles in prM-E interaction and VLP production. Our results indicate that negatively charged prM-E125 residue at the prM-H domain affected VLP production via one or more interactions with positively charged E-K93 and E-H246 residues at EDII. Exchanges of oppositely charged residue side chains at prM-E125/E-K93 and prM-E125/E-H246 are recoverable for VLP production. The prM-E125 and E-H246 residues are conserved and that the positive charge of the E-K93 residue is preserved in different flavivirus groups. These findings suggest that the electrostatic attractions of prM-E125, E-K93, and E-H246 residues are important to flavivirus VLP production and that inhibiting these interactions is a potential strategy for blocking flavivirus infections. IMPORTANCE Molecular interaction between E and prM proteins of Japanese encephalitis virus is a major driving force for virus-like particle (VLP) production. The current high-resolution structures available for prM-E complexes do not include the membrane proximal stem region of prM. The prM stem region contains an N-terminal loop and a helix domain (prM-H). Since the prM-H domain is topologically close to domain II of the E protein (EDII), this study was to determine molecular interactions between the prM-H domain and EDII. We found that the molecular interactions between prM-E125 residue and positively charged E-K93 and E-H246 residues at EDII are critical for VLP production. More importantly, the prM-E125 and E-H246 residues are conserved and the positive charge of the E-K93 residue is preserved in different flavivirus groups. Our findings help refine the structure and molecular interactions on the flavivirus surface and reveal a potential strategy for blocking flavivirus infections by inhibiting these electrostatic interactions.
Peng, J.-G., Wu, S.-C.
Glutamic Acid at Residue 125 of the prM Helix Domain Interacts with Positively Charged Amino Acids in E Protein Domain II for Japanese Encephalitis Virus-Like-Particle Production [Structure and Assembly]
Date de mise en ligne : Mardi 01 juillet 2014 Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens caused by infectious laryngotracheitis virus (ILTV). The disease is controlled mainly through biosecurity and vaccination with live attenuated strains of ILTV and vectored vaccines based on turkey herpesvirus (HVT) and fowlpox virus (FPV). The current live attenuated vaccines (chicken embryo origin [CEO] and tissue culture origin [TCO]), although effective, can regain virulence, whereas HVT- and FPV-vectored ILTV vaccines are less efficacious than live attenuated vaccines. Therefore, there is a pressing need to develop safer and more efficacious ILTV vaccines. In the present study, we generated Newcastle disease virus (NDV) recombinants, based on the LaSota vaccine strain, expressing glycoproteins B (gB) and D (gD) of ILTV using reverse genetics technology. These recombinant viruses, rLS/ILTV-gB and rLS/ILTV-gD, were slightly attenuated in vivo yet retained growth dynamics, stability, and virus titers in vitro that were similar to those of the parental LaSota virus. Expression of ILTV gB and gD proteins in the recombinant virus-infected cells was detected by immunofluorescence assay. Vaccination of specific-pathogen-free chickens with these recombinant viruses conferred significant protection against virulent ILTV and velogenic NDV challenges. Immunization of commercial broilers with rLS/ILTV-gB provided a level of protection against clinical disease similar to that provided by the live attenuated commercial vaccines, with no decrease in body weight gains. The results of the study suggested that the rLS/ILTV-gB and -gD viruses are safe, stable, and effective bivalent vaccines that can be mass administered via aerosol or drinking water to large chicken populations. IMPORTANCE This paper describes the development and evaluation of novel bivalent vaccines against chicken infectious laryngotracheitis (ILT) and Newcastle disease (ND), two of the most economically important infectious diseases of poultry. The current commercial ILT vaccines are either not safe or less effective. Therefore, there is a pressing need to develop safer and more efficacious ILT vaccines. In the present study, we generated Newcastle disease virus (NDV) recombinants expressing glycoproteins B (gB) and D (gD) of infectious laryngotracheitis virus (ILTV) using reverse genetics technology. These recombinant viruses were safe, stable, and immunogenic and replicated efficiently in birds. Vaccination of chickens with these recombinant viruses conferred complete protection against ILTV and NDV challenge. These novel bivalent vaccines can be mass administered via aerosol or drinking water to large chicken populations at low cost, which will have a direct impact on poultry health, fitness, and performance.
Zhao, W., Spatz, S., Zhang, Z., Wen, G., Garcia, M., Zsak, L., Yu, Q.
Newcastle Disease Virus (NDV) Recombinants Expressing Infectious Laryngotracheitis Virus (ILTV) Glycoproteins gB and gD Protect Chickens against ILTV and NDV Challenges [Vaccines and Antiviral Agents]
Date de mise en ligne : Mardi 01 juillet 2014 Human immunodeficiency virus type 1 (HIV-1) infection in the central nervous system (CNS) is characterized by replication in macrophages or brain microglia that express low levels of the CD4 receptor and is the cause of HIV-associated dementia and related cognitive and motor disorders that affect 20 to 30% of treatment-naive patients with AIDS. Independent viral envelope evolution in the brain has been reported, with the need for robust replication in resident CD4low cells, as well as CD4-negative cells, such as astrocytes, proposed as a major selective pressure. We previously reported giant-cell encephalitis in subtype B and C R5 simian-human immunodeficiency virus (SHIV)-infected macaques (SHIV-induced encephalitis [SHIVE]) that experienced very high chronic viral loads and progressed rapidly to AIDS, with varying degrees of macrophage or microglia infection and activation of these immune cells, as well as astrocytes, in the CNS. In this study, we characterized envelopes (Env) amplified from the brains of subtype B and C R5 SHIVE macaques. We obtained data in support of an association between severe neuropathological changes, robust macrophage and microglia infection, and evolution to CD4 independence. Moreover, the degree of Env CD4 independence appeared to correlate with the extent of astrocyte infection in vivo. These findings further our knowledge of the CNS viral population phenotypes that are associated with the severity of HIV/SHIV-induced neurological injury and improve our understanding of the mechanism of HIV-1 cellular tropism and persistence in the brain. IMPORTANCE Human immunodeficiency virus type 1 (HIV-1) infection of astrocytes in the brain has been suggested to be important in HIV persistence and neuropathogenesis but has not been definitively demonstrated in an animal model of HIV-induced encephalitis (HIVE). Here, we describe a new nonhuman primate (NHP) model of R5 simian-human immunodeficiency virus (SHIV)-induced encephalitis (SHIVE) with several classical HIVE features that include astrocyte infection. We further show an association between severe neuropathological changes, robust resident microglia infection, and evolution to CD4 independence of viruses in the central nervous system (CNS), with expansion to infection of truly CD4-negative cells in vivo. These findings support the use of the R5 SHIVE models to study the contribution of the HIV envelope and viral clades to neurovirulence and residual virus replication in the CNS, providing information that should guide efforts to eradicate HIV from the body.
Zhuang, K., Leda, A. R., Tsai, L., Knight, H., Harbison, C., Gettie, A., Blanchard, J., Westmoreland, S., Cheng-Mayer, C.
Emergence of CD4 Independence Envelopes and Astrocyte Infection in R5 Simian-Human Immunodeficiency Virus Model of Encephalitis [Virus-Cell Interactions]
Date de mise en ligne : Mardi 01 juillet 2014 Herpes simplex virus 2 (HSV-2) subunit antigen vaccines targeting virus entry molecules have failed to prevent genital herpes in human trials. Our approach is to include a virus entry molecule and add antigens that block HSV-2 immune evasion. HSV-2 glycoprotein C (gC2) is an immune evasion molecule that inhibits complement. We previously reported that adding gC2 to gD2 improved vaccine efficacy compared to the efficacy of either antigen alone in mice and guinea pigs. Here we demonstrate that HSV-2 glycoprotein E (gE2) functions as an immune evasion molecule by binding the IgG Fc domain. HSV-2 gE2 is synergistic with gC2 in protecting the virus from antibody and complement neutralization. Antibodies produced by immunization with gE2 blocked gE2-mediated IgG Fc binding and cell-to-cell spread. Mice immunized with gE2 were only partially protected against HSV-2 vaginal challenge in mice; however, when gE2 was added to gC2/gD2 to form a trivalent vaccine, neutralizing antibody titers with and without complement were significantly higher than those produced by gD2 alone. Importantly, the trivalent vaccine protected the dorsal root ganglia (DRG) of 32/33 (97%) mice between days 2 and 7 postchallenge, compared with 27/33 (82%) in the gD2 group. The HSV-2 DNA copy number was significantly lower in mice immunized with the trivalent vaccine than in those immunized with gD2 alone. The extent of DRG protection using the trivalent vaccine was better than what we previously reported for gC2/gD2 immunization. Therefore, gE2 is a candidate antigen for inclusion in a multivalent subunit vaccine that attempts to block HSV-2 immune evasion. IMPORTANCE Herpes simplex virus is the most common cause of genital ulcer disease worldwide. Infection results in emotional distress for infected individuals and their partners, is life threatening for infants exposed to herpes during childbirth, and greatly increases the risk of individuals acquiring and transmitting HIV infection. A vaccine that prevents genital herpes infection will have major public health benefits. Our vaccine approach includes strategies to prevent the virus from evading immune attack. Mice were immunized with a trivalent vaccine containing an antigen that induces antibodies to block virus entry and two antigens that induce antibodies that block immune evasion from antibody and complement. Immunized mice demonstrated no genital disease, and 32/33 (97%) animals had no evidence of infection of dorsal root ganglia, suggesting that the vaccine may prevent the establishment of latency and recurrent infections.
Awasthi, S., Huang, J., Shaw, C., Friedman, H. M.
Blocking Herpes Simplex Virus 2 Glycoprotein E Immune Evasion as an Approach To Enhance Efficacy of a Trivalent Subunit Antigen Vaccine for Genital Herpes [Vaccines and Antiviral Agents]
Date de mise en ligne : Mardi 01 juillet 2014 The molecular mechanism of the hepatic tropism of hepatitis C virus (HCV) remains incompletely defined. In vitro hepatic differentiation of pluripotent stem cells produces hepatocyte-like cells (HLCs) permissive for HCV infection, providing an opportunity for studying liver development and host determinants of HCV susceptibility. We previously identified the transition stage of HCV permissiveness and now investigate whether a host protein whose expression is induced during this transition stage is important for HCV infection. We suppressed the expression of a liver-specific protein, cell death-inducing DFFA-like effector b (CIDEB), and performed hepatocyte function and HCV infection assays. We also used a variety of cell-based assays to dissect the specific step of the HCV life cycle that potentially requires CIDEB function. We found CIDEB to be an essential cofactor for HCV entry into hepatocytes. Genetic interference with CIDEB in stem cells followed by hepatic differentiation leads to HLCs that are refractory to HCV infection, and infection time course experiments revealed that CIDEB functions in a late step of HCV entry, possibly to facilitate membrane fusion. The role of CIDEB in mediating HCV entry is distinct from those of the well-established receptors, as it is not required for HCV pseudoparticle entry. Finally, HCV infection effectively downregulates CIDEB protein through a posttranscriptional mechanism. IMPORTANCE This study identifies a hepatitis C virus (HCV) entry cofactor that is required for HCV infection of hepatocytes and potentially facilitates membrane fusion between viral and host membranes. CIDEB and its interaction with HCV may open up new avenues of investigation of lipid droplets and viral entry.
Wu, X., Lee, E. M., Hammack, C., Robotham, J. M., Basu, M., Lang, J., Brinton, M. A., Tang, H.
Cell Death-Inducing DFFA-Like Effector b Is Required for Hepatitis C Virus Entry into Hepatocytes [Virus-Cell Interactions]
Date de mise en ligne : Mardi 01 juillet 2014 Some animal influenza A viruses (IAVs) bind not only to N-acetylneuraminic acid (Neu5Ac) but also to N-glycolylneuraminic acid (Neu5Gc), which has been discussed as a virus receptor. Human cells cannot synthesize Neu5Gc due to dysfunction of the CMP-Neu5Ac hydroxylase (CMAH) gene, which converts CMP-Neu5Ac to CMP-Neu5Gc. However, exogenous Neu5Gc from Neu5Gc-rich dietary sources is able to be metabolically incorporated into surfaces of tissue cells and may be related to enhancement of the infectivity and severity of IAV. Here, we investigated the receptor function of Neu5Gc on IAV infection in Neu5Gc-expressing cells by transfection of the monkey CMAH gene into human cells or by incubation with human cells in the presence of N-glycolylmannosamine. Expression of Neu5Gc on human cells clearly suppressed infectivity of IAVs that possess Neu5Gc binding ability. Furthermore, there was no difference in infectivity of a transfectant virus that included the wild-type HA gene from A/Memphis/1/1971 (H3N2), which shows no Neu5Gc binding, between parent MCF7 cells and cells stably expressing the monkey CMAH gene (CMAH-MCF7 cells). On the other hand, cell entry of the transfectant virus that included the Neu5Gc-binding HA gene with a single mutation to Tyr at position Thr155 was arrested at the stage of internalization from the plasma membrane of the CMAH-MCF7 cells. These results indicate that expression of Neu5Gc on the surface of human epithelial cells suppresses infection of IAVs that possess Neu5Gc binding ability. Neu5Gc is suggested to work as a decoy receptor of Neu5Gc-binding IAVs but not a functional receptor for IAV infection. IMPORTANCE Influenza A viruses (IAVs) bind to the host cell surfaces through sialic acids at the terminal of glycoconjugates. For IAV binding to sialic acids, some IAVs bind not only to N-acetylneuraminic acid (Neu5Ac) as a receptor but also to N-glycolylneuraminic acid (Neu5Gc). Neu5Gc has been discussed as a receptor of human and animal IAVs. Our results showed that Neu5Gc expression on human epithelial cells suppresses infection of IAVs that possess Neu5Gc binding ability. Neu5Gc is suggested to be a "decoy receptor" of Neu5Gc-binding IAVs but not a functional receptor for IAV infection. Human cells cannot synthesize Neu5Gc because of dysfunction of the CMP-N-acetylneuraminic acid hydroxylase gene but can exogenously and metabolically incorporate Neu5Gc from dietary sources. The expression of Neu5Gc on human epithelial cells by taking in exogenous Neu5Gc from Neu5Gc-rich dietary sources may be related to restriction of the infection of IAVs that have acquired Neu5Gc binding ability.
Takahashi, T., Takano, M., Kurebayashi, Y., Masuda, M., Kawagishi, S., Takaguchi, M., Yamanaka, T., Minami, A., Otsubo, T., Ikeda, K., Suzuki, T.
N-Glycolylneuraminic Acid on Human Epithelial Cells Prevents Entry of Influenza A Viruses That Possess N-Glycolylneuraminic Acid Binding Ability [Virus-Cell Interactions]
Date de mise en ligne : Mardi 01 juillet 2014 West Nile virus (WNV) is an enveloped virus with a single-stranded positive-sense RNA genome from the Flaviviridae family. WNV is spread by mosquitoes and able to infect humans, causing encephalitis and meningitis that can be fatal; it therefore presents a significant risk for human health. In insects, innate response to RNA virus infection mostly relies on RNA interference and JAK/SAT pathways; however, some evidence indicates that it can also involve microRNAs (miRNAs). miRNAs are small noncoding RNAs that regulate gene expression at posttranscriptional level and play an important role in a number of processes, including immunity and antiviral response. In this study, we focus on the miRNA-mediated response to WNV in mosquito cells. We demonstrate that in response to WNV infection the expression of a mosquito-specific miRNA, aae-miR-2940, is selectively downregulated in Aedes albopictus cells. This miRNA is known to upregulate the metalloprotease m41 FtsH gene, which we have also shown to be required for efficient WNV replication. Correspondingly, downregulation of aae-miR-2940 reduced the metalloprotease level and restricted WNV replication. Thus, we have identified a novel miRNA-dependent mechanism of antiviral response to WNV in mosquitoes. IMPORTANCE A detailed understanding of vector-pathogen interactions is essential to address the problems posed by vector-borne diseases. Host and viral miRNAs play an important role in regulating expression of viral and host genes involved in endogenous processes, including antiviral response. There has been no evidence to date for the role of mosquito miRNAs in response to flaviviruses. In this study, we show that downregulation of aae-miR-2940 in mosquito cells acts as a potential antiviral mechanism in the mosquito host to inhibit WNV replication by repressing the expression of the metalloprotease m41 FtsH gene, which is required for efficient WNV replication. This is the first identification of an miRNA-dependent antiviral mechanism in mosquitoes, which inhibits replication of WNV. Our findings should facilitate identification of targets in the mosquito genome that can be utilized to suppress vector population and/or limit WNV replication.
Slonchak, A., Hussain, M., Torres, S., Asgari, S., Khromykh, A. A.
Expression of Mosquito MicroRNA Aae-miR-2940-5p Is Downregulated in Response to West Nile Virus Infection To Restrict Viral Replication [Cellular Response to Infection]
Date de mise en ligne : Mardi 01 juillet 2014 The possibility that vaccination with adenovirus (AdV) vectors increased mucosal T cell activation remains a central hypothesis to explain the potential enhancement of HIV acquisition within the Step trial. Modeling this within rhesus macaques is complicated because human adenoviruses, including human adenovirus type 5 (HAdV-5), are not endogenous to macaques. Here, we tested whether vaccination with a rhesus macaque-derived adenoviral vector (simian adenovirus 7 [SAdV-7]) enhances mucosal T cell activation within rhesus macaques. Following intramuscular SAdV-7 vaccination, we observed a pronounced increase in SAdV-7-specific CD4+ T cell responses in peripheral blood and, more dramatically, in rectal mucosa tissue. Vaccination also induced a significant increase in the frequency of activated memory CD4+ T cells in SAdV-7- and HAdV-5-vaccinated animals in the rectal mucosa but not in peripheral blood. These fluctuations within the rectal mucosa were also associated with a pronounced decrease in the relative frequency of naive resting CD4+ T cells. Together, these results indicate that peripheral vaccination with an AdV vector can increase the activation of mucosal CD4+ T cells, potentially providing an experimental model to further evaluate the role of host-vector interactions in increased HIV acquisition after AdV vector vaccination. IMPORTANCE The possibility that vaccination with a human adenovirus 5 vector increased mucosal T cell activation remains a central hypothesis to explain the potential enhancement of human immunodeficiency virus (HIV) acquisition within the Step trial. In this study, we tested whether vaccination with a rhesus macaque-derived adenoviral vector in rhesus macaques enhances mucosal CD4+ T cell activation, the main cell target of simian immunodeficiency virus (SIV)/HIV. The results showed that vaccination with an adenoviral vector indeed increases activation of mucosal CD4+ T cells and potentially increases susceptibility to SIV infection.
Bukh, I., Calcedo, R., Roy, S., Carnathan, D. G., Grant, R., Qin, Q., Boyd, S., Ratcliffe, S. J., Veeder, C. L., Bellamy, S. L., Betts, M. R., Wilson, J. M.
Increased Mucosal CD4+ T Cell Activation in Rhesus Macaques following Vaccination with an Adenoviral Vector [Vaccines and Antiviral Agents]
Date de mise en ligne : Mardi 01 juillet 2014 Interleukin-6 (IL-6) plays an important role in the development and progression of inflammatory responses, autoimmune diseases, and cancers. Many viral infections, including Theiler's murine encephalomyelitis virus (TMEV), result in the vigorous production of IL-6. However, the role of IL-6 in the development of virus-induced inflammatory responses is unclear. The infection of susceptible mice with TMEV induces the development of chronic demyelinating disease, which is considered a relevant infectious model for multiple sclerosis. In this study, we demonstrate that resistant C57BL/6 mice carrying an IL-6 transgene (IL-6 Tg) develop a TMEV-induced demyelinating disease accompanied by an increase in viral persistence and an elevated Th17 cell response in the central nervous system. Either IL-6 or IL-17 induced the expression of Bcl-2 and Bcl-xL at a high concentration. The upregulated expression of prosurvival molecules in turn inhibited target cell destruction by virus-specific CD8+ T cells. More interestingly, IL-6 and IL-17 synergistically promoted the expression of these prosurvival molecules, preventing cellular apoptosis at a much lower (<5-fold) concentration. The signals involved in the synergy appear to include the activation of both STAT3 and NF-B via distinct cytokine-dependent pathways. Thus, the excessive IL-6 promotes the generation of Th17 cells, and the resulting IL-6 and IL-17 synergistically promote viral persistence by protecting virus-infected cells from apoptosis and CD8+ T cell-mediated target destruction. These results suggest that blocking both IL-6 and IL-17 functions are important considerations for therapies of chronic viral diseases, autoimmune diseases, and cancers. IMPORTANCE This study indicates that an excessive level of IL-6 cytokine produced following viral infection promotes the development of IL-17-producing pathogenic helper T cells. We demonstrate here for the first time that IL-6 together with IL-17 synergistically enhances the expression of survival molecules to hinder critical host defense mechanisms removing virus-infected cells. This finding has an important implication in controlling not only chronic viral infections but also autoimmune diseases and cancers, which are associated with prolonged cell survival.
Hou, W., Jin, Y.-H., Kang, H. S., Kim, B. S.
Interleukin-6 (IL-6) and IL-17 Synergistically Promote Viral Persistence by Inhibiting Cellular Apoptosis and Cytotoxic T Cell Function [Pathogenesis and Immunity]
Date de mise en ligne : Mardi 01 juillet 2014 The maintenance of latent Kaposi's sarcoma-associated herpesvirus (KSHV) genomes is mediated in cis by their terminal repeats (TR). A KSHV genome can have 16 to 50 copies of the 801-bp TR, each of which harbors a 71-bp-long minimal replicator element (MRE). A single MRE can support replication in transient assays, and the presence of as few as two TRs appears to support establishment of KSHV-derived plasmids. Why then does KSHV have such redundancy and heterogeneity in the number of TRs? By determining the abilities of KSHV-derived plasmids containing various numbers of the TRs and MREs to be established and maintained in the long term, we have found that plasmids with fewer than 16 TRs or those with tandem repeats of the MREs are maintained inefficiently, as shown by both their decreased abilities to support formation of colonies and their instability, resulting in frequent rearrangements yielding larger plasmids during and after establishment. These defects often can be overcome by adding the Epstein-Barr virus (EBV) partitioning element, FR (i.e., family of repeats), in cis to these plasmids. In addition we have found that the spacing between MREs is important for their functions, too. Thus, two properties of KSHV's origin of latent replication essential for the efficient establishment and maintenance of viral plasmids stably are (i) the presence of approximately 16 copies of the TR, which are needed for efficient partitioning, and (ii) the presence of at least 2 MRE units separated by 801 bp of center-to-center spacing, which are required for efficient synthesis. IMPORTANCE KSHV is a human tumor virus that maintains its genome as a plasmid in lymphoid tumor cells. Each plasmid DNA molecule encodes many origins of synthesis. Here we show that these many origins provide an essential advantage to KSHV, allowing the DNAs to be maintained without rearrangement. We find also that the correct spacing between KSHV's origins of DNA synthesis is required for them to support synthesis efficiently. The identification of these properties illuminates plasmid replication in mammalian cells and should lead to the development of rational means to inhibit these tumorigenic replicons.
Shrestha, P., Sugden, B.
Identification of Properties of the Kaposi's Sarcoma-Associated Herpesvirus Latent Origin of Replication That Are Essential for the Efficient Establishment and Maintenance of Intact Plasmids [Genome Replication and Regulation of Viral Gene Expression]
Date de mise en ligne : Mardi 01 juillet 2014 In recent decades, Raman spectroscopy has entered the biological and medical fields. It enables nondestructive analysis of structural details at the molecular level and has been used to study viruses and their constituents. Here, we used Raman spectroscopy to study echovirus 1 (EV1), a small, nonenveloped human pathogen, in two different uncoating states induced by heat treatments. Raman signals of capsid proteins and RNA genome were observed from the intact virus, the uncoating intermediate, and disrupted virions. Transmission electron microscopy data revealed general structural changes between the studied particles. Compared to spectral characteristics of proteins in the intact virion, those of the proteins of the heat-treated particles indicated reduced α-helix content with respect to β-sheets and coil structures. Changes observed in tryptophan and tyrosine signals suggest an increasingly hydrophilic environment around these residues. RNA signals revealed a change in the environment of the genome and in its conformation. The ionized-carbonyl vibrations showed small changes between the intact virion and the uncoating intermediate, which points to cleavage of salt bridges in the protein structure during the uncoating process. In conclusion, our data reveal distinguishable Raman signatures of the intact, intermediate, and disrupted EV1 particles. These changes indicate structural, chemical, and solute-solvent alterations in the genome and in the capsid proteins and lay the essential groundwork for investigating the uncoating of EV1 and related viruses in real time. IMPORTANCE In order to combat virus infection, we need to know the details of virus uncoating. We present here the novel Raman signatures for opened and intact echovirus 1. This gives hope that the signatures may be used in the near future to evaluate the ambient conditions in endosomes leading to virus uncoating using, e.g., coherent anti-Stokes Raman spectroscopy (CARS) imaging. These studies will complement structural studies on virus uncoating. In addition, Raman/CARS imaging offers the possibility of making dynamic live measurements in vitro and in cells which are impossible to measure by, for example, cryo-electron tomography. Furthermore, as viral Raman spectra can be overwhelmed with various contaminants, our study is highly relevant in demonstrating the importance of sample preparation for Raman spectroscopy in the field of virology.
Ruokola, P., Dadu, E., Kazmertsuk, A., Hakkanen, H., Marjomaki, V., Ihalainen, J. A.
Raman Spectroscopic Signatures of Echovirus 1 Uncoating [Structure and Assembly]
Date de mise en ligne : Mardi 01 juillet 2014 The fidelity of human immunodeficiency virus (HIV) reverse transcriptase (RT) has been a subject of intensive investigation. The mutation frequencies for the purified enzyme in vitro vary widely but are typically in the 10–4 range (per nucleotide addition), making the enzyme severalfold less accurate than most polymerases, including other RTs. This has often been cited as a factor in HIV's accelerated generation of genetic diversity. However, cellular experiments suggest that HIV does not have significantly lower fidelity than other retroviruses and shows a mutation frequency in the 10–5 range. In this report, we reconcile, at least in part, these discrepancies by showing that HIV RT fidelity in vitro is in the same range as cellular results from experiments conducted with physiological (for lymphocytes) concentrations of free Mg2+ (~0.25 mM) and is comparable to Moloney murine leukemia virus (MuLV) RT fidelity. The physiological conditions produced mutation rates that were 5 to 10 times lower than those obtained under typically employed in vitro conditions optimized for RT activity (5 to 10 mM Mg2+). These results were consistent in both commonly used lacZα complementation and steady-state fidelity assays. Interestingly, although HIV RT showed severalfold-lower fidelity under high-Mg2+ (6 mM) conditions, MuLV RT fidelity was insensitive to Mg2+. Overall, the results indicate that the fidelity of HIV replication in cells is compatible with findings of experiments carried out in vitro with purified HIV RT, providing more physiological conditions are used. IMPORTANCE Human immunodeficiency virus rapidly evolves through the generation and subsequent selection of mutants that can circumvent the immune response and escape drug therapy. This process is fueled, in part, by the presumably highly error-prone HIV polymerase reverse transcriptase (RT). Paradoxically, results of studies examining HIV replication in cells indicate an error frequency that is ~10 times lower than the rate for RT in the test tube, which invokes the possibility of factors that make RT more accurate in cells. This study brings the cellular and test tube results in closer agreement by showing that HIV RT is not more error prone than other RTs and, when assayed under physiological magnesium conditions, has a much lower error rate than in typical assays conducted using conditions optimized for enzyme activity.
Achuthan, V., Keith, B. J., Connolly, B. A., DeStefano, J. J.
Human Immunodeficiency Virus Reverse Transcriptase Displays Dramatically Higher Fidelity under Physiological Magnesium Conditions In Vitro [Genome Replication and Regulation of Viral Gene Expression]
Date de mise en ligne : Mardi 01 juillet 2014 Activation of the ATM (ataxia telangiectasia-mutated kinase)-dependent DNA damage response (DDR) is necessary for productive replication of human papillomavirus 31 (HPV31). We previously found that DNA repair and homologous recombination (HR) factors localize to sites of HPV replication, suggesting that ATM activity is required to recruit factors to viral genomes that can productively replicate viral DNA in a recombination-dependent manner. The Mre11-Rad50-Nbs1 (MRN) complex is an essential component of the DDR that is necessary for ATM-mediated HR repair and localizes to HPV DNA foci. In this study, we demonstrate that the HPV E7 protein is sufficient to increase levels of the MRN complex and also interacts with MRN components. We have found that Nbs1 depletion blocks productive viral replication and results in decreased localization of Mre11, Rad50, and the principal HR factor Rad51 to HPV DNA foci upon differentiation. Nbs1 contributes to the DDR by acting as an upstream activator of ATM in response to double-strand DNA breaks (DSBs) and as a downstream effector of ATM activity in the intra-S-phase checkpoint. We have found that phosphorylation of ATM and its downstream target Chk2, as well as SMC1 (structural maintenance of chromosome 1), is maintained upon Nbs1 knockdown in differentiating cells. Given that ATM and Chk2 are required for productive replication, our results suggest that Nbs1 contributes to viral replication outside its role as an ATM activator, potentially through ensuring localization of DNA repair factors to viral genomes that are necessary for efficient productive replication. IMPORTANCE The mechanisms that regulate human papillomavirus (HPV) replication during the viral life cycle are not well understood. Our finding that Nbs1 is necessary for productive replication even in the presence of ATM (ataxia telangiectasia-mutated kinase) and Chk2 phosphorylation offers evidence that Nbs1 contributes to viral replication downstream of facilitating ATM activation. Nbs1 is required for the recruitment of Mre11 and Rad50 to viral genomes, suggesting that the MRN complex plays a direct role in facilitating productive viral replication, potentially through the processing of substrates that are recognized by the key homologous recombination (HR) factor Rad51. The discovery that E7 increases levels of MRN components, and MRN complex formation, identifies a novel role for E7 in facilitating productive replication. Our study not only identifies DNA repair factors necessary for HPV replication but also provides a deeper understanding of how HPV utilizes the DNA damage response to regulate viral replication.
Anacker, D. C., Gautam, D., Gillespie, K. A., Chappell, W. H., Moody, C. A.
Productive Replication of Human Papillomavirus 31 Requires DNA Repair Factor Nbs1 [Genome Replication and Regulation of Viral Gene Expression]
Date de mise en ligne : Mardi 01 juillet 2014 The human papillomavirus (HPV) E1 helicase promotes viral DNA replication through its DNA unwinding activity and association with host factors. The E1 proteins from anogenital HPV types interact with the cellular WD repeat-containing factor UAF1 (formerly known as p80). Specific amino acid substitutions in E1 that impair this interaction inhibit maintenance of the viral episome in immortalized keratinocytes and reduce viral DNA replication by up to 70% in transient assays. In this study, we determined by affinity purification of UAF1 that it interacts with three deubiquitinating enzymes in C33A cervical carcinoma cells: USP1, a nuclear protein, and the two cytoplasmic enzymes USP12 and USP46. Coimmunoprecipitation experiments indicated that E1 assembles into a ternary complex with UAF1 and any one of these three USPs. Moreover, expression of E1 leads to a redistribution of USP12 and USP46 from the cytoplasm to the nucleus. Chromatin immunoprecipitation studies further revealed that E1 recruits these threes USPs to the viral origin in association with UAF1. The function of USP1, USP12, and USP46 in viral DNA replication was investigated by overproduction of catalytically inactive versions of these enzymes in transient assays. All three dominant negative USPs reduced HPV31 DNA replication by up to 60%, an effect that was specific, as it was not observed in assays performed with a truncated E1 lacking the UAF1-binding domain or with bovine papillomavirus 1 E1, which does not bind UAF1. These results highlight the importance of the USP1, USP12, and USP46 deubiquitinating enzymes in anogenital HPV DNA replication. IMPORTANCE Human papillomaviruses are small DNA tumor viruses that induce benign and malignant lesions of the skin and mucosa. HPV types that infect the anogenital tract are the etiological agents of cervical cancer, the majority of anal cancers, and a growing proportion of head-and-neck cancers. Replication of the HPV genome requires the viral protein E1, a DNA helicase that also interacts with host factors to promote viral DNA synthesis. We previously reported that the E1 helicase from anogenital HPV types associates with the WD40 repeat-containing protein UAF1. Here, we show that UAF1 bridges the interaction of E1 with three deubiquitinating enzymes, USP1, USP12, and USP46. We further show that these deubiquitinases are recruited by E1/UAF1 to the viral origin of DNA replication and that overexpression of catalytically inactive versions of these enzymes reduces viral DNA replication. These results highlight the need for an E1-associated deubiquitinase activity in anogenital HPV genome replication.
Lehoux, M., Gagnon, D., Archambault, J.
E1-Mediated Recruitment of a UAF1-USP Deubiquitinase Complex Facilitates Human Papillomavirus DNA Replication [Genome Replication and Regulation of Viral Gene Expression]
Date de mise en ligne : Mardi 01 juillet 2014 The family Arenaviridae includes a number of viruses of public health importance, such as the category A hemorrhagic fever viruses Lassa virus, Junin virus, Machupo virus, Guanarito virus, and Sabia virus. Current chemotherapy for arenavirus infection is limited to the nucleoside analogue ribavirin, which is characterized by considerable toxicity and treatment failure. Using Pichinde virus as a model arenavirus, we attempted to design glycoprotein-derived fusion inhibitors similar to the FDA-approved anti-HIV peptide enfuvirtide. We have identified a GP2-derived peptide, AVP-p, with antiviral activity and no acute cytotoxicity. The 50% inhibitory dose (IC50) for the peptide is 7 μM, with complete inhibition of viral plaque formation at approximately 20 μM, and its antiviral activity is largely sequence dependent. AVP-p demonstrates activity against viruses with the Old and New World arenavirus viral glycoprotein complex but not against enveloped viruses of other families. Unexpectedly, fusion assays reveal that the peptide induces virus-liposome fusion at neutral pH and that the process is strictly glycoprotein mediated. As observed in cryo-electron micrographs, AVP-p treatment causes morphological changes consistent with fusion protein activation in virions, including the disappearance of prefusion glycoprotein spikes and increased particle diameters, and fluorescence microscopy shows reduced binding by peptide-treated virus. Steady-state fluorescence anisotropy measurements suggest that glycoproteins are destabilized by peptide-induced alterations in viral membrane order. We conclude that untimely deployment of fusion machinery by the peptide could render virions less able to engage in on-pathway receptor binding or endosomal fusion. AVP-p may represent a potent, highly specific, novel therapeutic strategy for arenavirus infection. IMPORTANCE Because the only drug available to combat infection by Lassa virus, a highly pathogenic arenavirus, is toxic and prone to treatment failure, we identified a peptide, AVP-p, derived from the fusion glycoprotein of a nonpathogenic model arenavirus, which demonstrates antiviral activity and no acute cytotoxicity. AVP-p is unique among self-derived inhibitory peptides in that it shows broad, specific activity against pseudoviruses bearing Old and New World arenavirus glycoproteins but not against viruses from other families. Further, the peptide's mechanism of action is highly novel. Biochemical assays and cryo-electron microscopy indicate that AVP-p induces premature activation of viral fusion proteins through membrane perturbance. Peptide treatment, however, does not increase the infectivity of cell-bound virus. We hypothesize that prematurely activated virions are less fit for receptor binding and membrane fusion and that AVP-p may represent a viable therapeutic strategy for arenavirus infection.
Spence, J. S., Melnik, L. I., Badani, H., Wimley, W. C., Garry, R. F.
Inhibition of Arenavirus Infection by a Glycoprotein-Derived Peptide with a Novel Mechanism [Virus-Cell Interactions]
Date de mise en ligne : Mardi 01 juillet 2014 The Bunyaviridae constitute a large family of enveloped animal viruses, many of which are important emerging pathogens. How bunyaviruses enter and infect mammalian cells remains largely uncharacterized. We used two genome-wide silencing screens with distinct small interfering RNA (siRNA) libraries to investigate host proteins required during infection of human cells by the bunyavirus Uukuniemi virus (UUKV), a late-penetrating virus. Sequence analysis of the libraries revealed that many siRNAs in the screens inhibited infection by silencing not only the intended targets but additional genes in a microRNA (miRNA)-like manner. That the 7-nucleotide seed regions in the siRNAs can cause a perturbation in infection was confirmed by using synthetic miRNAs (miRs). One of the miRs tested, miR-142-3p, was shown to interfere with the intracellular trafficking of incoming viruses by regulating the v-SNARE VAMP3, a strong hit shared by both siRNA screens. Inactivation of VAMP3 by the tetanus toxin led to a block in infection. Using fluorescence-based techniques in fixed and live cells, we found that the viruses enter VAMP3+ endosomal vesicles 5 min after internalization and that colocalization was maximal 15 min thereafter. At this time, LAMP1 was associated with the VAMP3+ virus-containing endosomes. In cells depleted of VAMP3, viruses were mainly trapped in LAMP1-negative compartments. Together, our results indicated that UUKV relies on VAMP3 for penetration, providing an indication of added complexity in the trafficking of viruses through the endocytic network. IMPORTANCE Bunyaviruses represent a growing threat to humans and livestock globally. Unfortunately, relatively little is known about these emerging pathogens. We report here the first human genome-wide siRNA screens for a bunyavirus. The screens resulted in the identification of 562 host cell factors with a potential role in cell entry and virus replication. To demonstrate the robustness of our approach, we confirmed and analyzed the role of the v-SNARE VAMP3 in Uukuniemi virus entry and infection. The information gained lays the basis for future research into the cell biology of bunyavirus infection and new antiviral strategies. In addition, by shedding light on serious caveats in large-scale siRNA screening, our experimental and bioinformatics procedures will be valuable in the comprehensive analysis of past and future high-content screening data.
Meier, R., Franceschini, A., Horvath, P., Tetard, M., Mancini, R., von Mering, C., Helenius, A., Lozach, P.-Y.
Genome-Wide Small Interfering RNA Screens Reveal VAMP3 as a Novel Host Factor Required for Uukuniemi Virus Late Penetration [Virus-Cell Interactions]
Date de mise en ligne : Mardi 01 juillet 2014 Naturally occurring Newcastle disease virus (NDV) strains vary greatly in virulence. The presence of multibasic residues at the proteolytic cleavage site of the fusion (F) protein has been shown to be a primary determinant differentiating virulent versus avirulent strains. However, there is wide variation in virulence among virulent strains. There also are examples of incongruity between cleavage site sequence and virulence. These observations suggest that additional viral factors contribute to virulence. In this study, we evaluated the contribution of each viral gene to virulence individually and in different combinations by exchanging genes between velogenic (highly virulent) strain GB Texas (GBT) and mesogenic (moderately virulent) strain Beaudette C (BC). These two strains are phylogenetically closely related, and their F proteins contain identical cleavage site sequences, 112RRQKRF117. A total of 20 chimeric viruses were constructed and evaluated in vitro, in 1-day-old chicks, and in 2-week-old chickens. The results showed that both the envelope-associated and polymerase-associated proteins contribute to the difference in virulence between rBC and rGBT, with the envelope-associated proteins playing the greater role. The F protein was the major individual contributor and was sometimes augmented by the homologous M and HN proteins. The dramatic effect of F was independent of its cleavage site sequence since that was identical in the two strains. The polymerase L protein was the next major individual contributor and was sometimes augmented by the homologous N and P proteins. The leader and trailer regions did not appear to contribute to the difference in virulence between BC and GBT. IMPORTANCE This study is the first comprehensive and systematic study of NDV virulence and pathogenesis. Genetic exchanges between a mesogenic and a velogenic strain revealed that the fusion glycoprotein is the major virulence determinant regardless of the identical virulence protease cleavage site sequence present in both strains. The contribution of the large polymerase protein to NDV virulence is second only to that of the fusion glycoprotein. The identification of virulence determinants is of considerable importance, because of the potential to generate better live attenuated NDV vaccines. It may also be possible to apply these findings to other paramyxoviruses.
Paldurai, A., Kim, S.-H., Nayak, B., Xiao, S., Shive, H., Collins, P. L., Samal, S. K.
Evaluation of the Contributions of Individual Viral Genes to Newcastle Disease Virus Virulence and Pathogenesis [Pathogenesis and Immunity]
Date de mise en ligne : Mardi 01 juillet 2014 Severe acute respiratory syndrome-related coronavirus (SARS-CoV) is an emerging pathogen that causes severe respiratory illness. Whole UV-inactivated SARS-CoV (UV-V), bearing multiple epitopes and proteins, is a candidate vaccine against this virus. However, whole inactivated SARS vaccine that includes nucleocapsid protein is reported to induce eosinophilic infiltration in mouse lungs after challenge with live SARS-CoV. In this study, an ability of Toll-like receptor (TLR) agonists to reduce the side effects of UV-V vaccination in a 6-month-old adult BALB/c mouse model was investigated, using the mouse-passaged Frankfurt 1 isolate of SARS-CoV. Immunization of adult mice with UV-V, with or without alum, resulted in partial protection from lethal doses of SARS-CoV challenge, but extensive eosinophil infiltration in the lungs was observed. In contrast, TLR agonists added to UV-V vaccine, including lipopolysaccharide, poly(U), and poly(I·C) (UV-V+TLR), strikingly reduced excess eosinophilic infiltration in the lungs and induced lower levels of interleukin-4 and -13 and eotaxin in the lungs than UV-V-immunization alone. Additionally, microarray analysis showed that genes associated with chemotaxis, eosinophil migration, eosinophilia, and cell movement and the polarization of Th2 cells were upregulated in UV-V-immunized but not in UV-V+TLR-immunized mice. In particular, CD11b+ cells in the lungs of UV-V-immunized mice showed the upregulation of genes associated with the induction of eosinophils after challenge. These findings suggest that vaccine-induced eosinophil immunopathology in the lungs upon SARS-CoV infection could be avoided by the TLR agonist adjuvants. IMPORTANCE Inactivated whole severe acute respiratory syndrome-related coronavirus (SARS-CoV) vaccines induce neutralizing antibodies in mouse models; however, they also cause increased eosinophilic immunopathology in the lungs upon SARS-CoV challenge. In this study, the ability of adjuvant Toll-like receptor (TLR) agonists to reduce the side effects of UV-inactivated SARS-CoV vaccination in a BALB/c mouse model was tested, using the mouse-passaged Frankfurt 1 isolate of SARS-CoV. We found that TLR stimulation reduced the high level of eosinophilic infiltration that occurred in the lungs of mice immunized with UV-inactivated SARS-CoV. Microarray analysis revealed that genes associated with chemotaxis, eosinophil migration, eosinophilia, and cell movement and the polarization of Th2 cells were upregulated in UV-inactivated SARS-CoV-immunized mice. This study may be helpful for elucidating the pathogenesis underlying eosinophilic infiltration resulting from immunization with inactivated vaccine.
Iwata-Yoshikawa, N., Uda, A., Suzuki, T., Tsunetsugu-Yokota, Y., Sato, Y., Morikawa, S., Tashiro, M., Sata, T., Hasegawa, H., Nagata, N.
Effects of Toll-Like Receptor Stimulation on Eosinophilic Infiltration in Lungs of BALB/c Mice Immunized with UV-Inactivated Severe Acute Respiratory Syndrome-Related Coronavirus Vaccine [Vaccines and Antiviral Agents]
Date de mise en ligne : Mardi 01 juillet 2014 Cowpox viruses (CPXV) cause hemorrhagic lesions ("red pocks") on infected chorioallantoic membranes (CAM) of embryonated chicken eggs, while most other members of the genus Orthopoxvirus produce nonhemorrhagic lesions ("white pocks"). Cytokine response modifier A (CrmA) of CPXV strain Brighton Red (BR) is necessary but not sufficient for the induction of red pocks. To identify additional viral proteins involved in the induction of hemorrhagic lesions, a library of single-gene CPXV knockout mutants was screened. We identified 10 proteins that are required for the formation of hemorrhagic lesions, which are encoded by CPXV060, CPXV064, CPXV068, CPXV069, CPXV074, CPXV136, CPXV168, CPXV169, CPXV172, and CPXV199. The genes are the homologues of F12L, F15L, E2L, E3L, E8R, A4L, A33R, A34R, A36R, and B5R of vaccinia virus (VACV). Mutants with deletions in CPXV060, CPXV168, CPXV169, CPXV172, or CPXV199 induced white pocks with a comet-like shape on the CAM. The homologues of these five genes in VACV encode proteins that are involved in the production of extracellular enveloped viruses (EEV) and the repulsion of superinfecting virions by actin tails. The homologue of CPXV068 in VACV is also involved in EEV production but is not related to actin tail induction. The other genes encode immunomodulatory proteins (CPXV069 and crmA) and viral core proteins (CPXV074 and CPXV136), and the function of the product of CPXV064 is unknown. IMPORTANCE It has been known for a long time that cowpox virus induces hemorrhagic lesions on chicken CAM, while most of the other orthopoxviruses produce nonhemorrhagic lesions. Although cowpox virus CrmA has been proved to be responsible for the hemorrhagic phenotype, other proteins causing this phenotype remain unknown. Recently, we generated a complete single-gene knockout bacterial artificial chromosome (BAC) library of cowpox virus Brighton strain. Out of 183 knockout BAC clones, 109 knockout viruses were reconstituted. The knockout library makes possible high-throughput screening for studying poxvirus replication and pathogenesis. In this study, we screened all 109 single-gene knockout viruses and identified 10 proteins necessary for inducing hemorrhagic lesions. The identification of these genes gives a new perspective for studying the hemorrhagic phenotype and may give a better understanding of poxvirus virulence.
Xu, Z., Zikos, D., Tamošiūnaite, A., Klopfleisch, R., Osterrieder, N., Tischer, B. K.
Identification of 10 Cowpox Virus Proteins That Are Necessary for Induction of Hemorrhagic Lesions (Red Pocks) on Chorioallantoic Membranes [Pathogenesis and Immunity]
Date de mise en ligne : Mardi 01 juillet 2014 Generalized immune activation during HIV infection is associated with an increased risk of cardiovascular disease, neurocognitive disease, osteoporosis, metabolic disorders, and physical frailty. The mechanisms driving this immune activation are poorly understood, particularly for individuals effectively treated with antiretroviral medications. We hypothesized that viral characteristics such as sequence diversity may play a role in driving HIV-associated immune activation. We therefore sequenced proviral DNA isolated from peripheral blood mononuclear cells from HIV-infected individuals on fully suppressive antiretroviral therapy. We performed phylogenetic analyses, calculated viral diversity and divergence in the env and pol genes, and determined coreceptor tropism and the frequency of drug resistance mutations. Comprehensive immune profiling included quantification of immune cell subsets, plasma cytokine levels, and intracellular signaling responses in T cells, B cells, and monocytes. These antiretroviral therapy-treated HIV-infected individuals exhibited a wide range of diversity and divergence in both env and pol genes. However, proviral diversity and divergence in env and pol, coreceptor tropism, and the level of drug resistance did not significantly correlate with markers of immune activation. A clinical history of virologic failure was also not significantly associated with levels of immune activation, indicating that a history of virologic failure does not inexorably lead to increased immune activation as long as suppressive antiretroviral medications are provided. Overall, this study demonstrates that latent viral diversity is unlikely to be a major driver of persistent HIV-associated immune activation. IMPORTANCE Chronic immune activation, which is associated with cardiovascular disease, neurologic disease, and early aging, is likely to be a major driver of morbidity and mortality in HIV-infected individuals. Although treatment of HIV with antiretroviral medications decreases the level of immune activation, levels do not return to normal. The factors driving this persistent immune activation, particularly during effective treatment, are poorly understood. In this study, we investigated whether characteristics of the latent, integrated HIV provirus that persists during treatment are associated with immune activation. We found no relationship between latent viral characteristics and immune activation in treated individuals, indicating that qualities of the provirus are unlikely to be a major driver of persistent inflammation. We also found that individuals who had previously failed treatment but were currently effectively treated did not have significantly increased levels of immune activation, providing hope that past treatment failures do not have a lifelong "legacy" impact.
Liang, E. C., Sceats, L., Bayless, N. L., Strauss-Albee, D. M., Kubo, J., Grant, P. M., Furman, D., Desai, M., Katzenstein, D. A., Davis, M. M., Zolopa, A. R., Blish, C. A.
Association between Latent Proviral Characteristics and Immune Activation in Antiretrovirus-Treated Human Immunodeficiency Virus Type 1-Infected Adults [Pathogenesis and Immunity]
Date de mise en ligne : Mardi 01 juillet 2014 Natural transmission of prion diseases depends upon the spread of prions from the nervous system to excretory or secretory tissues, but the mechanism of prion transport in axons and into peripheral tissue is unresolved. Here, we examined the temporal and spatial movement of prions from the brain stem along cranial nerves into skeletal muscle as a model of axonal transport and transynaptic spread. The disease-specific isoform of the prion protein, PrPSc, was observed in nerve fibers of the tongue approximately 2 weeks prior to PrPSc deposition in skeletal muscle. Initially, PrPSc deposits had a small punctate pattern on the edge of muscle cells that colocalized with synaptophysin, a marker for the neuromuscular junction (NMJ), in >50% of the cells. At later time points PrPSc was widely distributed in muscle cells, but <10% of prion-infected cells exhibited PrPSc deposition at the NMJ, suggesting additional prion replication and dissemination within muscle cells. In contrast to the NMJ, PrPSc was not associated with synaptophysin in nerve fibers but was found to colocalize with LAMP-1 and cathepsin D during early stages of axonal spread. We propose that PrPSc-bound endosomes can lead to membrane recycling in which PrPSc is directed to the synapse, where it either moves across the NMJ into the postsynaptic muscle cell or induces PrPSc formation on muscle cells across the NMJ. IMPORTANCE Prion diseases are transmissible and fatal neurodegenerative diseases in which prion dissemination to excretory or secretory tissues is necessary for natural disease transmission. Despite the importance of this pathway, the cellular mechanism of prion transport in axons and into peripheral tissue is unresolved. This study demonstrates anterograde spread of prions within nerve fibers prior to infection of peripheral synapses (i.e., neuromuscular junction) and infection of peripheral tissues (i.e., muscle cells). Within nerve fibers prions were associated with the endosomal-lysosomal pathway prior to entry into muscle cells. Since early prion spread is anterograde and endosome-lysosomal movement within axons is primarily retrograde, these findings suggest that endosome-bound prions may have an alternate fate that directs prions to the peripheral synapse.
Shearin, H., Bessen, R. A.
Axonal and Transynaptic Spread of Prions [Prions]
Date de mise en ligne : Mardi 01 juillet 2014 Hepatitis B virus (HBV) quasispecies contain a large number of variants that serve as a reservoir for viral selection under antiviral treatment and the immune response, leading to the acute exacerbation and subsequent development of liver failure. However, there is no clear experimental evidence for a significant role of HBV quasispecies in viral pathogenesis. In the present study, HBV sequences were amplified from a patient with severe liver disease and used for construction of HBV replication-competent plasmids. Western blotting, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence staining were performed to analyze the expression, secretion, and subcellular localization of viral proteins in vitro. Viral replication intermediates were detected by Southern blotting. HBV gene expression and replication and the induction of specific immune responses in an HBV hydrodynamic injection (HI) mouse model were investigated. The results demonstrated that two naturally occurring HBV variants, SH and SH-DPS, were identified. The variant SH-DPS expressed only a nonexportable hepatitis B virus surface antigen (HBsAg) with abnormal intracellular accumulation. The coexistence of the HBV variants at a ratio of 1 to 4 (SH to SH-DPS) increased HBV replication. Significantly stronger intrahepatic cytotoxic T lymphocyte (CTL) responses and antibody responses specific to HBsAg were induced in mice by the HBV variants when coapplied by HI. These findings uncovered an unexpected aspect of HBV quasispecies: the coexistence of different variants can significantly modulate specific host immune responses, representing a novel mechanism for the immunopathogenesis of HBV infection. IMPORTANCE Hepatitis B virus (HBV) is an important human pathogen. HBV quasispecies with genetically heterogenous variants are thought to play a role in the progression of HBV-associated liver diseases. So far, direct evidence is available in only a few cases to confirm the proposed role of HBV variants in the pathogenesis. We report here that the coexistence of two naturally occurring HBV variants at a ratio of 1 to 4 increased HBV replication and induced significantly stronger intrahepatic cytotoxic T lymphocyte responses and antibody responses specific to HBV surface antigen (HBsAg) in mice. Our discovery uncovered an unexpected aspect of HBV quasispecies: the coexistence of different variants can significantly modulate specific host immune responses and may enhance immune-mediated liver damage under some circumstances, representing a novel mechanism for the immunopathogenesis of HBV infection.
Cao, L., Wu, C., Shi, H., Gong, Z., Zhang, E., Wang, H., Zhao, K., Liu, S., Li, S., Gao, X., Wang, Y., Pei, R., Lu, M., Chen, X.
Coexistence of Hepatitis B Virus Quasispecies Enhances Viral Replication and the Ability To Induce Host Antibody and Cellular Immune Responses [Pathogenesis and Immunity]
Date de mise en ligne : Mardi 01 juillet 2014 Apoptosis is a tightly regulated process that plays a crucial role in the removal of virus-infected cells, a process controlled by both pro- and antiapoptotic members of the Bcl-2 family. The proapoptotic proteins Bak and Bax are regulated by antiapoptotic Bcl-2 proteins and are also activated by a subset of proteins known as BH3-only proteins that perform dual functions by directly activating Bak and Bax or by sequestering and neutralizing antiapoptotic family members. Numerous viruses express proteins that prevent premature host cell apoptosis. Vaccinia virus encodes F1L, an antiapoptotic protein essential for survival of infected cells that bears no discernible sequence homology to mammalian cell death inhibitors. Despite the limited sequence similarities, F1L has been shown to adopt a novel dimeric Bcl-2-like fold that enables hetero-oligomeric binding to both Bak and the proapoptotic BH3-only protein Bim that ultimately prevents Bak and Bax homo-oligomerization. However, no structural data on the mode of engagement of F1L and its Bcl-2 counterparts are available. Here we solved the crystal structures of F1L in complex with two ligands, Bim and Bak. Our structures indicate that F1L can engage two BH3 ligands simultaneously via the canonical Bcl-2 ligand binding grooves. Furthermore, by structure-guided mutagenesis, we generated point mutations within the binding pocket of F1L in order to elucidate the residues responsible for both Bim and Bak binding and prevention of apoptosis. We propose that the sequestration of Bim by F1L is primarily responsible for preventing apoptosis during vaccinia virus infection. IMPORTANCE Numerous viruses have adapted strategies to counteract apoptosis by encoding proteins responsible for sequestering proapoptotic components. Vaccinia virus, the prototypical member of the family Orthopoxviridae, encodes a protein known as F1L that functions to prevent apoptosis by interacting with Bak and the BH3-only protein Bim. Despite recent structural advances, little is known regarding the mechanics of binding between F1L and the proapoptotic Bcl-2 family members. Utilizing three-dimensional structures of F1L bound to host proapoptotic proteins, we generated variants of F1L that neutralize Bim and/or Bak. We demonstrate that during vaccinia virus infection, engagement of Bim and Bak by F1L is crucial for subversion of host cell apoptosis.
Campbell, S., Thibault, J., Mehta, N., Colman, P. M., Barry, M., Kvansakul, M.
Structural Insight into BH3 Domain Binding of Vaccinia Virus Antiapoptotic F1L [Virus-Cell Interactions]
Date de mise en ligne : Mardi 01 juillet 2014 The dietary exposure of the human population to the prions responsible for the bovine spongiform encephalopathy (BSE) epizooty has led to the emergence of variant Creutzfeldt-Jakob disease (vCJD). This fatal, untreatable neurodegenerative disorder is a growing public health concern because the prevalence of the infection seems much greater than the disease incidence and because secondary transmission of vCJD by blood transfusion or use of blood products has occurred. A current limitation in variant CJD risk assessment is the lack of quantitative information on the infectivity of contaminated tissues. To address this limitation, we tested the potential of a transgenic mouse line overexpressing human prion protein (PrP), which was previously reported to propagate vCJD prions. Endpoint titration of vCJD infectivity in different tissues was evaluated by two different methods: (i) the "classical" bioassay, based on the appearance of clinical symptoms and the detection of pathological prion protein in tissues of the inoculated mouse, and (ii) a shortened bioassay based on the detection of the protein in the mouse spleen at defined time points. The two methods proved equally sensitive in quantifying infectivity, even after very-low-dose inoculation of infected material, but the time schedule was shortened from ~2.5 years to ~1 year with the spleen bioassay. Compared to the "gold-standard" RIII model routinely used for endpoint titration of vCJD/BSE prions, either method improved the sensitivity by >2 orders of magnitude and allowed reevaluating the infectious titer of spleen from a vCJD individual at disease end stage to >1,000-fold-higher values. IMPORTANCE Here, we provide key reevaluation of the infectious titer of variant CJD brain and spleen tissues. The highly sensitive, accelerated spleen-based assay should thus constitute a key advance for variant CJD epidemiological and risk assessment purposes and should greatly facilitate future titration studies, including, for example, those aimed at validating decontamination procedures. The overlooked notion that the lymphoid tissue exhibits a higher capacity than the brain to replicate prions even after low-dose infection raises new questions about the molecular and/or cellular determinant(s) involved, a key issue regarding potent silent carriers of variant CJD in the lymphoid tissue.
Halliez, S., Reine, F., Herzog, L., Jaumain, E., Haik, S., Rezaei, H., Vilotte, J.-L., Laude, H., Beringue, V.
Accelerated, Spleen-Based Titration of Variant Creutzfeldt-Jakob Disease Infectivity in Transgenic Mice Expressing Human Prion Protein with Sensitivity Comparable to That of Survival Time Bioassay [Prions]
Date de mise en ligne : Mardi 01 juillet 2014 Kaposi's sarcoma-associated herpesvirus (KSHV) ORF6 is homologous to the herpes simplex virus 1 (HSV-1) ICP8 and Epstein-Barr virus (EBV) BALF2 proteins. Here, we describe its single-stranded DNA (ssDNA) binding properties. Based on previous findings with ICP8 and BALF2, a 60-amino-acid C-terminal deletion mutant of Orf6 was generated, and the protein was purified to explore the function of the C terminus in ssDNA binding. We showed that full-length ORF6 binds cooperatively to M13 ssDNA, disrupting its secondary structure and extending it to a length equivalent to that of duplex M13 DNA. The width of the ORF6-ssDNA filament is 9 nm, and a 7.3-nm repeat can be distinguished along the filament axis. Fluorescence polarization analysis revealed that the wild-type and C-terminal mutant ORF6 proteins bind equally well to short ssDNA substrates, with dissociation constant (Kd) values of 2.2 x 10–7M and 1.5 x 10–7M, respectively. These values were confirmed by electrophoretic mobility shift assay (EMSA) analysis, which also suggested that binding by the full-length protein may involve both monomers and small multimers. While no significant difference in affinities of binding between full-length ORF6 and the C-terminal deletion mutant were observed with the short DNAs, binding of the C-terminal mutant protein to M13 ssDNA showed a clear lack of cooperativity as seen by electron microscopy (EM). Incubation of a duplex DNA containing a long single-stranded tail with double-helical ORF6 protein filaments revealed that the ssDNA segment can be enveloped within the protein filament without disrupting the filament structure. IMPORTANCE This work describes the biochemical characterization of the single-stranded DNA binding protein of KSHV, ORF6, central to viral DNA replication in infected cells. A C-terminal deletion mutant protein was generated to aid in understanding the role of the C terminus in DNA binding. Here we analyze the binding of the wild-type and mutant proteins to short oligomeric and longer genomic ssDNA substrates. Although it is capable of interacting with the short substrates, the inability of mutant ORF6 to form oligomers in solution hindered it from fully covering the long genomic substrates. We previously showed that ORF6 forms long filaments in solution, and we showed here that these can absorb ssDNA without disruption of the filament structure. This work will provide an important basis for future studies by us and/or others.
Ozgur, S., Griffith, J.
Interaction of Kaposi's Sarcoma-Associated Herpesvirus ORF6 Protein with Single-Stranded DNA [Genome Replication and Regulation of Viral Gene Expression]
Date de mise en ligne : Mardi 01 juillet 2014 Hepatitis E virus (HEV) causes both the endemic and epidemic spread of acute hepatitis in many parts of the world. HEV open reading frame 3 (ORF3) encodes a 13-kDa multifunctional protein (vp13) that is essential for HEV infection of animals. The exact role of vp13 in HEV infection remains unclear. In this study, vp13 was found to enhance interferon (IFN) production induced by poly(I · C), a synthetic analog of double-stranded RNA. Poly(I · C) treatment induced a higher level of IFN-β mRNA in HeLa cells stably expressing vp13 than in control cells. Using a luciferase reporter construct driven by the IFN-β promoter, we demonstrated that vp13 enhanced retinoic acid-inducible gene I (RIG-I)-dependent luciferase expression. This enhancement was found to be due to both an increased level of RIG-I protein and its activation. The levels of both endogenous and exogenous RIG-I were increased by vp13 by extension of the half-life of RIG-I. Additionally, vp13 interacts with the RIG-I N-terminal domain and enhances its K63-linked ubiquitination, which is essential for RIG-I activation. Analysis of vp13 deletion constructs suggested that the C-terminal domain of vp13 was essential for the enhancement of RIG-I signaling. In HEV-infected hepatoma cells, wild-type HEV led to a higher level of RIG-I and more poly(I · C)-induced IFN-β expression than did ORF3-null mutants. Analysis of vp13 from four HEV genotypes showed that vp13 from genotype I and III strains boosted RIG-I signaling, while vp13 from genotype II and IV strains had a minimal effect. These results indicate that vp13 enhances RIG-I signaling, which may play a role in HEV invasion. IMPORTANCE Hepatitis E virus (HEV) is a significant pathogen causing hepatitis in many parts of the world, yet it is understudied compared with other viral hepatitis pathogens. Here we found that the HEV open reading frame 3 product, vp13, enhances interferon induction stimulated by a synthetic analog of double-stranded RNA. This enhancement may play a role in HEV invasion, as vp13 is essential for HEV infection in vivo. The results of this study provide insights into virus-cell interactions during HEV infection. In addition to revealing its possible roles in HEV interference with cellular signaling, these results suggest that the second half of the vp13 sequence can be ligated into the genomes of attenuated live viruses to induce an innate immune response for better protective immunity, as well as a marker for differentiation of vaccinated animals from those infected with the corresponding wild-type viruses.
Nan, Y., Ma, Z., Wang, R., Yu, Y., Kannan, H., Fredericksen, B., Zhang, Y.-J.
Enhancement of Interferon Induction by ORF3 Product of Hepatitis E Virus [Virus-Cell Interactions]
Date de mise en ligne : Mardi 01 juillet 2014 Viral ribonucleocapsids harboring the viral genomic RNA are used as the template for viral mRNA synthesis and replication of the viral genome by viral RNA-dependent RNA polymerase (RdRp). Here we show that hantavirus nucleocapsid protein (N protein) interacts with RdRp in virus-infected cells. We mapped the RdRp binding domain at the N terminus of N protein. Similarly, the N protein binding pocket is located at the C terminus of RdRp. We demonstrate that an N protein-RdRp interaction is required for RdRp function during the course of virus infection in the host cell.
Cheng, E., Wang, Z., Mir, M. A.
Interaction between Hantavirus Nucleocapsid Protein (N) and RNA-Dependent RNA Polymerase (RdRp) Mutants Reveals the Requirement of an N-RdRp Interaction for Viral RNA Synthesis [Genome Replication and Regulation of Viral Gene Expression]
Date de mise en ligne : Mardi 01 juillet 2014 The N terminus of arenavirus L protein contains an endonuclease presumably involved in "cap snatching." Here, we employed the Lassa virus replicon system to map other L protein sites that might be involved in this mechanism. Residues Phe-1979, Arg-2018, Phe-2071, Asp-2106, Trp-2173, Tyr-2179, Arg-2200, and Arg-2204 were important for viral mRNA synthesis but dispensable for genome replication. Thus, the C terminus of L protein is involved in the mRNA synthesis process, potentially by mediating cap binding.
Lehmann, M., Pahlmann, M., Jerome, H., Busch, C., Lelke, M., Gunther, S.
Role of the C Terminus of Lassa Virus L Protein in Viral mRNA Synthesis [Genome Replication and Regulation of Viral Gene Expression]