Hot off the presses! Apr 25 Virology

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Latest Articles Include:

• Editorial Board
  - Virology 387(1):IFC (2009)
  
• The high-risk HPV E6 oncoprotein preferentially targets phosphorylated nuclear forms of hDlg
  - Virology 387(1):1-4 (2009)
  High-risk mucosal HPV E6 oncoproteins target a number of PDZ domain-containing substrates for proteasome mediated degradation. One of these, Discs Large (Dlg), is involved in the regulation of cell polarity and proliferation control. Previous studies had suggested that Dlg when hyperphosphorylated by osmotic shock, or when present in the nucleus could be preferentially targeted by E6. In this study we use phospho-specific antibodies directed against Dlg phosphorylated at residues S158 and S442 to show that these two observations are, in fact, linked. Dlg, when phosphorylated on S158 and S442 by CDK1 or CDK2, shows a preferential nuclear accumulation. However, these forms of Dlg are absent in cells derived from HPV-induced cervical cancers. Upon either proteasome inhibition or siRNA ablation of E6 expression, we see specific rescue of these phosphorylated forms of Dlg. These results demonstrate that nuclear forms of Dlg phosphorylated on its CDK phospho-acceptor sites h! as enhanced susceptibility to E6-induced degradation and place previous studies on the stress-induced phosphorylation of Dlg into a relevant biological context.
• Identification of a lipid kinase as a host factor involved in hepatitis C virus RNA replication
  - Virology 387(1):5-10 (2009)
  A functional screen of an adenovirus-delivered shRNA library that targets not, vert, similar 4500 host genes was performed to identify cellular factors that regulate hepatitis C virus (HCV) sub-genomic RNA replication. Seventy-three hits were further examined by siRNA oligonucleotide-directed knockdown, and silencing of the PI4KA gene was demonstrated to have a significant effect on the replication of a HCV genotype 1b replicon. Using transient siRNA oligonucleotide transfections and stable shRNA knockdown clones in HuH-7 cells, the PI4KA gene was shown to be essential for the replication of all HCV genotypes tested (1a, 1b and 2a) but not required for bovine viral diarrhea virus (BVDV) RNA replication.
• Single-particle cryo-electron microscopy of Rift Valley fever virus
  - Virology 387(1):11-15 (2009)
  Rift Valley fever virus (RVFV; Bunyaviridae; Phlebovirus) is an emerging human and veterinary pathogen causing acute hepatitis in ruminants and has the potential to cause hemorrhagic fever in humans. We report a three-dimensional reconstruction of RVFV vaccine strain MP-12 (RVFV MP-12) by cryo-electron microcopy using icosahedral symmetry of individual virions. Although the genomic core of RVFV MP-12 is apparently poorly ordered, the glycoproteins on the virus surface are highly symmetric and arranged on a T = 12 icosahedral lattice. Our RVFV MP-12 structure allowed clear identification of inter-capsomer contacts and definition of possible glycoprotein arrangements within capsomers. This structure provides a detailed model for phleboviruses, opens new avenues for high-resolution structural studies of the bunyavirus family, and aids the design of antiviral diagnostics and effective subunit vaccines.
• Mamu-Alow asterisk01/Kb transgenic and MHC Class I knockout mice as a tool for HIV vaccine development
  - Virology 387(1):16-28 (2009)
  We have developed a murine model expressing the rhesus macaque (RM) Mamu-Alow asterisk01 MHC allele to characterize immune responses and vaccines based on antigens of importance to human disease processes. Towards that goal, transgenic (Tg) mice expressing chimeric RM (α1 and α2 Mamu-Alow asterisk01 domains) and murine (α3, transmembrane, and cytoplasmic H-2Kb domains) MHC Class I molecules were derived by transgenesis of the H-2KbDb double MHC Class I knockout strain. After immunization of Mamu-Alow asterisk01/Kb Tg mice with rVV-SIVGag–Pol, the mice generated CD8+ T-cell IFN-γ responses to several known Mamu-Alow asterisk01 restricted epitopes from the SIV Gag and Pol antigen sequence. Fusion peptides of highly recognized CTL epitopes from SIV Pol and Gag and a strong T-help epitope were shown to be immunogenic and capable of limiting an rVV-SIVGag–Pol challenge. Mamu-Alow asterisk01/Kb Tg mice provide a model system to study the Mamu-Alow asterisk01 restrict! ed T-cell response for various infectious diseases which are applicable to a study in RM.
• Characterization and subcellular localization of an RNA silencing suppressor encoded by Rice stripe tenuivirus
  - Virology 387(1):29-40 (2009)
  Rice stripe virus (RSV) is a single-stranded (ss) RNA virus belonging to the genus Tenuivirus. RSV is present in many East Asian countries and causes severe diseases in rice fields, especially in China. In this study, we analyzed six proteins encoded by the virus for their abilities to suppress RNA silencing in plant using a green fluorescent protein (GFP)-based transient expression assay. Our results indicate that NS3 encoded by RSV RNA3, but not other five RSV encoded proteins, can strongly suppress local GFP silencing in agroinfiltrated Nicotiana benthamiana leaves. NS3 can reverse the GFP silencing, it can also prevent long distance spread of silencing signals which have been reported to be necessary for inducing systemic silencing in host plants. The NS3 protein can significantly reduce the levels of small interfering RNAs (siRNAs) in silencing cells, and was found to bind 21-nucleotide ss-siRNA, siRNA duplex and long ssRNA but not long double-stranded (ds)-RNA. B! oth N and C terminal of the NS3 protein are critical for silencing suppression, and mutation of the putative nuclear localization signal decreases its local silencing suppression efficiency and blocks its systemic silencing suppression. The NS3-GFP fusion protein and NS3 were shown to accumulate predominantly in nuclei of onion, tobacco and rice cells through transient expression assay or immunocytochemistry and electron microscopy. In addition, transgenic rice and tobacco plants expressing the NS3 did not show any apparent alteration in plant growth and morphology, although NS3 was proven to be a pathogenicity determinant in the PVX heterogenous system. Taken together, our results demonstrate that RSV NS3 is a suppressor of RNA silencing in planta, possibly through sequestering siRNA molecules generated in cells that are undergoing gene silencing.
• Activation of the lytic program of the Epstein–Barr virus in Burkitt's lymphoma cells leads to a two steps downregulation of expression of the proapoptotic protein BimEL, one of which is EBV-late-gene expression dependent
  - Virology 387(1):41-49 (2009)
  The Epstein–Barr virus (EBV) generally latently infects its target cells with expression of genes conferring resistance to apoptosis. However, the modulation of apoptotic signals during lytic cycle remains poorly understood. We show here that resulting from viral reactivation in the EBV-positive Mutu-I and Akata Burkitt's lymphoma cell lines, a two steps proteasome-dependent downregulation of expression of the proapoptotic protein BimEL occurs. The first drop might be EBV-independent, is ERK 1/2 dependent, and BimEL is phosphorylated on Ser69. A second dramatic drop of the BimEL level observed during the lytic cycle is dependent of EBV-late-gene expression, ERK 1/2 independent, and no further phosphorylation of BimEL on Ser69 occurred. These results demonstrate for the first time, that the lytic cycle contributes to downregulation of BimEL and then could add to protection against apoptosis.
• A procedure for systematic identification of bacteriophage–host interactions of P. aeruginosa phages
  - Virology 387(1):50-58 (2009)
  Immediately after bacteriophage infection, phage early proteins establish optimal conditions for phage infection, often through a direct interaction with host-cell proteins. We implemented a yeast two-hybrid approach for Pseudomonas aeruginosa phages as a first step in the analysis of these – often uncharacterized – proteins. A 24-fold redundant prey library of P. aeruginosa PAO1 (7.32 × 106 independent clones), was screened against early proteins (gp1 to 9) of phiKMV, a P. aeruginosa-infecting member of the Podoviridae; interactions were verified using an independent in vitro assay. None resembles previously known bacteriophage–host interactions, as the three identified target malate synthase G, a regulator of a secretion system and a regulator of nitrogen assimilation. Although at least two-bacteriophage infections are non-essential to phiKMV infection, their disruption has an influence on infection efficiency. This methodology allows systematic analysis of ph! age proteins and is applicable as an interaction analysis tool for P. aeruginosa.
• CD4+ NK cells can be productively infected with HIV, leading to downregulation of CD4 expression and changes in function
  - Virology 387(1):59-66 (2009)
  NK cells mediate the innate immune response, and HIV-infected individuals demonstrate altered NK cell phenotype and function. We find that CD4+ NK cells are susceptible to HIV infection; this could account for the NK cell dysfunction seen in HIV-infected individuals. CD4+ NK cells express CXCR4 and can be infected with X4-tropic viruses and some primary R5-utilizing viral isolates. Treatment with the CXCR4 ligands AMD3100 and SDF-1α partially blocks infection with X4-tropic virus, treatment with anti-CCL Igs upregulates CCR5 surface expression and enables infection with HIV-Bal. HIV infection of NK cells results in CD4 downregulation and the production of infectious virus. HIV-infected CD4+ NK cells mediate NK cell cytotoxicity, however, HIV infection is associated with decreased chemotaxis towards IL-16. Thus, HIV infection of CD4+ NK cells could account for the NK cell dysfunction observed in HIV-infected individuals. Furthermore infected NK cells could serve as a v! iral reservoir of HIV in vivo.
• Antiviral activity of carbohydrate-binding agents and the role of DC-SIGN in dengue virus infection
  - Virology 387(1):67-75 (2009)
  Dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) is an important binding receptor for dengue virus (DENV) that recognizes N-glycosylation sites on the viral E-glycoprotein. DENV cannot bind nor infect the human B-cell line Raji/0. However, DENV productively infects Raji/DC-SIGN+ cells that constitutively express DC-SIGN on their surface. IL-4-treated monocytes, expressing high levels of DC-SIGN, are also susceptible for DENV infection. Several carbohydrate-binding agents (CBAs), such as the plant lectins HHA, GNA (mannose-specific) and UDA (N-acetylglucosamine-specific), inhibited dose-dependently the binding of DENV and subsequently viral replication in Raji/DC-SIGN+ cells (EC50: 0.1–2.2 μM). These CBAs were clearly more active against DENV in IL-4-treated monocytes (EC50: 4–56 nM). However, the CBAs were devoid of antiviral activity in DENV-susceptible Vero-B (DC-SIGN−) cells, demonstrating cell type-dependent differenc! es in viral entry mechanisms.
• A comprehensive analysis of recruitment and transactivation potential of K-Rta and K-bZIP during reactivation of Kaposi's sarcoma-associated herpesvirus
  - Virology 387(1):76-88 (2009)
  Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma. K-Rta and K-bZIP are two major viral transcription factors that control reactivation of this virus. Here we report a genome-wide analysis of transcriptional capacity by evaluation of a comprehensive library of 83 putative KSHV promoters. In reporter assays, 34 viral promoters were activated by K-Rta, whereas K-bZIP activated 21 promoters. When K-Rta and K-bZIP were combined, 3 K-Rta responsive promoters were repressed by K-bZIP. The occupancy of K-Rta and K-bZIP across KSHV promoters was analyzed by chromatin immunoprecipitation with a viral promoter-chip in BCBL-1 cells. In addition, acetylation of local histones was examined to determine accessibility of promoters during latency and reactivation. Finally, 10 promoters were selected to study the dynamics of transcription factor recruitment. This study provides a comprehensive overview of the responsiveness of KSHV promoters to K! -Rta and K-bZIP, and describes key chromatin changes during viral reactivation.
• Hybrid of baculovirus and galactosylated PEI for efficient gene carrier
  - Virology 387(1):89-97 (2009)
  Baculovirus, containing an appropriate eukaryotic promoter, is considered an attractive approach for an efficient and safe gene delivery vehicle. However, the drawbacks of baculovirus, such as the lack of specificity and the inactivation of baculovirus by the complement system in human serum, negatively affect efficient gene delivery. Therefore, a hybrid system utilizing the positive aspects of both viral and non-viral vector systems would be useful to overcome the obstacles of either system alone. In this study, we constructed a hybrid system composed of baculovirus (B) and galactosylated polyethylenimine (GP)/DNA complexes through electrostatic interaction. The resulting GP/B hybrid had suitable physicochemical properties and low cytotoxicity for use in gene therapy. Furthermore, the GP/B significantly enhanced transduction efficiency and showed good cell-specificity compared to either viral or non-viral vector systems. These results suggest that the GP/B hybrid syst! em can be used in gene therapy to enhance transduction efficiency and hepatocyte specificity.
• Latently-infected CD4+ T cells are enriched for HIV-1 Tat variants with impaired transactivation activity
  - Virology 387(1):98-108 (2009)
  The ability of HIV to establish latent infection in CD4+ lymphocytes represents a major barrier to the eradication of HIV. It is not clear what mechanisms favor latent over productive infection, but prior studies have suggested a role for the viral transcription factor Tat or its RNA target, TAR. Using samples from five individuals who were started on ART within 6 months of infection and achieved a viral load < 50 (suppressed), we isolated one- and two-exon tat RNA from HIV propagated ex vivo from baseline plasma and from co-cultures of CD4+ T cells obtained at baseline and suppressed time points. Compared to virus from the baseline plasma (mostly from productively-infected CD4+ T cells), virus from the baseline and suppressed co-cultures (mostly from latently-infected cells) had more Tat variants with impaired transactivation activity. These findings suggest that impaired activity in the Tat–TAR axis may contribute to the establishment of latent infection in CD4+ T ! cells.
• Heterogeneity in the capsid protein of bovine enteric caliciviruses belonging to a new genus
  - Virology 387(1):109-116 (2009)
  Some bovine enteric caliciviruses form a new genus in the family Caliciviridae. In this study, Bayesian phylogenetic analysis of 31 full length capsid sequences from Europe, North America and Asia revealed that this new genus had four currently circulating lineages that showed both temporal and geographical distribution. These groupings were supported by the distribution of the frequency of pair-wise distances. However, the nucleotide and amino acid heterogeneity was low, with a maximum nucleotide and amino acid divergence of 16.7% and 8.4%, respectively. Most variability was found between amino acid residues 288 and 420 of the capsid protein and the sequence motifs observed in this region supported the division of the four lineages. Homology modelling using the structure of the San Miguel sea lion capsid indicated that most variation occurred in the predicted P2 domain and thus, may affect antigenic sites on the surface of the capsid of this newly described genus.
• Peripheral dendritic cells are essential for both the innate and adaptive antiviral immune responses in the central nervous system
  - Virology 387(1):117-126 (2009)
  Intranasal application of vesicular stomatitis virus (VSV) causes acute infection of the central nervous system (CNS). However, VSV encephalitis is not invariably fatal, suggesting that the CNS may contain a professional antigen-presenting cell (APC) capable of inducing or propagating a protective antiviral immune response. To examine this possibility, we first characterized the cellular elements that infiltrate the brain as well as the activation status of resident microglia in the brains of normal and transgenic mice acutely ablated of peripheral dendritic cells (DCs) in vivo. VSV encephalitis was characterized by a pronounced infiltrate of myeloid cells (CD45highCD11b+) and CD8+ T cells containing a subset that was specific for the immunodominant VSV nuclear protein epitope. This T cell response correlated temporally with a rapid and sustained upregulation of MHC class I expression on microglia, whereas class II expression was markedly delayed. Ablation of periphera! l DCs profoundly inhibited the inflammatory response as well as infiltration of virus-specific CD8+ T cells. Unexpectedly, the VSV-induced interferon-gamma (IFN-γ) response in the CNS remained intact in DC-deficient mice. Thus, both the inflammatory and certain components of the adaptive primary antiviral immune response in the CNS are dependent on peripheral DCs in vivo.
• Fragile X mental retardation protein restricts replication of human immunodeficiency virus type 1
  - Virology 387(1):127-135 (2009)
  Gag protein is the major structural component of human immunodeficiency virus type 1 (HIV-1) particles and drives virus assembly on cellular membranes. This function of Gag is attributed to its intrinsic self-multimerization ability as well as its interaction with cellular factors such as TSG101 that binds to the PTAP sequence in the p6 region of Gag and promotes HIV-1 budding through recruiting the ESCRT (endosomal sorting complex required for transport). As a result of its essential role in virus assembly, Gag also becomes the target of cellular restriction factors such as APOBEC3G and Trim5α. In this study, we report that the fragile X mental retardation protein (FMRP) interacts with HIV-1 Gag and is packaged into virus particles. Although knockdown of FMRP does not markedly affect production of virus particles, infectivity of HIV-1 virions was significantly decreased. Consistent with this observation, overexpression of the wild type FMRP, but not the FMRP mutants ! that lack the KH1 or the KH2 domains, led to 2- to 3-fold reduction of virus infectivity. Together, these results suggest that FMRP diminishes HIV-1 infectivity through association with viral Gag protein and virus particles.
• Myxoma virus selectively disrupts type I interferon signaling in primary human fibroblasts by blocking the activation of the Janus kinase Tyk2
  - Virology 387(1):136-146 (2009)
  Poxviruses currently are known to disrupt Jak-STAT signal transduction induced by interferon (IFN) through two distinct mechanisms: (1) secreted poxviral IFN decoy receptors that prevent the initiation of IFN signaling from type I or II receptors at the cell surface; and (2) poxviral phosphatase that dephosphorylates STAT1 intracellularly. Here, we report a novel mechanism by which poxviruses can inhibit Jak-STAT signaling in response to type I IFN. Myxoma virus (MV) is a highly species-restricted member of the poxvirus family that infects only rabbits under the natural setting. Interestingly, primary human fibroblasts support a permissive MV infection that is only partially sensitive to the antiviral state induced by type I IFN. In this study we show that when type I IFN is added to primary human fibroblasts following MV infection, the tyrosine phosphorylation of the Janus kinase Tyk2 is specifically blocked, thereby preventing the subsequent activation of downstream ! STAT1 and STAT2. In stark contrast, type II IFN-induced activation of Jak1, Jak2 and STAT1 remains largely unaffected in MV-infected human fibroblasts. Unlike the de-activation of STAT1 by the poxvirus phosphatase, which is delivered into the cell by the input virions, the Tyk2 inhibition by MV infection requires new viral gene expression. Thus, our study documents a previously unrecognized immune evasion mechanism exploited by a poxvirus to selectively disrupt the type I IFN-Jak-STAT signaling cascade.
• Neutralizing antibody responses to subtype B and C adjuvanted HIV envelope protein vaccination in rabbits
  - Virology 387(1):147-156 (2009)
  Improving the potency, breadth, and durability of neutralizing antibody responses to HIV are major challenges for HIV vaccine development. To address these challenges, the studies described evaluate in rabbits the titers, breadth, and epitope specificities of antibody responses elicited by HIV envelope subunit vaccines adjuvanted with MF59 with or without CpG oligodeoxynucleotide (ODN). Animals were immunized with trimeric o-gp140ΔV2 derived from subtype B HIV-1SF162 or subtype C HIV-1TV1, or proteins from both strains. Immunization with SF162 or TV1 with MF59/CpG elicited higher titers of binding and neutralizing antibodies to SF162 than monovalent immunization with MF59 alone (P < 0.01). Bivalent immunization increased binding and neutralizing antibody titers over single envelope immunization in MF59 (P < 0.01). Bivalent immunization also improved neutralization breadth. Epitope mapping indicated neutralizing activity in rabbits was directed to V3 and V4. Overall, o! ur data suggests that a multivalent vaccination approach with MF59 and CpG can enhance humoral responses to HIV-1.
• Murine Polyomavirus encodes a microRNA that cleaves early RNA transcripts but is not essential for experimental infection
  - Virology 387(1):157-167 (2009)
  MicroRNAs are small regulatory RNAs that post-transcriptionally regulate gene expression and can be encoded by viral as well as cellular genomes. The functions of most viral miRNAs are unknown and few have been studied in an in vivo context. Here we show that the murine polyomavirus (PyV) encodes a precursor microRNA that is processed into two mature microRNAs, both of which are active at directing the cleavage of the early PyV mRNAs. Furthermore, we identify a deletion mutant of polyomavirus that is defective in encoding the microRNAs. This mutant replicates normally and transforms cultured cells with efficiencies comparable to wildtype PyV. The miRNA mutant is competent to establish a transient infection of mice following parenteral inoculation, and is cleared post infection at approximately the same rate as the wildtype virus. In addition, under these laboratory conditions, we observe no differences in anti-viral CD8 T cell responses. These results indicate that PyV! miRNA expression is not essential for infection of cultured cells or experimentally inoculated mice, and raise the possibility that its role in natural infection might involve aspects of acquisition or spread that are not recapitulated by experimental inoculation.
• Association between genomic heterogeneity of hepatitis B virus and intrauterine infection
  - Virology 387(1):168-175 (2009)
  Hepatitis B virus (HBV) intrauterine infection remains to be an important cause for a large number of persistent hepatitis B surface antigen (HBsAg) positive carriers in areas with a high HBV prevalence, particularly in China and Southeast Asia. In this study, the possible association between the HBV genomic heterogeneity and intrauterine infection was investigated by comparing the quasi species isolated from eight pairs of HBsAg-positive mothers and their neonates, who were infected intrauterinely with HBV, with clones from eight HBsAg-positive mothers whose neonates were not infected with HBV. The proportion of clones with specific mutations was compared among different subject groups, and phylogenetic analysis was performed to evaluate the significance of specific mutations. It was observed that the core promoter with conserved major functional regions and conserved hepatitis B e antigen (HBeAg) might be beneficial to HBV maternal–fetal transmission. Particularly,! A1762T/G1764A mutations seemed to be disadvantageous for fetal infection. It was also shown that amino acid substitutions located in the immune epitopes of HBsAg were strongly associated with intrauterine HBV transmission. The clones with mutations such as amino acid P110S in preS1 region, P36L in preS2 region and C107R in S region might infect fetuses more readily. In addition, positively selected site analysis confirmed the above results.
• Host cell sumoylation level influences papillomavirus E2 protein stability
  - Virology 387(1):176-183 (2009)
  The stability of papillomavirus E2 proteins is regulated by proteasomal degradation, and regulation of degradation could contribute to the higher expression levels of E2 proteins observed in suprabasal layers of differentiated skin. We have recently shown that the E2 proteins are modified by sumoylation [Wu Y-C, Roark AA, Bian X-L, Wilson, VG (2008) Virol 378:329–338], and that sumoylation levels are up-regulated during keratinocyte differentiation [Deyrieux AF, Rosas-Acosta G, Ozbun MA, Wilson VG (2007) J Cell Sci 120:125–136]. These observations, coupled with the known ability of sumoylation to prevent proteasomal degradation of certain proteins, suggested that this modification might contribute to stabilizing E2 proteins in suprabasal keratinocytes. Conditions that increased overall sumoylation were found to increase the intracellular amounts of the HPV11, 16, and 18 E2 proteins. No effect of sumoylation was seen on E2 transcripts, and the increased levels of E2! proteins resulted from a greatly increased half-life for the E2 proteins. In vitro studies confirmed that sumoylation could block the proteasomal degradation of the 16E2 protein. Interestingly, this stabilization effect was indirect as it did not require sumoylation of 16E2 itself and must be acting through sumoylation of a cellular target(s). This sumoylation-dependent, indirect stabilization of E2 proteins is a novel process that may couple E2 levels to changes in the cellular environment. Specifically, our results suggest that the levels of papillomavirus E2 protein could be up-regulated in differentiating keratinocytes in response to the increased overall sumoylation that accompanies differentiation.
• Involvement of Bombyx mori nucleopolyhedrovirus ORF41 (Bm41) in BV production and ODV envelopment
  - Virology 387(1):184-192 (2009)
  Bombyx mori nucleopolyhedrovirus (BmNPV) ORF41 (Bm41), homologous to Ac52, is a gene present in most lepidopteran nucleopolyhedroviruses. Bm41 transcripts and encoded protein in BmNPV-infected cells can be detected from 3 and 6 h post-infection, respectively. Immunoassays have shown that Bm41 is not a viral structural protein and is detected in both the nuclei and cytoplasm of infected cells. A Bm41-disrupted virus (vBmDe) and a repaired virus (vBmRe) were generated to investigate the function of Bm41. The results showed that Bm41 was essential for viral replication, and the disruption of Bm41 resulted in a much lower viral titer. Transmission electron microscopy revealed that disruption of Bm41 affected normal nucleocapsid envelopment and polyhedra formation in the nucleus. The disruption of Bm41 might severely affect odv-ec27 and polyhedrin expression. The disrupted virus reduced BmNPV infectivity in an LD50 bioassay and took 18–23 h longer to kill larvae than wild! -type virus in an LT50 bioassay.
• Impairment in reactivation of a latency associated transcript (LAT)-deficient HSV-2 is not solely dependent on the latent viral load or the number of CD8+ T cells infiltrating the ganglia
  - Virology 387(1):193-199 (2009)
  The HSV latency-associated transcript (LAT) is abundantly expressed during virus latency. Previous studies have shown that the latent viral load and CD8+ T cells in ganglia influence the rate of reactivation of HSV. While LAT is important for efficient reactivation and establishment of latency, it is uncertain how LAT affects either the HSV latent viral load or CD8+ T cell infiltration of ganglia. We infected mice with LAT-deficient or LAT-restored HSV-2 at a wide range of inocula. We found that the reduced rate of spontaneous ex-vivo reactivation of the LAT-deficient virus was not associated with a higher number of CD8+ T cells in the ganglia. Reactivation rates were lower for LAT-deficient than LAT restored HSV-2 even when the latent viral loads were similar, indicating that differences in reactivation were not solely dependent on the latent viral load. Therefore, LAT likely has additional functions important for reactivation.
• Influence of extended mutations of the HIV-1 transframe protein p6low asterisk on Nef-dependent viral replication and infectivity in vitro
  - Virology 387(1):200-210 (2009)
  The HIV-1 transframe protein p6low asterisk known to modulate HIV-1 protease activation has been suggested to interact with the viral pathogenicity factor Nef. However, a potential interaction site in p6low asterisk has not been mapped so far. To evaluate effects of p6low asterisk modification on viral replication in light of Nef function, clustered substitutions were introduced into the central p6low asterisk region of the infectious provirus NL4-3 and virus growth and composition of the various mutants was analyzed in different cell cultures in the presence or absence of Nef. Whereas clustered p6low asterisk substitutions did neither affect particle incorporation of Nef, nor precursor maturation or viral infectivity, a simultaneous substitution of 40 of the total 56 p6low asterisk residues significantly diminished viral infectivity and replication in a Nef-independent manner. Furthermore, this extended modification was not capable of rescuing the negative effects of ! a transdominant Nef mutant on particle production suggesting that the proposed target for Nef interaction in Gag-Pol is located outside the modified p6low asterisk region. In sum these data strongly argue against a functional connection of the central p6low asterisk region and Nef during viral life cycle.
• The 5′UTR-specific mutation in VEEV TC-83 genome has a strong effect on RNA replication and subgenomic RNA synthesis, but not on translation of the encoded proteins
  - Virology 387(1):211-221 (2009)
  Venezuelan equine encephalitis virus (VEEV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. Viruses in the VEEV serocomplex continuously circulate in the Central and South America. The only currently available attenuated strain VEEV TC-83 is being used only for vaccination of at-risk laboratory workers and military personnel. Its attenuated phenotype was shown to rely only on two point mutations, one of which, G3A, was found in the 5′ untranslated region (5′UTR) of the viral genome. Our data demonstrate that the G3A mutation strongly affects the secondary structure of VEEV 5′UTR, but has only a minor effect on translation. The indicated mutation increases replication of the viral genome, downregulates transcription of the subgenomic RNA, and, thus, affects the ratio of genomic and subgenomic RNA synthesis. These findings and the previously reported G3A-induced, higher sensitivity of VEEV TC-83 to IFN-α/β suggest a plausi! ble explanation for its attenuated phenotype.
• West Nile virus envelope protein glycosylation is required for efficient viral transmission by Culex vectors
  - Virology 387(1):222-228 (2009)
  Many, but not all, strains of West Nile virus (WNV) contain a single N-linked glycosylation site on their envelope (E) proteins. Previous studies have shown that E-glycosylated strains are more neuroinvasive in mice than non-glycosylated strains. E protein glycosylation also appears to play a role in attachment and entry of WNV into host cells in vitro; however, studies examining how E protein glycosylation affects the interactions of WNV with its mosquito vectors in vivo have not yet been performed. We mutated the E protein glycosylation site from NYS to IYS in a previously described full-length clone of the NY99 genotype of WNV (WT), resulting in a virus that lacked the glycan at aa154. WNV-N154I replicated less efficiently than WNV-WT in Culex mosquito tissues, although the extent of the decrease was greater in Cx. pipiens than in Cx. tarsalis. Following peroral infection, mosquitoes infected with WNV-N154I were less likely to transmit virus than those infected with! WNV-WT. Interestingly, all but one of the mosquitoes infected with WNV-N154I transmitted a revertant virus, suggesting that there is strong selective pressure toward E protein glycosylation. Together these data suggest that loss of the glycan at aa154 on the WNV E protein can severely restrict viral spread in the mosquito vector.
• Estimating the date of origin of an HIV-1 circulating recombinant form
  - Virology 387(1):229-234 (2009)
  HIV is capable of frequent genetic exchange through recombination. Despite the pandemic spread of HIV-1 recombinants, their times of origin are not well understood. We investigate the epidemic history of a HIV-1 circulating recombinant form (CRF) by estimating the time of the recombination event that lead to the emergence of CRF33_01B, a recently described recombinant descended from CRF01_AE and subtype B. The gag, pol and env genes were analyzed using a combined coalescent and relaxed molecular clock model, implemented in a Bayesian Markov chain Monte Carlo framework. Using linked genealogical trees we calculated the time interval between the common ancestor of CRF33_01B and the ancestors it shares with closely related parental lineages. The recombination event that generated CRF33_01B (trec) occurred sometime between 1991 and 1993, suggesting that recombination is common in the early evolutionary history of HIV-1. The proof-of-concept approach provides a new tool for! the investigation of HIV molecular epidemiology and evolution.
• Trans-species amplification of PrPCWD and correlation with rigid loop 170N
  - Virology 387(1):235-243 (2009)
  Chronic wasting disease (CWD) is an efficiently transmitted spongiform encephalopathy of cervids. Whether CWD could represent a threat to non-cervid species remains speculative. Here we show that brain homogenates from several CWD-susceptible non-cervid species, such as ferrets and hamsters, support amplification of PrPCWD by sPMCA, whereas brain homogenates from CWD-resistant species, such as laboratory mice and transgenic mice expressing human PrPC [Tg(HuPrP) mice], do not. We also investigated whether several North American species that share the environment with cervids would support amplification of PrPCWD by sPMCA. Three native rodent species, including voles and field mice, supported PrPCWD amplification, whereas other species (e.g. prairie dog, coyote) did not. Analysis of PrP sequences suggests that an ability to support amplification of PrPCWD in trans-species sPMCA is correlated with the presence of asparagine at position 170 of the substrate species PrP. Se! rial PMCA may offer insights into species barriers to transmission of CWD.
• Corrigendum to "The tale of a modern animal plague: Tracing the evolutionary history and determining the time-scale for foot and mouth disease virus" [Virology 382 (2008) 250–256]
  - Virology 387(1):244 (2009)