Mise ŕ jour le : 07-02-2012
Les derniers abstracts de la revue JVI Current Issue : |
Date de mise en ligne : Jeudi 26 janvier 2012
Articles of Significant Interest Selected from This Issue by the Editors [Spotlight]
Date de mise en ligne : Jeudi 26 janvier 2012 Hepatitis B virus (HBV) is an important pathogen that chronically infects more men than women. To understand the molecular mechanism of this gender disparity, we analyzed HBV replication in transgenic mice that carried the HBV genome with or without the ability to express the HBV X protein (HBx). We found that gender had no effect on HBV surface antigen (HBsAg), DNA, and RNA levels in mice before puberty, but its effect on HBV after puberty was apparent, with HBV replicating approximately twice more efficiently in male mice than in female mice whether or not HBx was expressed. The castration of male mice resulted in a reduction of HBV HBsAg, DNA, and RNA levels, which could be partially restored by the injection of the androgen agonist R1881, indicating a positive role of androgen in HBV replication. The introduction of HBV genomic DNA and androgen receptor (AR) short hairpin RNA (shRNA) into the liver of naïve mice by hydrodynamic injection revealed that the effect of androgen on HBV was dependent on its receptor, which apparently could also stimulate HBV replication via an androgen-independent pathway. Further studies indicated that the two previously identified androgen response elements (AREs) in the HBV genome could indeed mediate the effect of androgen on HBV RNA transcription and DNA replication in vivo. These effects of androgen and its receptor on HBV thus provide an explanation for why men have a higher risk of HBV infection than women.
Tian, Y., Kuo, C.-f., Chen, W.-l., Ou, J.-h. J.
Enhancement of Hepatitis B Virus Replication by Androgen and Its Receptor in Mice [Virus-Cell Interactions]
Date de mise en ligne : Jeudi 26 janvier 2012 The Wnt/β-catenin pathway is involved in diverse cell functions governing development and disease. β-Catenin, a central mediator of this pathway, binds to members of the TCF/LEF family of transcription factors to modulate hundreds of genes. Active Wnt/β-catenin/TCF-4 signaling plays a significant role in repression of HIV-1 replication in multiple cell targets, including astrocytes. To determine the mechanism by which active β-catenin/TCF-4 leads to inhibition of HIV replication, we knocked down β-catenin or TCF/LEF members in primary astrocytes and astrocytomas transiently transfected with an HIV long terminal repeat (LTR)-luciferase reporter that contained an integrated copy of the HIV LTR-luciferase construct. Knockdown of either β-catenin or TCF-4 induced LTR activity by 2- to 3-fold under both the episomal and integrated conditions. This knockdown also increased presence of serine 2-phosphorylated RNA polymerase II (Pol II) on the HIV LTR as well as enhanced its processivity. Knockdown of β-catenin/TCF-4 also impacted tethering of other transcription factors on the HIV promoter. Specifically, knockdown of TCF-4 enhanced binding of C/EBPβ, C/EBP, and NF-B to the HIV LTR, while β-catenin knockdown increased binding of C/EBPβ and C/EBP but had no effect on NF-B. Approximately 150 genes in astrocytes were impacted by β-catenin knockdown, including genes involved in inflammation/immunity, uptake/transport, vesicular transport/exocytosis, apoptosis/cellular stress, and cytoskeleton/trafficking. These findings indicate that modulation of the β-catenin/TCF-4 axis impacts the basal level of HIV transcription in astrocytes, which may drive low level/persistent HIV in astrocytes that can contribute to ongoing neuroinflammation, and this axis also has profound effects on astrocyte biology.
Narasipura, S. D., Henderson, L. J., Fu, S. W., Chen, L., Kashanchi, F., Al-Harthi, L.
Role of {beta}-Catenin and TCF/LEF Family Members in Transcriptional Activity of HIV in Astrocytes [Genome Replication and Regulation of Viral Gene Expression]
Date de mise en ligne : Jeudi 26 janvier 2012 Theiler's murine encephalomyelitis virus (TMEV) results in a persistent central nervous system infection (CNS) and immune-mediated demyelination in mice. TMEV largely persists in macrophages (Ms) in the CNS, and infected Ms in vitro undergo apoptosis, whereas the infection of other rodent cells produces necrosis. We have found that necrosis is the dominant form of cell death in BeAn virus-infected BHK-21 cells but that ~20% of cells undergo apoptosis. Mcl-1 was highly expressed in BHK-21 cells, and protein levels decreased upon infection, consistent with onset of apoptosis. In infected BHK-21 cells in which Mcl-1 expression was knocked down using silencing RNAs there was a 3-fold increase in apoptotic cell death compared to parental cells. The apoptotic program switched on by BeAn virus is similar to that in mouse Ms, with hallmarks of activation of the intrinsic apoptotic pathway in a tumor suppressor protein p53-dependent manner. Infection of stable Mcl-1-knockdown cells led to restricted virus titers and increased physical to infectious particle (PFU) ratios, with additional data suggesting that a late step in the viral life cycle after viral RNA replication, protein synthesis, and polyprotein processing is affected by apoptosis. Together, these results indicate that Mcl-1 acts as a critical prosurvival factor that protects against apoptosis and allows high yields of infectious virus in BHK-21 cells.
Arslan, S. Y., Son, K.-N., Lipton, H. L.
The Antiapoptotic Protein Mcl-1 Controls the Type of Cell Death in Theiler's Virus-Infected BHK-21 Cells [Virus-Cell Interactions]
Date de mise en ligne : Jeudi 26 janvier 2012 Cytokines play crucial roles in curtailing the propagation and spread of pathogens within the host. As obligate pathogens, gammaherpesviruses have evolved a plethora of mechanisms to evade host immune responses. We have previously shown that murine gammaherpesvirus 68 (HV68) induces the degradation of RelA, an essential subunit of the transcriptionally active NF-B dimer, to evade cytokine production. Here, we report that the immediately early gene product of HV68,
Dong, X., He, Z., Durakoglugil, D., Arneson, L., Shen, Y., Feng, P.
Murine Gammaherpesvirus 68 Evades Host Cytokine Production via Replication Transactivator-Induced RelA Degradation [Pathogenesis and Immunity]
Date de mise en ligne : Jeudi 26 janvier 2012 Influenza A virus NS1 protein has multiple functions in the infected cell during the virus life cycle. Identification of novel cellular factors that interact with NS1 and understanding their functions in virus infection are of great interest. Recombinant viruses carrying a tagged NS1 are valuable for investigation of interactions between NS1 and cellular factors in the context of virus infection. Here, we report the generation of replication-competent recombinant influenza A viruses bearing a Strep tag in the NS1 protein. Purification of a protein complex associated with Strep-tagged NS1 from virus-infected cells followed by mass spectrometry revealed a number of attractive host factors. Among them, we focused our study on RNA helicase A (RHA) in this report. Through biomedical and functional analyses, we demonstrated that RHA interacts with NS1 in an RNA-dependent manner. Knockdown of RHA resulted in a significant reduction on virus yield and polymerase activity in a minigenome assay. Our cell-free viral genome replication assay showed that viral RNA replication and transcription can be enhanced by addition of RHA, and the enhanced effect of RHA required its ATP-dependent helicase activity. In summary, we established a system to identify cellular factors that interact with NS1 protein during virus infection and furthermore demonstrated that RHA interacts with NS1 and enhances viral replication and transcription.
Lin, L., Li, Y., Pyo, H.-M., Lu, X., Raman, S. N. T., Liu, Q., Brown, E. G., Zhou, Y.
Identification of RNA Helicase A as a Cellular Factor That Interacts with Influenza A Virus NS1 Protein and Its Role in the Virus Life Cycle [Virus-Cell Interactions]
Date de mise en ligne : Jeudi 26 janvier 2012 The lymphocytic choriomeningitis virus (LCMV) system constitutes one of the most widely used models for the study of infectious disease and the regulation of virus-specific T cell immunity. However, with respect to the activity of costimulatory and associated regulatory pathways, LCMV-specific T cell responses have long been regarded as relatively independent and thus distinct from the regulation of T cell immunity directed against many other viral pathogens. Here, we have reevaluated the contribution of CD28-CD80/86 costimulation in the LCMV system by use of CD80/86-deficient mice, and our results demonstrate that a disruption of CD28-CD80/86 signaling compromises the magnitude, phenotype, and/or functionality of LCMV-specific CD8+ and/or CD4+ T cell populations in all stages of the T cell response. Notably, a profound inhibition of secondary T cell immunity in LCMV-immune CD80/86-deficient mice emerged as a composite of both defective memory T cell development and a specific requirement for CD80 but not CD86 in the recall response, while a related experimental scenario of CD28-dependent yet CD80/86-independent secondary CD8+ T cell immunity suggests the existence of a CD28 ligand other than CD80/86. Furthermore, we provide evidence that regulatory T cells (TREGs), the homeostasis of which is altered in CD80/86–/– mice, contribute to restrained LCMV-specific CD8+ T cell responses in the presence of CD80/86. Our observations can therefore provide a more coherent perspective on CD28-CD80/86 costimulation in antiviral T cell immunity that positions the LCMV system within a shared context of multiple defects that virus-specific T cells acquire in the absence of CD28-CD80/86 costimulation.
Eberlein, J., Davenport, B., Nguyen, T. T., Victorino, F., Sparwasser, T., Homann, D.
Multiple Layers of CD80/86-Dependent Costimulatory Activity Regulate Primary, Memory, and Secondary Lymphocytic Choriomeningitis Virus-Specific T Cell Immunity [Pathogenesis and Immunity]
Date de mise en ligne : Jeudi 26 janvier 2012 It is known that cytotoxic T lymphocytes (CTLs) recognizing HIV-1 escape mutants are elicited in HIV-1-infected individuals, but their role in the control of HIV-1 replication remains unclear. We investigated the antiviral ability of CTLs recognizing the HLA-A*24:02-restricted Gag28 -36 (KYKLKHIVW) epitope and/or its escape mutant (KYRLKHIVW) elicited in the early and chronic phases of the infection. Wild-type (WT)-epitope-specific CTLs, as well as cross-reactive CTLs recognizing both WT and K30R (3R) epitopes, which were predominantly elicited at early and/or chronic phases in HLA-A*24:02+ individuals infected with the WT virus, suppressed the replication of the WT virus but failed to suppress that of the 3R virus, indicating that the 3R virus was selected by these 2 types of CTLs. On the other hand, cross-reactive and 3R-specific CTLs, which were elicited in those infected with the 3R virus, did not suppress the replication of either WT or 3R virus, indicating that these CTLs did not contribute to the control of 3R virus replication. High accumulation of the 3R mutation was found in a Japanese population recently recruited. The selection and accumulation of this 3R mutation resulted from the antiviral ability of these Gag28-specific CTLs and high prevalence of HLA-A*24:02 in a Japanese population. The present study highlighted the mechanisms for the roles of cross-reactive and mutant-epitope-specific CTLs, as well as high accumulation of escape mutants, in an HIV-1-infected population.
Akahoshi, T., Chikata, T., Tamura, Y., Gatanaga, H., Oka, S., Takiguchi, M.
Selection and Accumulation of an HIV-1 Escape Mutant by Three Types of HIV-1-Specific Cytotoxic T Lymphocytes Recognizing Wild-Type and/or Escape Mutant Epitopes [Pathogenesis and Immunity]
Date de mise en ligne : Jeudi 26 janvier 2012 Alphaviruses, such as Sindbis virus, undergo dramatic changes in three-dimensional structure upon exposure to low pH, and such exposure can establish conditions allowing fusion of the virus membrane with a cell plasma membrane upon return to neutral pH. While exposure to low pH is not required for entry of Sindbis virus into vertebrate or invertebrate cells, the conformational changes occurring at low pH may mimic those occurring upon virus-receptor interaction. Here, we employed small-angle neutron scattering with contrast variation to probe how the structure of a mammalian-grown Sindbis virus responds to moderately acidic pH. Several changes took place throughout the virion structure when the pH decreased from 7.2 to 6.4. Specifically, the RNA in the virion core underwent a conformational change. Additionally, the protein was redistributed. A significant amount of protein moved from the layer containing the lipid bilayer to the exterior of the virion. The results improve our understanding of the pH-driven alteration of Sindbis virus structure.
He, L., Piper, A., Meilleur, F., Hernandez, R., Heller, W. T., Brown, D. T.
Conformational Changes in Sindbis Virus Induced by Decreased pH Are Revealed by Small-Angle Neutron Scattering [Structure and Assembly]
Date de mise en ligne : Jeudi 26 janvier 2012 Mason-Pfizer monkey virus (M-PMV), like some other betaretroviruses, encodes a G-patch domain (GPD). This glycine-rich domain, which has been predicted to be an RNA binding module, is invariably localized at the 3' end of the pro gene upstream of the pro-pol ribosomal frameshift sequence of genomic RNAs of betaretroviruses. Following two ribosomal frameshift events and the translation of viral mRNA, the GPD is present in both Gag-Pro and Gag-Pro-Pol polyproteins. During the maturation of the Gag-Pro polyprotein, the GPD transiently remains a C-terminal part of the protease (PR), from which it is then detached by PR itself. The destiny of the Gag-Pro-Pol-encoded GPD remains to be determined. The function of the GPD in the retroviral life cycle is unknown. To elucidate the role of the GPD in the M-PMV replication cycle, alanine-scanning mutational analysis of its most highly conserved residues was performed. A series of individual mutations as well as the deletion of the entire GPD had no effect on M-PMV assembly, polyprotein processing, and RNA incorporation. However, a reduction of the reverse transcriptase (RT) activity, resulting in a drop in M-PMV infectivity, was determined for all GPD mutants. Immunoprecipitation experiments suggested that the GPD is a part of RT and participates in its function. These data indicate that the M-PMV GPD functions as a part of reverse transcriptase rather than protease.
Krizova, I., Hadravova, R., Stokrova, J., Gunterova, J., Dolezal, M., Ruml, T., Rumlova, M., Pichova, I.
The G-Patch Domain of Mason-Pfizer Monkey Virus Is a Part of Reverse Transcriptase [Genome Replication and Regulation of Viral Gene Expression]
Date de mise en ligne : Jeudi 26 janvier 2012 Polioviruses (PVs) carrying a reporter gene are useful tools for studies of virus replication, particularly if the viral chimeras contain the polyprotein that provides all of the proteins necessary for a complete replication cycle. Replication in HeLa cells of a previously constructed poliovirus expressing the gene for Renilla luciferase (RLuc) fused to the N terminus of the polyprotein H2N-RLuc-P1-P2-P3-COOH (P1, structural domain; P2 and P3, nonstructural domains) led to the deletion of RLuc after only one passage. Here we describe a novel poliovirus chimera that expresses Gaussia luciferase (GLuc) inserted into the polyprotein between P1 and P2 (N2H-P1-GLuc-P2-P3-COOH). This chimera, termed PV-GLuc, replicated to 10% of wild-type yield. The reporter signal was fully retained for three passages and then gradually lost. After six passages the signal was barely detectable. On further passages, however, the GLuc signal reappeared, and after eight passages it had reached the same levels observed with the original PV-GLuc at the first passage. We demonstrated that this surprising observation was due to coevolution of defective interfering (DI) particles that had lost part or all of the capsid coding sequence (P1-GLuc-P2-P3) and wild-type-like viruses that had lost the GLuc sequence (P1-P2-P3). When used at low passage, PV-GLuc is an excellent tool for studying aspects of genome replication and morphogenesis. The GLuc protein was secreted from mammalian cells but, in agreement with published data, was not secreted from PV-GLuc-infected cells due to poliovirus-induced inhibition of cellular protein secretion. Published evidence indicates that individual expression of enterovirus polypeptide 3A, 2B, or 2BC in COS-1 cells strongly inhibits host protein secretion. In HeLa cells, however, expression of none of the poliovirus polypeptides, either singly or in pairs, inhibited GLuc secretion. Thus, inhibition of GLuc secretion in PV-infected HeLa cells is likely a result of the interaction between several viral and cellular proteins that are different from those in COS-1 cells.
Song, Y., Paul, A. V., Wimmer, E.
Evolution of Poliovirus Defective Interfering Particles Expressing Gaussia Luciferase [Genome Replication and Regulation of Viral Gene Expression]
Date de mise en ligne : Jeudi 26 janvier 2012 The human cytomegalovirus tegument protein UL69 has been shown to be required for efficient viral replication at low multiplicities of infection. Several functions have been associated with UL69, including its ability to regulate cell cycle progression, translation, and the export of viral transcripts from the nucleus to the cytoplasm. However, it remains unclear which, if any, of these activities contribute to the phenotype observed with the UL69 deletion mutant. UL69 has been shown to interact with the cellular protein SPT6. The functional significance of this interaction has never been examined in the context of an infection. To address this, we generated UL69 mutant viruses that were unable to interact with SPT6 and determined what effect these mutations had on virus replication. Abolishing UL69's ability to interact with the SPT6 protein inhibited virus replication to levels indistinguishable from those observed following infection with the UL69 deletion mutant. Surprisingly, abolishing UL69's interaction with SPT6 also resulted in the impairment of UL69 shuttling activity. Finally, we demonstrate that inhibition of SPT6 expression by short hairpin RNA (shRNA) knockdown inhibits wild-type virus replication. Taken together, our results demonstrate that UL69's ability to interact with SPT6 plays a critical role in viral replication.
Cygnar, D., Hagemeier, S., Kronemann, D., Bresnahan, W. A.
The Cellular Protein SPT6 Is Required for Efficient Replication of Human Cytomegalovirus [Virus-Cell Interactions]
Date de mise en ligne : Jeudi 26 janvier 2012 The group of closely related avian sarcoma and leukosis viruses (ASLVs) evolved from a common ancestor into multiple subgroups, A to J, with differential host range among galliform species and chicken lines. These subgroups differ in variable parts of their envelope glycoproteins, the major determinants of virus interaction with specific receptor molecules. Three genetic loci, tva, tvb, and tvc, code for single membrane-spanning receptors from diverse protein families that confer susceptibility to the ASLV subgroups. The host range expansion of the ancestral virus might have been driven by gradual evolution of resistance in host cells, and the resistance alleles in all three receptor loci have been identified. Here, we characterized two alleles of the tva receptor gene with similar intronic deletions comprising the deduced branch-point signal within the first intron and leading to inefficient splicing of tva mRNA. As a result, we observed decreased susceptibility to subgroup A ASLV in vitro and in vivo. These alleles were independently found in a close-bred line of domestic chicken and Indian red jungle fowl (Gallus gallus murghi), suggesting that their prevalence might be much wider in outbred chicken breeds. We identified defective splicing to be a mechanism of resistance to ASLV and conclude that such a type of mutation could play an important role in virus-host coevolution.
Reinisova, M., Plachy, J., Trejbalova, K., Senigl, F., Kucerova, D., Geryk, J., Svoboda, J., Hejnar, J.
Intronic Deletions That Disrupt mRNA Splicing of the tva Receptor Gene Result in Decreased Susceptibility to Infection by Avian Sarcoma and Leukosis Virus Subgroup A [Virus-Cell Interactions]
Date de mise en ligne : Jeudi 26 janvier 2012 Equine herpesvirus type 1 (EHV-1) and EHV-4 are genetically and antigenically very similar, but their pathogenic potentials are strikingly different. The differences in pathogenicity between both viruses seem to be reflected in cellular host range: EHV-1 can readily be propagated in many cell types of multiple species, while EHV-4 entry and replication appear to be restricted mainly to equine cells. The clear difference in cellular tropism may well be associated with differences in the gene products involved in virus entry and/or spread from cell to cell. Here we show that (i) most of the EHV-1 permissive cell lines became resistant to EHV-1 expressing EHV-4 glycoprotein D (gD4) and the opposite was observed for EHV-4 harboring EHV-1 gD (gD1). (ii) The absence of integrins did not inhibit entry into and replication of EHV-1 in CHO-K1 or peripheral blood mononuclear cells (PBMC). Furthermore, integrin-negative K562 cells did not acquire the ability to bind to gD1 when αVβ3 integrin was overexpressed. (iii) PBMC could be infected with similar efficiencies by both EHV-1 and EHV-4 in vitro. (iv) In contrast to results for equine fibroblasts and cells of endothelial or epithelial origin, we were unable to block entry of EHV-1 or EHV-4 into PBMC with antibodies directed against major histocompatibility complex class I (MHC-I), a result that indicates that these viruses utilize a different receptor(s) to infect PBMC. Cumulatively, we provide evidence that efficient EHV-1 and EHV-4 entry is dependent mainly on gD, which can bind to multiple cell surface receptors, and that gD has a defining role with respect to cellular host range of EHV-1 and EHV-4.
Azab, W., Osterrieder, N.
Glycoproteins D of Equine Herpesvirus Type 1 (EHV-1) and EHV-4 Determine Cellular Tropism Independently of Integrins [Virus-Cell Interactions]
Date de mise en ligne : Jeudi 26 janvier 2012 We previously established that at 3 years postseroconversion, ~30% of HIV-infected individuals have cross-reactive neutralizing activity (CrNA) in their sera. Here we studied the kinetics with which CrNA develops and how these relate to the development of autologous neutralizing activity as well as viral escape and diversification. For this purpose, sera from five individuals with CrNA and one elite neutralizer that were obtained at three monthly intervals in the first year after seroconversion and at multiple intervals over the disease course were tested for neutralizing activity against an established multiclade panel of six viruses. The same serum samples, as well as sera from three individuals who lacked CrNA, were tested for their neutralizing activities against autologous clonal HIV-1 variants from multiple time points covering the disease course from seroconversion onward. The elite neutralizer already had CrNA at 9.8 months postseroconversion, in contrast with the findings for the other five patients, in whom CrNA was first detected at 20 to 35 months postseroconversion and peaked around 35 months postseroconversion. In all patients, CrNA coincided with neutralizing activity against autologous viruses that were isolated <12 months postseroconversion, while viruses from later time points had already escaped autologous neutralizing activity. Also, the peak in gp160 sequence diversity coincided with the peak of CrNA titers. Individuals who lacked CrNA had lower peak autologous neutralizing titers, viral escape, and sequence diversity than individuals with CrNA. A better understanding of the underlying factors that determine the presence of CrNA or even an elite neutralizer phenotype may aid in the design of an HIV-1 vaccine.
Euler, Z., van den Kerkhof, T. L. G. M., van Gils, M. J., Burger, J. A., Edo-Matas, D., Phung, P., Wrin, T., Schuitemaker, H.
Longitudinal Analysis of Early HIV-1-Specific Neutralizing Activity in an Elite Neutralizer and in Five Patients Who Developed Cross-Reactive Neutralizing Activity [Pathogenesis and Immunity]
Date de mise en ligne : Jeudi 26 janvier 2012 The dynamics of the circulation and distribution of transmissible spongiform encephalopathy (TSE) agents in the blood of infected individuals remain largely unknown. This clearly limits the understanding of the role of blood in TSE pathogenesis and the development of a reliable TSE blood detection assay. Using two distinct sheep scrapie models and blood transfusion, this work demonstrates the occurrence of a very early and persistent prionemia. This ability to transmit disease by blood transfusion was correlated with the presence of infectivity in white blood cells (WBC) and peripheral blood mononucleated cells (PBMC) as detected by bioassay in mice overexpressing the ovine prion protein PrP (tg338 mice) and with the identification of abnormal PrP in WBC after using protein misfolding cyclic amplification (PMCA). Platelets and a large variety of leukocyte subpopulations also were shown to be infectious. The use of endpoint titration in tg338 mice indicated that the infectivity in WBC (per ml of blood) was 106.5-fold lower than that in 1 g of posterior brainstem sample. In both WBC and brainstem, infectivity displayed similar resistance to PK digestion. The data strongly support the concept that WBC are an accurate target for reliable TSE detection by PMCA. The presence of infectivity in short-life-span blood cellular elements raises the question of the origin of prionemia.
Lacroux, C., Vilette, D., Fernandez-Borges, N., Litaise, C., Lugan, S., Morel, N., Corbiere, F., Simon, S., Simmons, H., Costes, P., Weisbecker, J.-L., Lantier, I., Lantier, F., Schelcher, F., Grassi, J., Castilla, J., Andreoletti, O.
Prionemia and Leukocyte-Platelet-Associated Infectivity in Sheep Transmissible Spongiform Encephalopathy Models [Prions]
Date de mise en ligne : Jeudi 26 janvier 2012 Although O-mannosylated dystroglycan is a receptor for Lassa virus, a causative agent of Lassa fever, recent findings suggest the existence of an alternative receptor(s). Here we identified four molecules as receptors for Lassa virus: Axl and Tyro3, from the TAM family, and dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and liver and lymph node sinusoidal endothelial calcium-dependent lectin (LSECtin), from the C-type lectin family. These molecules enhanced the binding of Lassa virus to cells and mediated infection independently of dystroglycan. Axl- or Tyro3-mediated infection required intracellular signaling via the tyrosine kinase activity of Axl or Tyro3, whereas DC-SIGN- or LSECtin-mediated infection and binding were dependent on a specific carbohydrate and on ions. The identification of these four molecules as Lassa virus receptors advances our understanding of Lassa virus cell entry.
Shimojima, M., Stroher, U., Ebihara, H., Feldmann, H., Kawaoka, Y.
Identification of Cell Surface Molecules Involved in Dystroglycan-Independent Lassa Virus Cell Entry [Virus-Cell Interactions]
Date de mise en ligne : Jeudi 26 janvier 2012 Herpesvirus proteins pUL34 and pUL31 form a complex at the inner nuclear membrane (INM) which is necessary for efficient nuclear egress. Pseudorabies virus (PrV) pUL34 is a type II membrane protein of 262 amino acids (aa). The transmembrane region (TM) is predicted to be located between aa 245 and 261, leaving only one amino acid in the C terminus that probably extends into the perinuclear space. It is targeted to the nuclear envelope in the absence of other viral proteins, pointing to intrinsic localization motifs, and shows structural similarity to cellular INM proteins like lamina-associated polypeptide (Lap) 2ß and Emerin. To investigate which domains of pUL34 are relevant for localization and function, we constructed chimeric proteins by replacing parts of pUL34 with regions of cellular INM proteins. First the 18 C-terminal amino acids encompassing the TM were exchanged with TM regions and C-terminal domains of Lap2ß and Emerin or with the first TM region of the polytopic lamin B receptor (LBR), including the nine following amino acids. All resulting chimeric proteins complemented the replication defect of PrV-UL34, demonstrating that the substitution of the TM and the extension of the C-terminal domain does not interfere with the function of pUL34. Complementation was reduced but not abolished when the C-terminal 50 aa were replaced by corresponding Lap2ß sequences (pUL34-LapCT50). However, replacing the C-terminal 100 aa (pUL34-LapCT100) resulted in a nonfunctional protein despite continuing pUL31 binding, pointing to an important functional role of this region. The replacement of the N-terminal 100 aa (pUL34-LapNT100) had no effect on nuclear envelope localization but abrogated pUL31 binding and function.
Schuster, F., Klupp, B. G., Granzow, H., Mettenleiter, T. C.
Structural Determinants for Nuclear Envelope Localization and Function of Pseudorabies Virus pUL34 [Virus-Cell Interactions]
Date de mise en ligne : Jeudi 26 janvier 2012 The formation of replication compartments, the subnuclear structures in which the viral DNA genome is replicated, is a hallmark of herpesvirus infections. The localization of proteins and viral DNA within human cytomegalovirus replication compartments is not well characterized. Immunofluorescence analysis demonstrated the accumulation of the viral DNA polymerase subunit UL44 at the periphery of replication compartments and the presence of different populations of UL44 in infected cells. In contrast, the viral single-stranded-DNA binding protein UL57 was distributed throughout replication compartments. Using "click chemistry" to detect 5-ethynyl-2'-deoxyuridine (EdU) incorporation into replicating viral DNA and pulse-chase protocols, we found that viral DNA synthesis occurs at the periphery of replication compartments and that replicated viral DNA subsequently localizes to the interior of replication compartments. The interiors of replication compartments also contain regions in which UL44 and EdU-labeled DNA are absent. The treatment of cells with a viral DNA polymerase inhibitor reversibly caused the dispersal of both UL44 and EdU-labeled viral DNA from replication compartments, indicating that ongoing viral DNA synthesis is necessary to maintain the organization of replication compartments. Our results reveal a previously unappreciated complexity of the organization of human cytomegalovirus replication compartments.
Strang, B. L., Boulant, S., Chang, L., Knipe, D. M., Kirchhausen, T., Coen, D. M.
Human Cytomegalovirus UL44 Concentrates at the Periphery of Replication Compartments, the Site of Viral DNA Synthesis [Genome Replication and Regulation of Viral Gene Expression]
Date de mise en ligne : Jeudi 26 janvier 2012 Dengue virus (DENV) affects millions of people, causing more than 20,000 deaths annually. No effective treatment for the disease caused by DENV infection is currently available, partially due to the lack of knowledge on the basic aspects of the viral life cycle, including the molecular basis of the interaction between viral components and cellular compartments. Here, we characterized the properties of the interaction between the DENV capsid (C) protein and hepatic lipid droplets (LDs), which was recently shown to be essential for the virus replication cycle. Zeta potential analysis revealed a negative surface charge of LDs, with an average surface charge of –19 mV. The titration of LDs with C protein led to an increase of the surface charge, which reached a plateau at +13.7 mV, suggesting that the viral protein-LD interaction exposes the protein cationic surface to the aqueous environment. Atomic force microscopy (AFM)-based force spectroscopy measurements were performed by using C protein-functionalized AFM tips. The C protein-LD interaction was found to be strong, with a single (un)binding force of 33.6 pN. This binding was dependent on high intracellular concentrations of potassium ions but not sodium. The inhibition of Na+/K+-ATPase in DENV-infected cells resulted in the dissociation of C protein from LDs and a 50-fold inhibition of infectious virus production but not of RNA replication, indicating a biological relevance for the potassium-dependent interaction. Limited proteolysis of the LD surface impaired the C protein-LD interaction, and force measurements in the presence of specific antibodies indicated that perilipin 3 (TIP47) is the major DENV C protein ligand on the surface of LDs.
Carvalho, F. A., Carneiro, F. A., Martins, I. C., Assuncao-Miranda, I., Faustino, A. F., Pereira, R. M., Bozza, P. T., Castanho, M. A. R. B., Mohana-Borges, R., Da Poian, A. T., Santos, N. C.
Dengue Virus Capsid Protein Binding to Hepatic Lipid Droplets (LD) Is Potassium Ion Dependent and Is Mediated by LD Surface Proteins [Virus-Cell Interactions]
Date de mise en ligne : Jeudi 26 janvier 2012 Rift Valley fever (RVF) virus (RVFV) can cause severe human disease characterized by either acute-onset hepatitis, delayed-onset encephalitis, retinitis and blindness, or a hemorrhagic syndrome. The existing nonhuman primate (NHP) model for RVF utilizes an intravenous (i.v.) exposure route in rhesus macaques (Macaca mulatta). Severe disease in these animals is infrequent, and large cohorts are needed to observe significant morbidity and mortality. To overcome these drawbacks, we evaluated the infectivity and pathogenicity of RVFV in the common marmoset (Callithrix jacchus) by i.v., subcutaneous (s.c.), and intranasal exposure routes to more closely mimic natural exposure. Marmosets were more susceptible to RVFV than rhesus macaques and experienced higher rates of morbidity, mortality, and viremia and marked aberrations in hematological and chemistry values. An overwhelming infection of hepatocytes was a major consequence of infection of marmosets by the i.v. and s.c. exposure routes. Additionally, these animals displayed signs of hemorrhagic manifestations and neurological impairment. Based on our results, the common marmoset model more closely resembles severe human RVF disease and is therefore an ideal model for the evaluation of potential vaccines and therapeutics.
Smith, D. R., Bird, B. H., Lewis, B., Johnston, S. C., McCarthy, S., Keeney, A., Botto, M., Donnelly, G., Shamblin, J., Albarino, C. G., Nichol, S. T., Hensley, L. E.
Development of a Novel Nonhuman Primate Model for Rift Valley Fever [Pathogenesis and Immunity]
Date de mise en ligne : Jeudi 26 janvier 2012 Enterovirus 71 (EV71) is a neurotropic pathogen that has been consistently associated with the severe neurological forms of hand, foot, and mouth disease. The lack of a relevant animal model has hampered our understanding of EV71 pathogenesis, in particular the route and mode of viral dissemination. It has also hindered the development of effective prophylactic and therapeutic approaches, making EV71 one of the most pressing public health concerns in Southeast Asia. Here we report a novel mouse model of EV71 infection. We demonstrate that 2-week-old and younger immunodeficient AG129 mice, which lack type I and II interferon receptors, are susceptible to infection with a non-mouse-adapted EV71 strain via both the intraperitoneal (i.p.) and oral routes of inoculation. The infected mice displayed progressive limb paralysis prior to death. The dissemination of the virus was dependent on the route of inoculation but eventually resulted in virus accumulation in the central nervous systems of both animal groups, indicating a clear neurotropism of the virus. Histopathological examination revealed massive damage in the limb muscles, brainstem, and anterior horn areas. However, the minute amount of infectious viral particles in the limbs from orally infected animals argues against a direct viral cytopathic effect in this tissue and suggests that limb paralysis is a consequence of EV71 neuroinvasion. Together, our observations support that young AG129 mice display polio-like neuropathogenesis upon infection with a non-mouse-adapted EV71 strain, making this mouse model relevant for EV71 pathogenesis studies and an attractive platform for EV71 vaccine and drug testing.
Khong, W. X., Yan, B., Yeo, H., Tan, E. L., Lee, J. J., Ng, J. K. W., Chow, V. T., Alonso, S.
A Non-Mouse-Adapted Enterovirus 71 (EV71) Strain Exhibits Neurotropism, Causing Neurological Manifestations in a Novel Mouse Model of EV71 Infection [Pathogenesis and Immunity]
Date de mise en ligne : Jeudi 26 janvier 2012 The transmission of herpesviruses depends on viral shedding at mucosal surfaces. The salivary gland represents a major site of persistent viral replication for many viruses, including cytomegalovirus. We established a mouse model of salivary gland dysfunction after acute viral infection and investigated the cellular requirements for the loss of secretion. Murine cytomegalovirus (MCMV) infection severely impaired saliva secretion independently of salivary gland virus levels. Lymphocytes or circulating monocytes/macrophages were not required for secretory dysfunction. Dysfunction occurred before glandular inflammation, suggesting that a soluble mediator initiated the disruption of acinar cell function. Despite genetic differences in innate resistance to MCMV, NK cells protected the host against acinar atrophy and the loss of secretions under conditions of an exceedingly low virus inoculum. NK cells also modulated the type of glandular inflammation after infection, as they prevented an influx of Siglec-F+ polymorphonuclear leukocytes (PMNs). Therefore, beyond their recognized role in controlling MCMV replication, NK cells preserve organ integrity and function and regulate the innate inflammatory response within the gland.
Carroll, V. A., Lundgren, A., Wei, H., Sainz, S., Tung, K. S., Brown, M. G.
Natural Killer Cells Regulate Murine Cytomegalovirus-Induced Sialadenitis and Salivary Gland Disease [Pathogenesis and Immunity]
Date de mise en ligne : Jeudi 26 janvier 2012 To establish a cell culture system for chimeric hepatitis C virus (HCV) genotype 2b, we prepared a chimeric construct harboring the 5' untranslated region (UTR) to the E2 region of the MA strain (genotype 2b) and the region of p7 to the 3' UTR of the JFH-1 strain (genotype 2a). This chimeric RNA (MA/JFH-1.1) replicated and produced infectious virus in Huh7.5.1 cells. Replacement of the 5' UTR of this chimera with that from JFH-1 (MA/JFH-1.2) enhanced virus production, but infectivity remained low. In a long-term follow-up study, we identified a cell culture-adaptive mutation in the core region (R167G) and found that it enhanced virus assembly. We previously reported that the NS3 helicase (N3H) and the region of NS5B to 3' X (N5BX) of JFH-1 enabled replication of the J6CF strain (genotype 2a), which could not replicate in cells. To reduce JFH-1 content in MA/JFH-1.2, we produced a chimeric viral genome for MA harboring the N3H and N5BX regions of JFH-1, combined with a JFH-1 5' UTR replacement and the R167G mutation (MA/N3H+N5BX-JFH1/R167G). This chimeric RNA replicated efficiently, but virus production was low. After the introduction of four additional cell culture-adaptive mutations, MA/N3H+N5BX-JFH1/5am produced infectious virus efficiently. Using this chimeric virus harboring minimal regions of JFH-1, we analyzed interferon sensitivity and found that this chimeric virus was more sensitive to interferon than JFH-1 and another chimeric virus containing more regions from JFH-1 (MA/JFH-1.2/R167G). In conclusion, we established an HCV genotype 2b cell culture system using a chimeric genome harboring minimal regions of JFH-1. This cell culture system may be useful for characterizing genotype 2b viruses and developing antiviral strategies.
Murayama, A., Kato, T., Akazawa, D., Sugiyama, N., Date, T., Masaki, T., Nakamoto, S., Tanaka, Y., Mizokami, M., Yokosuka, O., Nomoto, A., Wakita, T.
Production of Infectious Chimeric Hepatitis C Virus Genotype 2b Harboring Minimal Regions of JFH-1 [Genome Replication and Regulation of Viral Gene Expression]
Date de mise en ligne : Jeudi 26 janvier 2012 Broad and potent neutralizing antibody (BNAb) responses are rare in people infected by human immunodeficiency virus type 1 (HIV-1). Clearly defining the nature of BNAb epitopes on HIV-1 envelope glycoproteins (Envs) targeted in vivo is critical for future directions of anti-HIV-1 vaccine development. Conventional techniques are successful in defining neutralizing epitopes in a small number of individual subjects but fail in studying large groups of subjects. Two independent methods were employed to investigate the nature of NAb epitopes targeted in 9 subjects, identified by the NIAID Center for HIV/AIDS Vaccine Immunology (CHAVI) 001 and 008 clinical teams, known to make a strong BNAb response. Neutralizing activity from 8/9 subjects was enhanced by enriching high-mannose N-linked glycan (HM-glycan) of HIV-1 glycoproteins on neutralization target viruses and was sensitive to specific glycan deletion mutations of HIV-1 glycoproteins, indicating that HM-glycan-dependent epitopes are targeted by BNAb responses in these subjects. This discovery adds to accumulating evidence supporting the hypothesis that glycans are important targets on HIV-1 glycoproteins for BNAb responses in vivo, providing an important lead for future directions in developing NAb-based anti-HIV-1 vaccines.
Lavine, C. L., Lao, S., Montefiori, D. C., Haynes, B. F., Sodroski, J. G., Yang, X., the NIAID Center for HIV/AIDS Vaccine Immunology (CHAVI)
High-Mannose Glycan-Dependent Epitopes Are Frequently Targeted in Broad Neutralizing Antibody Responses during Human Immunodeficiency Virus Type 1 Infection [Pathogenesis and Immunity]
Date de mise en ligne : Jeudi 26 janvier 2012 Natural killer (NK) cells and CD8+ T cells play a prominent role in the clearance of mouse cytomegalovirus (MCMV) infection. The role of NK cells in modulating the CD8+ T-cell response to MCMV infection is still the subject of intensive research. For analyzing the impact of NK cells on mounting of a CD8+ T-cell response and the contribution of these cells to virus control during the first days postinfection (p.i.), we used C57BL/6 mice in which NK cells are specifically activated through the Ly49H receptor engaged by the MCMV-encoded ligand m157. Our results indicate that the requirement for CD8+ T cells in early MCMV control inversely correlates with the engagement of Ly49H. While depletion of CD8+ T cells has only a minor effect on the early control of wild-type MCMV, CD8+ T cells are essential in the control of m157 virus. The frequencies of virus epitope-specific CD8+ T cells and their activation status were higher in mice infected with m157 virus. In addition, these mice showed elevated levels of alpha interferon (IFN-α) and several other proinflammatory cytokines as early as 1.5 days p.i. Although the numbers of conventional dendritic cells (cDCs) were reduced later during infection, particularly in m157-infected mice, they were not significantly affected at the peak of the cytokine response. Altogether, we concluded that increased antigen load, preservation of early cDCs' function, and higher levels of innate cytokines collectively account for an enhanced CD8+ T-cell response in C57BL/6 mice infected with a virus unable to activate NK cells via the Ly49H–m157 interaction.
Mitrovic, M., Arapovic, J., Jordan, S., Fodil-Cornu, N., Ebert, S., Vidal, S. M., Krmpotic, A., Reddehase, M. J., Jonjic, S.
The NK Cell Response to Mouse Cytomegalovirus Infection Affects the Level and Kinetics of the Early CD8+ T-Cell Response [Pathogenesis and Immunity]
Date de mise en ligne : Jeudi 26 janvier 2012 The small mRNA (SmRNA) of all Bunyaviridae encodes the nucleocapsid (N) protein. In 4 out of 5 genera in the Bunyaviridae, the smRNA encodes an additional nonstructural protein denominated NSs. In this study, we show that Andes hantavirus (ANDV) SmRNA encodes an NSs protein. Data show that the NSs protein is expressed in the context of an ANDV infection. Additionally, our results suggest that translation initiation from the NSs initiation codon is mediated by ribosomal subunits that have bypassed the upstream N protein initiation codon through a leaky scanning mechanism.
Vera-Otarola, J., Solis, L., Soto-Rifo, R., Ricci, E. P., Pino, K., Tischler, N. D., Ohlmann, T., Darlix, J.-L., Lopez-Lastra, M.
The Andes Hantavirus NSs Protein Is Expressed from the Viral Small mRNA by a Leaky Scanning Mechanism [Genome Replication and Regulation of Viral Gene Expression]
Date de mise en ligne : Jeudi 26 janvier 2012 The 134.5 protein of herpes simplex viruses (HSV) is essential for viral pathogenesis, where it precludes translational arrest mediated by double-stranded-RNA-dependent protein kinase (PKR). Paradoxically, inhibition of PKR alone is not sufficient for HSV to exhibit viral virulence. Here we report that 134.5 inhibits TANK binding kinase 1 (TBK1) through its amino-terminal sequences, which facilitates viral replication and neuroinvasion. Compared to wild-type virus, the 134.5 mutant lacking the amino terminus induces stronger antiviral immunity. This parallels a defect of 134.5 for interacting with TBK1 and reducing phosphorylation of interferon (IFN) regulatory factor 3. This activity is independent of PKR. Although resistant to IFN treatment, the 134.5 amino-terminal deletion mutant replicates at an intermediate level between replication of wild-type virus and that of the 134.5 null mutant in TBK1+/+ cells. However, such impaired viral growth is not observed in TBK1–/– cells, indicating that the interaction of 134.5 with TBK1 dictates HSV infection. Upon corneal infection, this mutant replicates transiently but barely invades the trigeminal ganglia or brain, which is a difference from wild-type virus and the 134.5 null mutant. Therefore, in addition to PKR, 134.5 negatively regulates TBK1, which contributes viral replication and spread in vivo.
Ma, Y., Jin, H., Valyi-Nagy, T., Cao, Y., Yan, Z., He, B.
Inhibition of TANK Binding Kinase 1 by Herpes Simplex Virus 1 Facilitates Productive Infection [Virus-Cell Interactions]
Date de mise en ligne : Jeudi 26 janvier 2012 Kaposi's sarcoma-associated herpesvirus and rhesus macaque rhadinovirus (RRV), two closely related gammaherpesviruses, are unique in their expression of viral homologs of cellular interferon regulatory factors (IRFs), termed viral IRFs (vIRFs). To assess the role of vIRFs during de novo infection, we have utilized the bacterial artificial chromosome clone of wild-type RRV17577 (WTBAC RRV) to generate a recombinant virus with all 8 of the vIRFs deleted (vIRF-ko RRV). The infection of primary rhesus fibroblasts and peripheral blood mononuclear cells (PBMCs) with vIRF-ko RRV resulted in earlier and increased induction of type I interferon (IFN) (IFN-α/β) and type II IFN (IFN-). Additionally, plasmacytoid dendritic cells maintained higher levels of IFN-α production in PBMC cultures infected with vIRF-ko RRV than in cultures infected with WTBAC RRV. Moreover, the nuclear accumulation of phosphorylated IRF-3, which is necessary for the induction of type I IFN, was also inhibited following WTBAC RRV infection. These findings demonstrate that during de novo RRV infection, vIRFs are inhibiting the induction of IFN at the transcriptional level, and one potential mechanism for this is the disruption of the activation and localization of IRF-3.
Robinson, B. A., Estep, R. D., Messaoudi, I., Rogers, K. S., Wong, S. W.
Viral Interferon Regulatory Factors Decrease the Induction of Type I and Type II Interferon during Rhesus Macaque Rhadinovirus Infection [Pathogenesis and Immunity]
Date de mise en ligne : Jeudi 26 janvier 2012 Since its initial identification in St. Petersburg, Russia, the recombinant hepatitis C virus (HCV) 2k/1b has been isolated from several countries throughout Eurasia. The 2k/1b strain is the only recombinant HCV to have spread widely, raising questions about the epidemiological background in which it first appeared. In order to further understand the circumstances by which HCV recombinants might be formed and spread, we estimated the date of the recombination event that generated the 2k/1b strain using a Bayesian phylogenetic approach. Our study incorporates newly isolated 2k/1b strains from Amsterdam, The Netherlands, and has employed a hierarchical Bayesian framework to combine information from different genomic regions. We estimate that 2k/1b originated sometime between 1923 and 1956, substantially before the first detection of the strain in 1999. The timescale and the geographic spread of 2k/1b suggest that it originated in the former Soviet Union at about the time that the world's first centralized national blood transfusion and storage service was being established. We also reconstructed the epidemic history of 2k/1b using coalescent theory-based methods, matching patterns previously reported for other epidemic HCV subtypes. This study demonstrates the practicality of jointly estimating dates of recombination from flanking regions of the breakpoint and further illustrates that rare genetic-exchange events can be particularly informative about the underlying epidemiological processes.
Raghwani, J., Thomas, X. V., Koekkoek, S. M., Schinkel, J., Molenkamp, R., van de Laar, T. J., Takebe, Y., Tanaka, Y., Mizokami, M., Rambaut, A., Pybus, O. G.
Origin and Evolution of the Unique Hepatitis C Virus Circulating Recombinant Form 2k/1b [Genetic Diversity and Evolution]
Date de mise en ligne : Jeudi 26 janvier 2012 The third component of human complement (C3) plays a central role in innate immune function as its activation is required to trigger classical as well as alternative complement pathways. In this study, we have observed that sera from patients chronically infected with hepatitis C virus (HCV) displayed significantly lower C3 levels than sera from healthy individuals. Liver biopsy specimens from the same patients also exhibited lower C3 mRNA expression than liver tissues from healthy donors. C3 mRNA level was reduced in hepatocytes upon infection with cell culture-grown HCV genotype 1a or 2a in vitro. Further analysis suggested that HCV core protein displayed a weak repression of C3 promoter activity by downregulating the transcription factor farnesoid X receptor (FXR). On the other hand, HCV NS5A protein strongly downregulated C3 promoter activity at the basal level or in the presence of interleukin-1β (IL-1β) as an inducer. In addition, the expression of the transcription factor CAAT/enhancer binding protein beta (C/EBP-β), which binds to the IL-1/IL-6 response element in the C3 promoter, was inhibited in liver biopsy specimens. Furthermore, expression of C/EBP-β was reduced in hepatocytes infected with cell culture-grown HCV, as well as in hepatocytes transfected with the NS5A genomic region of HCV. Together, these results underscore the role of HCV NS5A protein in impairing innate immune function.
Mazumdar, B., Kim, H., Meyer, K., Bose, S. K., Di Bisceglie, A. M., Ray, R. B., Ray, R.
Hepatitis C Virus Proteins Inhibit C3 Complement Production [Virus-Cell Interactions]
Date de mise en ligne : Jeudi 26 janvier 2012 During the 2009 H1N1 influenza virus pandemic (pdmH1N1) outbreak, it was found that most individuals lacked antibodies against the new pdmH1N1 virus, and only the elderly showed anti-hemagglutinin (anti-HA) antibodies that were cross-reactive with the new strains. Different studies have demonstrated that prior contact with the virus can confer protection against strains with some degree of dissimilarity; however, this has not been sufficiently explored within the context of a pdmH1N1 virus infection. In this study, we have found that a first infection with the A/Brisbane/59/2007 virus strain confers heterologous protection in ferrets and mice against a subsequent pdmH1N1 (A/Mexico/4108/2009) virus infection through a cross-reactive but non-neutralizing antibody mechanism. Heterologous immunity is abrogated in B cell-deficient mice but maintained in CD8–/– and perforin-1–/– mice. We identified cross-reactive antibodies from A/Brisbane/59/2007 sera that recognize non-HA epitopes in pdmH1N1 virus. Passive serum transfer showed that cross-reactive sH1N1-induced antibodies conferred protection in naive recipient mice during pdmH1N1 virus challenge. The presence or absence of anti-HA antibodies, therefore, is not the sole indicator of the effectiveness of protective cross-reactive antibody immunity. Measurement of additional antibody repertoires targeting the non-HA antigens of influenza virus should be taken into consideration in assessing protection and immunization strategies. We propose that preexisting cross-protective non-HA antibody immunity may have had an overall protective effect during the 2009 pdmH1N1 outbreak, thereby reducing disease severity in human infections.
Fang, Y., Banner, D., Kelvin, A. A., Huang, S. S. H., Paige, C. J., Corfe, S. A., Kane, K. P., Bleackley, R. C., Rowe, T., Leon, A. J., Kelvin, D. J.
Seasonal H1N1 Influenza Virus Infection Induces Cross-Protective Pandemic H1N1 Virus Immunity through a CD8-Independent, B Cell-Dependent Mechanism [Vaccines and Antiviral Agents]
Date de mise en ligne : Jeudi 26 janvier 2012 The Step Trial showed that the MRKAd5 HIV-1 subtype B Gag/Pol/Nef vaccine did not protect men from HIV infection or reduce setpoint plasma viral RNA (vRNA) levels but, unexpectedly, it did modestly enhance susceptibility to HIV infection in adenovirus type 5 (Ad5)-seropositive, uncircumcised men. As part of the process to understand the results of the Step Trial, we designed a study to determine whether rhesus macaques chronically infected with a host-range mutant Ad5 (Ad5hr) and then immunized with a replication defective Ad5 SIVmac239 Gag/Pol/Nef vaccine were more resistant or susceptible to SIV infection than unimmunized rhesus macaques challenged with a series of escalating dose penile exposures to SIVmac 251. The Ad5 SIV vaccine induced CD8+ T cell responses in 70% of the monkeys, which is similar to the proportion of humans that responded to the vaccine in the Step Trial. However, the vaccine did not protect vaccinated animals from penile SIV challenge. At the lowest SIV exposure dose (103 50% tissue culture infective doses), 2 of 9 Ad5-seropositive animals immunized with the Ad5 SIV vaccine became infected compared to 0 of 34 animals infected in the other animal groups (naive animals, Ad5-seropositive animals immunized with the empty Ad5 vector, Ad5-seronegative animals immunized with the Ad5 SIV vaccine, and Ad5-seronegative animals immunized with the empty Ad5 vector). Penile exposure to more concentrated virus inocula produced similar rates of infection in all animal groups. Although setpoint viral loads were unaffected in Step vaccinees, the Ad5 SIV-immunized animals had significantly lower acute-phase plasma vRNA levels compared to unimmunized animals. Thus, the results of the nonhuman primate (NHP) study described here recapitulate the lack of protection against HIV acquisition seen in the Step Trial and suggest a greater risk of infection in the Ad5-seropositive animals immunized with the Ad5 SIV vaccine. Further studies are necessary to confirm the enhancement of virus acquisition and to discern associated mechanisms.
Qureshi, H., Ma, Z.-M., Huang, Y., Hodge, G., Thomas, M. A., DiPasquale, J., DeSilva, V., Fritts, L., Bett, A. J., Casimiro, D. R., Shiver, J. W., Robert-Guroff, M., Robertson, M. N., McChesney, M. B., Gilbert, P. B., Miller, C. J.
Low-Dose Penile SIVmac251 Exposure of Rhesus Macaques Infected with Adenovirus Type 5 (Ad5) and Then Immunized with a Replication-Defective Ad5-Based SIV gag/pol/nef Vaccine Recapitulates the Results of the Phase IIb Step Trial of a Similar HIV-1 Vaccine [Pathogenesis and Immunity]
Date de mise en ligne : Jeudi 26 janvier 2012 It is known that respiratory syncytial virus (RSV) is the main cause of bronchiolitis and pneumonia in young children. RSV infection often leads to severe acute lung immunopathology, but the underlying immune mechanisms are not yet fully elucidated. Here, we found that RSV infection induced severe acute lung immune injury and promoted the accumulation and activation of lung natural killer (NK) cells at the early stage of infection in BALB/c mice. Activated lung NK cells highly expressed activating receptors NKG2D and CD27 and became functional NK cells by producing a large amount of gamma interferon (IFN-), which was responsible for acute lung immune injury. NK cell depletion significantly attenuated lung immune injury and reduced infiltration of total inflammatory cells and production of IFN- in bronchoalveolar lavage fluid (BALF). These data show that NK cells are involved in exacerbating the lung immune injury at the early stage of RSV infection via IFN- secretion.
Li, F., Zhu, H., Sun, R., Wei, H., Tian, Z.
Natural Killer Cells Are Involved in Acute Lung Immune Injury Caused by Respiratory Syncytial Virus Infection [Pathogenesis and Immunity]
Date de mise en ligne : Jeudi 26 janvier 2012 Tetherin/BST-2 forms a proteinaceous tether that restricts the release of a number of enveloped viruses following viral budding. Tetherin is an unusual membrane glycoprotein with two membrane anchors and an extended coiled-coil ectodomain. The ectodomain itself forms an imperfect coil that may undergo conformational shifts to accommodate membrane dynamics during the budding process. The coiled-coil ectodomain is required for restriction, but precisely how it contributes to the restriction of particle release remains under investigation. In this study, mutagenesis of the ectodomain was used to further define the role of the coiled-coil ectodomain in restriction. Scanning mutagenesis throughout much of the ectodomain failed to disrupt the ability of tetherin to restrict HIV particle release, indicating a high degree of plasticity. Targeted N- and C-terminal substitutions disrupting the coiled coil led to both a loss of restriction and an alteration of subcellular distribution. Two ectodomain mutants deficient in restriction were endocytosed inefficiently, and the levels of these mutants on the cell surface were significantly enhanced. An ectodomain mutant with four targeted serine substitutions (4S) failed to cluster in membrane microdomains, was deficient in restriction of particle release, and exhibited an increase in lateral mobility on the membrane. These results suggest that the tetherin ectodomain contributes to microdomain localization and to constrained lateral mobility. We propose that focal clustering of tetherin via ectodomain interactions plays a role in restriction of particle release.
Hammonds, J., Ding, L., Chu, H., Geller, K., Robbins, A., Wang, J.-J., Yi, H., Spearman, P.
The Tetherin/BST-2 Coiled-Coil Ectodomain Mediates Plasma Membrane Microdomain Localization and Restriction of Particle Release [Structure and Assembly]
Date de mise en ligne : Jeudi 26 janvier 2012 Herpes simplex virus 1 (HSV-1) causes a spectrum of disease, including herpes labialis, herpes keratitis, and herpes encephalitis, which can be lethal. Viral recognition by pattern recognition receptors plays a central role in cytokine production and in the generation of antiviral immunity. The relative contributions of different Toll-like receptors (TLRs) in the innate immune response during central nervous system infection with HSV-1 have not been fully characterized. In this study, we investigate the roles of TLR2, TLR9, UNC93B1, and the type I interferon (IFN) receptor in a murine model of HSV-1 encephalitis. TLR2 is responsible for detrimental inflammatory cytokine production following intracranial infection with HSV-1, and the absence of TLR2 expression leads to increased survival in mice. We prove that inflammatory cytokine production by microglial cells, astrocytes, neutrophils, and monocytes is mediated predominantly by TLR2. We also demonstrate that type I IFNs are absolutely required for survival following intracranial HSV-1 infection, as mice lacking the type I IFN receptor succumb rapidly following infection and have high levels of HSV in the brain. However, the absence of TLR9 does not impact survival, type I IFN levels, or viral replication in the brain following infection. The absence of UNC93B1 leads to a survival disadvantage but does not impact viral replication or type I IFN levels in the brain in HSV-1-infected mice. These results illustrate the complex but important roles that innate immune receptors play in host responses to HSV-1 during infection of the central nervous system.
Wang, J. P., Bowen, G. N., Zhou, S., Cerny, A., Zacharia, A., Knipe, D. M., Finberg, R. W., Kurt-Jones, E. A.
Role of Specific Innate Immune Responses in Herpes Simplex Virus Infection of the Central Nervous System [Pathogenesis and Immunity]
Date de mise en ligne : Jeudi 26 janvier 2012 The life cycle of adenoviruses is divided by convention into early and late phases, separated by the onset of viral genome replication. Early events include virus adsorption, transport of the genome into the nucleus, and the expression of early genes. After the onset of viral DNA replication, transcription of the major late transcription unit (MLTU) and thereby synthesis of late proteins is induced. These steps are controlled by an orchestra of regulatory processes and require import of the genome and numerous viral proteins into the nucleus, as well as active transport of viral transcripts and proteins from the nucleus to the cytoplasm. The latter is achieved by exploiting the shuttling functions of cellular transport receptors, which normally stimulate the nuclear export of cellular mRNA and protein cargos. A set of adenoviral early and late proteins contains a leucine-rich nuclear export signal of the HIV-1 Rev type, known to be recognized by the cellular export receptor CRM1. However, a role for CRM1-dependent export in supporting adenoviral replication has not been established. To address this issue in detail, we investigated the impact of two different CRM1 inhibitors on several steps of the adenoviral life cycle. Inhibition of CRM1 led to a reduction in viral early and late gene expression, viral genome replication, and progeny virus production. For the first time, our findings indicate that CRM1-dependent shuttling is required for the efficient export of adenoviral early mRNA.
Schmid, M., Gonzalez, R. A., Dobner, T.
CRM1-Dependent Transport Supports Cytoplasmic Accumulation of Adenoviral Early Transcripts [Genome Replication and Regulation of Viral Gene Expression]
Date de mise en ligne : Jeudi 26 janvier 2012 Most of an intravenous dose of species C adenovirus serotype 5 (Ad5) is destroyed by liver Kupffer cells. In contrast, another species C virus, Ad6, evades these cells to mediate more efficient liver gene delivery. Given that this difference in Kupffer cell interaction is mediated by the hypervariable (HVR) loops of the virus hexon protein, we genetically modified each of the seven HVRs of Ad5 with a cysteine residue to enable conditional blocking of these sites with polyethylene glycol (PEG). We show that these modifications do not affect in vitro virus transduction. In contrast, after intravenous injection, targeted PEGylation at HVRs 1, 2, 5, and 7 increased viral liver transduction up to 20-fold. Elimination or saturation of liver Kupffer cells did not significantly affect this increase in the liver transduction. In vitro, PEGylation blocked uptake of viruses via the Kupffer cell scavenger receptor SRA-II. These data suggest that HVRs 1, 2, 5, and 7 of Ad5 may be involved in Kupffer cell recognition and subsequent destruction. These data also demonstrate that this conditional genetic-chemical mutation strategy is a useful tool for investigating the interactions of viruses with host tissues.
Khare, R., Reddy, V. S., Nemerow, G. R., Barry, M. A.
Identification of Adenovirus Serotype 5 Hexon Regions That Interact with Scavenger Receptors [Virus-Cell Interactions]
Date de mise en ligne : Jeudi 26 janvier 2012 Myocarditis is indicated as the second leading cause of sudden death in young adults. Reovirus induces myocarditis in neonatal mice, providing a tractable model system for investigation of this important disease. Alpha/beta-interferon (IFN-α/β) treatment improves cardiac function and inhibits viral replication in patients with chronic myocarditis, and the host IFN-α/β response is a determinant of reovirus strain-specific differences in induction of myocarditis. Virus-induced IFN-β stimulates a signaling cascade that establishes an antiviral state and further induces IFN-α/β through an amplification loop. Reovirus strain-specific differences in induction of and sensitivity to IFN-α/β are associated with the viral M1, L2, and S2 genes. The reovirus M1 gene-encoded μ2 protein is a strain-specific repressor of IFN-β signaling, providing one possible mechanism for the variation in resistance to IFN and induction of myocarditis between different reovirus strains. We report here that μ2 amino acid 208 determines repression of IFN-β signaling and modulates reovirus induction of IFN-β in cardiac myocytes. Moreover, μ2 amino acid 208 determines reovirus replication, both in initially infected cardiac myocytes and after viral spread, by regulating the IFN-β response. Amino acid 208 of μ2 also influences the cytopathic effect in cardiac myocytes after spread. Finally, μ2 amino acid 208 modulates myocarditis in neonatal mice. Thus, repression of IFN-β signaling mediated by reovirus μ2 amino acid 208 is a determinant of the IFN-β response, viral replication and damage in cardiac myocytes, and myocarditis. These results demonstrate that a single amino acid difference between viruses can dictate virus strain-specific differences in suppression of the host IFN-β response and, consequently, damage to the heart.
Irvin, S. C., Zurney, J., Ooms, L. S., Chappell, J. D., Dermody, T. S., Sherry, B.
A Single-Amino-Acid Polymorphism in Reovirus Protein {mu}2 Determines Repression of Interferon Signaling and Modulates Myocarditis [Cellular Response to Infection]
Date de mise en ligne : Jeudi 26 janvier 2012 A rodent or other small animal model for HIV-1 has not been forthcoming, with the principal obstacles being species-specific restriction mechanisms and deficits in HIV-1 dependency factors. Some Carnivorans may harbor comparatively fewer impediments. For example, in contrast to mice, the domestic cat genome encodes essential nonreceptor HIV-1 dependency factors. All Feliformia species and at least one Caniformia species also lack a major lentiviral restriction mechanism (TRIM5α/TRIMCyp proteins). Here we investigated cells from two species in another carnivore family, the Mustelidae, for permissiveness to the HIV-1 life cycle. Mustela putorius furo (domesticated ferret) primary cells and cell lines did not restrict HIV-1, feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), or N-tropic murine leukemia virus (MLV) postentry and supported late HIV-1 life cycle steps comparably to human cells. The ferret TRIM5α gene exon 8, which encodes the B30.2 domain, was found to be pseudogenized. Strikingly, ferret (but not mink) cells engineered to express human HIV-1 entry receptors supported productive spreading replication, amplification, and serial passage of wild-type HIV-1. Nevertheless, produced virions had relatively reduced infectivity and the virus accrued G->A hypermutations, consistent with APOBEC3 protein pressure. Ferret cell-passaged HIV-1 also evolved amino acid changes in the capsid cyclophilin A binding loop. We conclude that the genome of this carnivore can provide essential nonreceptor HIV-1 dependency factors and that ferret APOBEC3 proteins with activity against HIV-1 are likely. Even so, unlike in cat cells, HIV-1 can replicate in ferret cells without vif substitution. The virus evolves in this novel nonprimate cell adaptive landscape. We suggest that further characterization of HIV-1 adaptation in ferret cells and delineation of Mustelidae restriction factor repertoires are warranted, with a view to the potential for an HIV-1 animal model.
Fadel, H. J., Saenz, D. T., Guevara, R., von Messling, V., Peretz, M., Poeschla, E. M.
Productive Replication and Evolution of HIV-1 in Ferret Cells [Virus-Cell Interactions]
Date de mise en ligne : Jeudi 26 janvier 2012 Sustained activation of the Raf/MEK/extracellular signal-regulated kinase (ERK) pathway in infected cells has been shown to be crucial for full replication efficiency of orthopoxviruses in cell culture. In infected cells, this pathway is mainly activated by the vaccinia virus growth factor (VGF), an epidermal growth factor (EGF)-like protein. We show here that chorioallantois vaccinia virus Ankara (CVA), but not modified vaccinia virus Ankara (MVA), induced sustained activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in infected human 293 cells, although both viruses direct secretion of functional VGF. A CVA mutant lacking the O1L gene (CVA-O1L) demonstrated that the O1 protein was required for sustained upregulation of the ERK1/2 pathway in 293 cells as well as in other mammalian cell lines. The highly conserved orthopoxvirus O1L gene encodes a predicted 78-kDa protein with a hitherto-unknown function. CVA-O1L showed reduced plaque size and an attenuated cytopathic effect (CPE) in infected cell cultures and reduced virulence and spread from lungs to ovaries in intranasally infected BALB/c mice. Reinsertion of an intact O1L gene into MVA, which in its original form harbors a fragmented O1L open reading frame (ORF), restored ERK1/2 activation in 293 cells but did not increase replication and spread of MVA in human or other mammalian cell lines. Thus, the O1 protein was crucial for sustained ERK1/2 activation in CVA- and MVA-infected human cells, complementing the autocrine function of VGF, and enhanced virulence in vivo.
Schweneker, M., Lukassen, S., Spath, M., Wolferstatter, M., Babel, E., Brinkmann, K., Wielert, U., Chaplin, P., Suter, M., Hausmann, J.
The Vaccinia Virus O1 Protein Is Required for Sustained Activation of Extracellular Signal-Regulated Kinase 1/2 and Promotes Viral Virulence [Virus-Cell Interactions]
Date de mise en ligne : Jeudi 26 janvier 2012 Japanese encephalitis virus (JEV) is the leading global cause of viral encephalitis. The JEV envelope protein (E) facilitates cellular attachment and membrane fusion and is the primary target of neutralizing antibodies. We have determined the 2.1-Å resolution crystal structure of the JEV E ectodomain refolded from bacterial inclusion bodies. The E protein possesses the three domains characteristic of flavivirus envelopes and epitope mapping of neutralizing antibodies onto the structure reveals determinants that correspond to the domain I lateral ridge, fusion loop, domain III lateral ridge, and domain I-II hinge. While monomeric in solution, JEV E assembles as an antiparallel dimer in the crystal lattice organized in a highly similar fashion as seen in cryo-electron microscopy models of mature flavivirus virions. The dimer interface, however, is remarkably small and lacks many of the domain II contacts observed in other flavivirus E homodimers. In addition, uniquely conserved histidines within the JEV serocomplex suggest that pH-mediated structural transitions may be aided by lateral interactions outside the dimer interface in the icosahedral virion. Our results suggest that variation in dimer structure and stability may significantly influence the assembly, receptor interaction, and uncoating of virions.
Luca, V. C., AbiMansour, J., Nelson, C. A., Fremont, D. H.
Crystal Structure of the Japanese Encephalitis Virus Envelope Protein [Structure and Assembly]
Date de mise en ligne : Jeudi 26 janvier 2012 The incorporation of viral envelope (Env) glycoproteins into nascent particles is an essential step in the production of infectious human immunodeficiency virus type 1 (HIV-1). This process has been shown to require interactions between Env and the matrix (MA) domain of the Gag polyprotein. Previous studies indicate that several residues in the N-terminal region of MA are required for Env incorporation. However, the precise mechanism by which Env proteins are acquired during virus assembly has yet to be fully defined. Here, we examine whether a highly conserved glutamate at position 99 in the C-terminal helix is required for MA function and HIV-1 replication. We analyze a panel of mutant viruses that contain different amino acid substitutions at this position using viral infectivity studies, virus-cell fusion assays, and immunoblotting. We find that E99V mutant viruses are defective for fusion with cell membranes and thus are noninfectious. We show that E99V mutant particles of HIV-1 strains LAI and NL4.3 lack wild-type levels of Env proteins. We identify a compensatory substitution in MA residue 84 and show that it can reverse the E99V-associated defects. Taken together, these results indicate that the C-terminal hydrophobic pocket of MA, which encompasses both residues 84 and 99, has a previously unsuspected and key role in HIV-1 Env incorporation.
Brandano, L., Stevenson, M.
A Highly Conserved Residue in the C-Terminal Helix of HIV-1 Matrix Is Required for Envelope Incorporation into Virus Particles [Structure and Assembly]
Date de mise en ligne : Jeudi 26 janvier 2012 A thorough understanding of the diversity of viruses in wildlife provides epidemiological baseline information about potential pathogens. Metagenomic analysis of the enteric viral flora revealed a new anellovirus and bocavirus species in pine martens and a new circovirus-like virus and geminivirus-related DNA virus in European badgers. In addition, sequences with homology to viruses from the families Paramyxo- and Picornaviridae were detected.
van den Brand, J. M. A., van Leeuwen, M., Schapendonk, C. M., Simon, J. H., Haagmans, B. L., Osterhaus, A. D. M. E., Smits, S. L.
Metagenomic Analysis of the Viral Flora of Pine Marten and European Badger Feces [Genetic Diversity and Evolution]
Date de mise en ligne : Jeudi 26 janvier 2012 The cutaneous beta human papillomavirus (beta HPV) types appear to be involved in skin carcinogenesis. However, only a few beta HPVs have been investigated so far. Here, we compared the properties of E6 and E7 oncoproteins from six uncharacterized beta HPVs (14, 22, 23, 24, 36, 49). Only HPV49 E6 and E7 immortalized primary human keratinocytes and efficiently deregulated the p53 and pRb pathways. Furthermore, HPV49 E6, similarly to E6 from the oncogenic HPV16, promoted p53 degradation.
Cornet, I., Bouvard, V., Campo, M. S., Thomas, M., Banks, L., Gissmann, L., Lamartine, J., Sylla, B. S., Accardi, R., Tommasino, M.
Comparative Analysis of Transforming Properties of E6 and E7 from Different Beta Human Papillomavirus Types [Transformation and Oncogenesis]
Date de mise en ligne : Jeudi 26 janvier 2012 The replication of many viruses involves the formation of higher-order structures or replication "factories." We show that the key replication enzyme of foot-and-mouth disease virus (FMDV), the RNA-dependent RNA polymerase, forms fibrils in vitro. Although there are similarities with previously characterized poliovirus polymerase fibrils, FMDV fibrils are narrower, are composed of both protein and RNA, and, importantly, are seen only when all components of an elongation assay are present. Furthermore, an inhibitory RNA aptamer prevents fibril formation.
Bentham, M., Holmes, K., Forrest, S., Rowlands, D. J., Stonehouse, N. J.
Formation of Higher-Order Foot-and-Mouth Disease Virus 3Dpol Complexes Is Dependent on Elongation Activity [Genome Replication and Regulation of Viral Gene Expression]
Date de mise en ligne : Jeudi 26 janvier 2012 The 2009 pandemic influenza virus (pdm/09) has been frequently introduced to pigs and has reassorted with other swine viruses. Recently, H3N2 reassortants with pdm/09-like internal genes were isolated in Guangxi and Hong Kong, China. Genetic and epidemiological analyses suggest that these viruses have circulated in swine for some time. This is the first evidence that swine reassortant viruses with pdm/09-like genes may have become established in the field, altering the landscape of human and swine influenza.
Fan, X., Zhu, H., Zhou, B., Smith, D. K., Chen, X., Lam, T. T.- Y., Poon, L. L. M., Peiris, M., Guan, Y.
Emergence and Dissemination of a Swine H3N2 Reassortant Influenza Virus with 2009 Pandemic H1N1 Genes in Pigs in China [Genetic Diversity and Evolution]
Date de mise en ligne : Jeudi 26 janvier 2012 Porcine parvovirus (PPV) isolate PPV2010 has recently emerged in China. Herein, we analyze the complete genome sequence of PPV2010. Our results indicate that the genome of PPV2010 bears mixed characteristics of virulent PPV and vaccine strains. Importantly, PPV2010 has the potential to be a naturally attenuated candidate vaccine strain.
Cui, J., Wang, X., Ren, Y., Cui, S., Li, G., Ren, X.
Genome Sequence of Chinese Porcine Parvovirus Strain PPV2010 [Genome Announcements]
Date de mise en ligne : Jeudi 26 janvier 2012 Emiliania huxleyi virus 202 (EhV-202) is a member of the Coccolithoviridae, a group of viruses that infect the marine coccolithophorid Emiliania huxleyi. EhV-202 has a 160- to 180-nm-diameter icosahedral structure and a genome of approximately 407 kbp, consisting of 485 coding sequences (CDSs). Here we describe the genomic features of EhV-202, together with a draft genome sequence and its annotation, highlighting the homology and heterogeneity of this genome in comparison with the EhV-86 reference genome.
Nissimov, J. I., Worthy, C. A., Rooks, P., Napier, J. A., Kimmance, S. A., Henn, M. R., Ogata, H., Allen, M. J.
Draft Genome Sequence of the Coccolithovirus Emiliania huxleyi Virus 202 [Genome Announcements]
Date de mise en ligne : Jeudi 26 janvier 2012 Bacteriophages are the most numerous biological entities in the biosphere, and although their genetic diversity is high, it remains ill defined. Mycobacteriophages—the viruses of mycobacterial hosts—provide insights into this diversity as well as tools for manipulating Mycobacterium tuberculosis. We report here the complete genome sequences of 138 new mycobacteriophages, which—together with the 83 mycobacteriophages previously reported—represent the largest collection of phages known to infect a single common host, Mycobacterium smegmatis mc2 155.
Hatfull, G. F., the Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science Program, the KwaZulu-Natal Research Institute for Tuberculosis and HIV Mycobacterial Genetics Course Students, the Phage Hunters Integrating Research and Education Program
Complete Genome Sequences of 138 Mycobacteriophages [Genome Announcements]
Date de mise en ligne : Jeudi 26 janvier 2012
Liegeois, F., Verrier, D., Greenwood, E., Schmidt, F., Heeney, J., Rouet, F.
Control of Simian Immunodeficiency Virus SIVmnd-1 RNA Plasma Viremia after Coinfection or Superinfection with SIVmnd-1 in SIVmnd-2-Infected Mandrills and Vice Versa [Letters to the Editor]
Date de mise en ligne : Jeudi 26 janvier 2012
Souquiere, S., Apetrei, C., Chahroudi, A., Pandrea, I., Silvestri, G., Roques, P.
Reply to "Control of Simian Immunodeficiency Virus SIVmnd-1 RNA Plasma Viremia after Coinfection or Superinfection with SIVmnd-1 in SIVmnd-2-Infected Mandrills and Vice Versa" [Letters to the Editor]