- Volume 93, Issue 5, 2012

In order to investigate molecular mechanisms of internal ribosome entry site (IRES)-mediated translation in classical swine fever virus (CSFV), an important pathogen of pigs, the expression level of NS3 was evaluated in the context of genomic RNAs and reporter RNA fragments. All data showed that the NS5A protein has an inhibitory effect on IRES-mediated translation and that NS5B proteins suppress the inhibitory effect of NS5A on viral translation, but CSFV NS5B GDD mutants do not. Furthermore, glutathione S-transferase pull-down assay and immunoprecipitation analysis, associated with deletion and alanine-scanning mutations, were performed. Results showed that NS5B interacts with NS5A and that the region aa 390–414, located in the C-terminal half of NS5A, is important for binding of NS5B to NS5A. Furthermore, amino acids K399, T401, E406 and L413 in the region were found to be essential for NS5A–NS5B interaction, virus rescue and infection. The above-mentioned region and four amino acids were observed to overlap with the site responsible for inhibition of IRES-mediated translation by the NS5A protein. We also found that aa 63–72, aa 637–653 and the GDD motif of NS5B were necessary for the interaction between NS5A and NS5B. These findings suggest that the repression activity of the NS5B protein toward the role of NS5A in translation might be achieved by NS5A–NS5B interaction, for which aa 390–414 of NS5A and aa 63–72, aa 637–653 and the GDD motif of NS5B are indispensable. This is important for understanding the role of NS5A–NS5B interaction in the virus life cycle.

Cap-dependent and internal ribosomal entry site (IRES)-mediated translation are regulated differently within cells. Viral IRES-mediated translation often remains active when cellular cap-dependent translation is severely impaired under cellular stresses induced by virus infection. To investigate how cellular stresses influence the efficiency of viral IRES-mediated translation, we used a bicistronic luciferase reporter construct harbouring IRES elements from the following viruses: encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV) or human rhinovirus (HRV). NIH3T3 cells transfected with these bicistronic reporter constructs were subjected to different cellular stresses. Increased translation initiation was only observed under amino acid starvation when EMCV or FMDV IRES elements were present. To identify cellular mechanisms that promoted viral IRES-mediated translation, we tested the involvement of eukaryotic initiation factor 4E-binding protein (4E-BP), general control non-depressed 2 (GCN2) and eukaryotic initiation factor 2B (eIF2B), as these are known to be modulated under amino acid starvation. Knockdown of 4E-BP1 impaired the promotion of EMCV and FMDV IRES-mediated translation under amino acid starvation, whereas GCN2 and eIF2B were not involved. To further investigate how 4E-BP1 regulates translation initiated by EMCV and FMDV IRES elements, we used a phosphoinositide kinase-3 inhibitor (LY294002), an mTOR inhibitor (Torin1) or leucine starvation to mimic 4E-BP1 dephosphorylation induced by amino acid starvation. 4E-BP1 dephosphorylation induced by the treatments was not sufficient to promote viral IRES-mediated translation. These results suggest that 4E-BP1 regulates EMCV and FMDV IRES-mediated translation under amino acid starvation, but not via its dephosphorylation.

The cellular protein tetherin is thought to act as a ‘leash’ that anchors many enveloped viruses to the plasma membrane and prevents their release. We found that replication of multiple strains of influenza A virus was generally insensitive to alteration of tetherin levels, as assessed by output titre or scanning electron microscopy of cell-associated virions. This included human, swine, avian and equine isolates, strains that form filamentous or spherical particles and viruses that lack the M2 or NS1 proteins. Levels of cell-surface tetherin were not reduced by influenza infection, but tetherin and the viral haemagglutinin co-localized on the plasma membrane. However, tetherin could not be detected in filamentous virions, suggesting that influenza may possess a mechanism to exclude it from virions. Overall, if influenza does encode a specific antagonist of tetherin, it is not M2 or NS1 and we find no evidence for a role in host range specificity.

The viral haemagglutinin (HA) and the viral polymerase complex determine the replication fitness of a highly virulent variant of influenza A virus strain A/PR/8/34 (designated hvPR8) and its high pathogenicity in mice. We report here that the HA of the hvPR8 differs from the HA of a low virulent strain (lvPR8) by the efficiency of receptor binding and membrane fusion. hvPR8 bound to 2,6-linked as well as 2,3-linked sialic acid-containing receptors, whereas lvPR8 bound exclusively to 2,3-linked sialic acids with high avidity. Remarkably, hvPR8 infected its target cells faster than lvPR8 and tolerated an elevated pH for efficient membrane fusion. In spite of these differences, both viruses targeted type II but not type I pneumocytes in the lung of infected mice. The HA of hvPR8 differs from that of lvPR8 by 16 aa substitutions and one insertion. Mutational analyses revealed that amino acid at HA position 190 (H3 numbering) primarily determined the specificity of receptor binding, while the insertion at position 133 influenced the avidity of receptor binding. Both amino acid positions also strongly influenced viral virulence. Furthermore, leucine at position 78 and glutamine at position 354 were critical determinants of increased fusion activity and virulence of hvPR8. Our data suggest that the HA of hvPR8 enhances virulence by mediating optimal receptor binding and membrane fusion thereby promoting rapid and efficient viral entry into host cells.

The emergence of drug-resistant strains of influenza virus has catalysed a search for new antiviral agents to supplement or replace existing drugs. Following the success of the human immunodeficiency virus entry blocker Enfuvirtide, there has been a resurgence of interest in peptide-based antivirals. In this paper, we report on the discovery of a novel family of peptides (FluPep, FP) that function as inhibitors of influenza A virus infection. The prototype peptide (FP1, also known as Tkip) interacts with haemagglutinin and inhibits the binding of the virus to cell membranes. Using a plaque-reduction assay, we have demonstrated that a variety of influenza A virus subtypes (including H1N1, H3N2 and H5N1) are inhibited by FluPep and its derivatives at nanomolar concentrations. By truncating FluPep we have identified a minimal sequence of 6 aa that binds to haemagglutinin and inhibits infection. Using a mouse model of intranasal influenza virus infection, we observed potent inhibition of virus infection when peptide is given at the time of virus administration. These data indicate that FluPep is a highly effective anti-influenza agent with the potential to translate to the clinic.

Although the T-cell-mediated immune response to influenza virus has been studied extensively, little information is available on the direct interaction between influenza virus and T-cells that pertains to severe diseases in humans and animals. To address these issues, we utilized the BALB/c mouse model combined with primary T-cells infected with A/WSN/33 influenza virus to investigate whether influenza virus has an affinity for T-cells in vivo. We observed that small proportions of CD4+ T-cells and CD8+ T-cells in spleen and thymus expressed viral proteins in infected mice. A significant proportion of mouse primary T-cells displayed expression of α-2,6 sialic acid-linked influenza virus receptor and were infected directly by influenza A virus. These experiments reveal that there exists a population of T-cells that is susceptible to influenza A virus infection. Furthermore, we employed human Jurkat T-cells to investigate the virus–T-cell interaction, with particular emphasis on understanding whether Itk (interleukin-2-inducible T-cell kinase), a Tec family tyrosine kinase that regulates T-cell activation, is involved in virus infection of T-cells. Interestingly, influenza virus infection resulted in an increased recruitment of Itk to the plasma membrane and an increased level of phospholipase C-γ1 (PLC-γ1) phosphorylation, suggesting that Itk/PLC-γ1 signalling is activated by the virus infection. We demonstrated that depletion of Itk inhibited the replication of influenza A virus, whereas overexpression of Itk increased virus replication. These results indicate that Itk is required for efficient replication of influenza virus in infected T-cells.

Influenza viruses readily mutate by accumulating point mutations and also by reassortment in which they acquire whole gene segments from another virus in a co-infected host. The NS1 gene is a major virulence factor of influenza A virus. The effects of changes in NS1 sequence depend on the influenza polymerase constellation. Here, we investigated the consequences of a virus with the polymerase of pandemic H1N1 2009 acquiring an NS gene segment derived from a seasonal influenza A H3N2 virus, a combination that might arise during natural reassortment of viruses that currently circulate in humans. We generated recombinant influenza viruses with surface HA and NA genes and matrix M gene segment from A/PR/8/34 virus, but different combinations of polymerase and NS genes. Thus, any changes in phenotype were not due to differences in receptor use, entry, uncoating or virus release. In Madin–Darby canine kidney (MDCK) cells, the virus with the NS gene from the H3N2 parent showed enhanced replication, probably a result of increased control of the interferon response. However, in mice the same virus was attenuated in comparison with the virus containing homologous pH1N1 polymerase and NS genes. Levels of viral RNA during single-cycles of replication were lower for the virus with H3N2 NS, and this virus reached lower titres in the lungs of infected mice. Thus, virus with pH1N1 polymerase genes did not increase its virulence by acquiring the H3N2 NS gene segment, and MDCK cells were a poor predictor of the outcome of infection in vivo.

This study is the first report of experimental infection and transmission of Menangle virus (MenPV) in pigs. Isolated in 1997 from piglets that were stillborn at a large commercial piggery in New South Wales, Australia, MenPV is a recently identified paramyxovirus of bat origin that causes severe reproductive disease in pigs and an influenza-like illness, with a rash, in humans. Although successfully eradicated from the infected piggery, the virus was only isolated from affected fetuses and stillborn piglets during the period of reproductive disease, and thus the mode of transmission between pigs was not established. To investigate the pathogenesis of MenPV, we undertook time-course studies in 6-week-old pigs following intranasal administration of a low-passage, non-plaque-purified isolate from the lung of an infected stillborn piglet. Viraemia was of short duration and low titre, as determined by real-time RT-PCR and virus isolation. Following an incubation period of 2–3 days, virus was shed in nasal and oral secretions, faeces and urine, typically for less than 1 week. Cessation of shedding correlated with the development of neutralizing antibodies in sera. Secondary lymphoid organs and intestine were identified, using quantitative real-time RT-PCR, as major sites of viral replication and dissemination, and this was confirmed by positive immunolabelling of viral antigen within various lymphoid tissues and intestinal epithelium. These data provide new insights into the pathogenesis of MenPV in weaned pigs, and will facilitate future control and eradication programmes should it ever re-emerge in the pig population.

To better understand the pathogenicity and infectivity of a natural reassortant CGRn9415 generated from Hantaan virus (HTNV) and Seoul virus (SEOV), CGRn9415, HTNV 76–118 and SEOV L99 were used to infect newborn Kunming (KM) mice and newborn Wistar rats. In KM mice, there was no statistical difference between the death rate with CGRn9415 and that of L99, while 76–118 killed all mice even at low dosage; CGRn9415 killed all infected rats similar to L99 at the dosage of 105 f.f.u., while no death occurred in rats infected with 76–118 even as high as 2×105 f.f.u., suggesting that the reassortant CGRn9415 possesses similar pathogenicity as L99. Furthermore, the reassortant CGRn9415 could establish a persistent infection in both KM mice and Wistar rats more easily than 76–118 or L99. These data suggest that the reassorted hantavirus behaves more like SEOV as far as the pathogenicity is concerned.

Phylogenetic analyses can give new insights into the evolutionary history of viruses, especially of viruses with segmented genomes. However, sequence information for many viral families or genera is still limited and phylogenies based on single or short genome fragments can be misleading. We report the first genetic analysis of all three genome segments of Wyeomyia group viruses Wyeomyia, Taiassui, Macaua, Sororoca, Anhembi and Cachoeira Porteira (BeAr328208) in the genus Orthobunyavirus of the family Bunyaviridae. In addition, Tucunduba and Iaco viruses were identified as members of the Wyeomyia group. Features of Wyeomyia group members that distinguish them from other viruses in the Bunyamwera serogroup and from other orthobunyaviruses, including truncated NSs sequences that may not counteract the host’s interferon response, were characterized. Our findings also suggest genome reassortment within the Wyeomyia group, identifying Macaua and Tucunduba viruses as M-segment reassortants that, in the case of Tucunduba virus, may have altered pathogenicity, stressing the need for whole-genome sequence information to facilitate characterization of orthobunyaviruses and their phylogenetic relationships.

A global phylogeny for chelonid fibropapilloma-associated herpesvirus (CFPHV), the most likely aetiological agent of fibropapillomatosis (FP) in sea turtles, was inferred, using dated sequences, through Bayesian Markov chain Monte Carlo analysis and used to estimate the virus evolutionary rate independent of the evolution of the host, and to resolve the phylogenetic positions of new haplotypes from Puerto Rico and the Gulf of Guinea. Four phylogeographical groups were identified: eastern Pacific, western Atlantic/eastern Caribbean, mid-west Pacific and Atlantic. The latter comprises the Gulf of Guinea and Puerto Rico, suggesting recent virus gene flow between these two regions. One virus haplotype from Florida remained elusive, representing either an independent lineage sharing a common ancestor with all other identified virus variants or an Atlantic representative of the lineage giving rise to the eastern Pacific group. The virus evolutionary rate ranged from 1.62×10−4 to 2.22×10−4 substitutions per site per year, which is much faster than what is expected for a herpesvirus. The mean time for the most recent common ancestor of the modern virus variants was estimated at 192.90–429.71 years ago, which, although more recent than previous estimates, still supports an interpretation that the global FP pandemic is not the result of a recent acquisition of a virulence mutation(s). The phylogeographical pattern obtained seems partially to reflect sea turtle movements, whereas altered environments appear to be implicated in current FP outbreaks and in the modern evolutionary history of CFPHV.

Broad cell tropism contributes to the pathogenesis of human cytomegalovirus (HCMV), but the extent to which cell type influences HCMV gene expression is unclear. A bespoke HCMV DNA microarray was used to monitor the transcriptome activity of the low passage Merlin strain of HCMV at 12, 24, 48 and 72 h post-infection, during a single round of replication in human fetal foreskin fibroblast cells (HFFF-2s), human retinal pigmented epithelial cells (RPE-1s) and human astrocytoma cells (U373MGs). In order to correlate transcriptome activity with concurrent biological responses, viral cytopathic effect, growth kinetics and genomic loads were examined in the three cell types. The temporal expression pattern of viral genes was broadly similar in HFFF-2s and RPE-1s, but dramatically different in U373MGs. Of the 165 known HCMV protein-coding genes, 41 and 48 were differentially regulated in RPE-1s and U373MGs, respectively, compared with HFFF-2s, and 22 of these were differentially regulated in both RPE-1s and U373MGs. In RPE-1s, all differentially regulated genes were downregulated, but, in U373MGs, some were down- and others upregulated. Differentially regulated genes were identified among the immediate-early, early, early late and true-late viral gene classes. Grouping of downregulated genes according to function at landmark stages of the replication cycle led to the identification of potential bottleneck stages (genome replication, virion assembly, and virion maturation and release) that may account for cell type-dependent viral growth kinetics. The possibility that cell type-specific differences in expressed cellular factors are responsible for modulation of viral gene expression is discussed.

Epstein–Barr virus (EBV) replicates in superficial differentiated cells of oral hairy leukoplakia (OHL). Differentiation of squamous epithelial cells depends on B-lymphocyte-induced maturation protein 1 (Blimp1). Here we show that expression of the EBV immediate-early protein BZLF1 is restricted to Blimp1-positive epithelial cells in OHL. Luciferase assays revealed Blimp1-dependent induction of the BZLF1 promoter Zp in epithelial cell lines. Expression of ZEB1, a negative regulator of Zp, and of Xbp-1, which mediates the Blimp1 effect on Zp in B-cells, was not affected by enforced Blimp1 expression. Moreover, Xbp-1 protein expression was not detected in differentiated epithelial cells of OHL. Thus, Blimp1 induces BZLF1 expression in epithelial cells independently of ZEB1 and Xbp-1. In contrast to epithelial cells of OHL, BZLF1 expression was also observed in Blimp1-negative lymphoid cells in infectious mononucleosis tonsils, suggesting that EBV replication in B-cells may be induced independently of terminal differentiation.

The Epstein–Barr virus (EBV) C promoter (Cp) regulates several genes required for B-cell proliferation in latent EBV infection. The family of repeats (FR) region of the latent origin of plasmid replication (oriP) functions as an Epstein–Barr nuclear antigen 1 (EBNA1)-dependent distant enhancer of Cp activity, and the enhancer–promoter interaction is mediated by a higher-order multi-protein complex containing several copies of EBNA1. Using DNA-affinity purification with a 170 bp region of the Cp in combination with mass spectrometry, we identified the cell cycle-regulatory protein E2F1, the E2F-binding protein ARID3A, and the B-cell-specific transcription factor Oct-2 as components of this multi-protein complex. Binding of the three factors to the FR region of oriP was determined by DNA-affinity and immunoblot analysis. Co-immunoprecipitation and proximity ligation analysis revealed that the three factors, E2F1, ARID3A and Oct-2, interact with each other as well as with EBNA1 in the nuclei of EBV-positive cells. Using the chromatin immunoprecipitation assay, we showed that E2F1 and Oct-2 interacted with the FR part of oriP and the Cp, but the ARID3A interaction was, however, only detected at the Cp. Our findings support the hypothesis that EBNA1 initiates transcription at the Cp via interactions between multiple EBNA1 homodimers and cellular transcription factors in a large molecular machinery that forms a dynamic interaction between Cp and FR.

Although ORF23 is conserved among gammaherpesviruses, its role during infection is unknown. Here, we studied the expression of ORF23 of murine gammaherpesvirus 68 (MHV-68) and its role during infection. ORF23 mRNA was detected in infected cells as a late transcript. The ORF23 protein product could be expressed and detected as an N-terminally FLAG-tagged protein by Western blot and indirect immunofluorescence. To investigate the role of ORF23 in the infection cycle of a gammaherpesvirus, we constructed an ORF23 deletion mutant of MHV-68. The analysis of the ORF23 deletion mutant suggested that ORF23 of MHV-68 is neither essential for replication in cell culture nor for lytic or latent infection in vivo. A phenotype of the ORF23 deletion mutant, reflected by a moderate reduction in lytic replication and latency amplification, was only detectable in the face of direct competition to the parental virus.

The oncogenic E6 proteins produced by high-risk human papillomaviruses (HPVs) are invariably expressed in cervical carcinomas and are multifunctional proteins capable of affecting host-cell proliferation by binding and deregulating key host molecules such as p53. High-risk HPVs, including HPV16, have the unique ability to splice the E6 viral transcript, resulting in the production of a truncated E6 protein known as E6*I whose precise biological function is unclear. This study explored the changes in gene expression of the cervical cancer C33A cell line stably expressing HPV16 E6*I (16E6*I) and observed the upregulation of ten genes. Two of these genes were aldo-keto reductases (AKR1Cs), AKR1C1 and AKR1C3, which have been implicated in drug resistance. The results demonstrated that expression of 16E6*I, but not full-length E6, specifically increased AKR1C1 transcript levels although it did not alter AKR1C2 transcript levels. HPV16 E7 alone also had the ability to cause a moderate increase in AKR1C3 at both mRNA and protein levels. Site-directed mutagenesis of 16E6*I revealed that transactivation activity was abolished in R8A, R10A and T17A 16E6*I mutants without altering their intracellular localization patterns. Loss of transactivation activity of the 16E6*I mutants resulted in a significant loss of AKR1C expression and a decrease in drug resistance. Analysis of the AKR1C1 promoter revealed that, unlike the E6 protein, 16E6*I does not mediate transactivation activity solely through Sp1-binding sites. Taken together, it was concluded that 16E6*I has a novel function in upregulating expression of AKR1C and, in concert with E7, has implications for drug treatment in HPV-mediated cervical cancer.

Banana bunchy top virus (BBTV; family Nanoviridae, genus Babuvirus) is a multi-component, ssDNA virus, which causes widespread banana crop losses throughout tropical Africa and Australasia. We determined the full genome sequences of 12 BBTV isolates from the Kingdom of Tonga and analysed these together with previously determined BBTV sequences to show that reassortment and both inter- and intra-component recombination have all been relatively frequent occurrences during BBTV evolution. We found that whereas DNA-U3 components display evidence of complex inter- and intra-component recombination, all of the South Pacific DNA-R components have a common intra-component recombinant origin spanning the replication-associated protein gene. Altogether, the DNA-U3 and DNA-M components display a greater degree of inter-component recombination than the DNA-R, -S, -C and -M components. The breakpoint distribution of the inter-component recombination events reveals a primary recombination hotspot around the 5′ side of the common region major and, in accordance with recombination hotspots detectable in related ssDNA viruses, a secondary recombination hotspot near the origin of virion-strand replication.