- Volume 91, Issue 8, 2010

The Rev protein of human immunodeficiency viruses (HIV) has long been recognized to be essential for the late phase of the virus replication cycle, due to its strong enhancement of expression of viral structural proteins. Surprisingly, a number of recent papers have demonstrated that Rev can also interfere with integration of the reverse-transcribed cDNA into the host-cell genome. This seems to be due to Rev's binding to integrase and LEDGF/p75, an important cellular cofactor of HIV-1 integration. As Rev is presumably expressed at sufficiently high levels only after the encoding genome has already integrated, the main function of Rev during the early phase might be to reduce genotoxicity due to excessive integration events after superinfection of the same cell by subsequent viruses. Other potential consequences for HIV-1 replication and evolution after co-infection of the same cell with two viruses are discussed.

A better understanding of human immunodeficiency virus type 1 drug-resistance evolution under the selective pressure of combination treatment is important for the design of long-term effective treatment strategies. We applied Bayesian network learning to sequences from patients treated with the reverse transcriptase inhibitor combination of zidovudine (AZT) and lamivudine (3TC) to identify the role of many treatment-selected mutations in the development of resistance. Based on the Bayesian network structure, an in vivo fitness landscape was built, reflecting the necessary selective pressure under treatment, to evolve naive sequences to sequences obtained from patients treated with the combination. This landscape, combined with an evolutionary model, was used to predict resistance evolution in longitudinal sequence pairs. In our analysis, mutations 41L, 70R, 184V and 215F/Y were identified as major resistance mutations to the combination of AZT and 3TC, as they were associated directly with treatment experience. The network also suggested a possible role in resistance development for a number of novel mutations. Estimated fitness, using the landscape, correlated significantly with in vitro resistance phenotype in genotype–phenotype pairs (R 2=0.70). Variation in predicted evolution under selective pressure correlated significantly with observed in vivo evolution during AZT plus 3CT treatment. In conclusion, we confirmed current knowledge on resistance development to the combination of AZT and 3CT, but additional novel mutations were identified. Moreover, a model to predict resistance evolution during AZT and 3CT treatment has been built and validated.

Genetically modified cells of haematopoietic and lymphocytic lineages could provide potentially curative treatments for a wide range of inherited and acquired diseases. However, this application is limited in mouse models by the low efficiency of lentiviral vectors. To facilitate the rapid production of high-titre helper-free retroviral vectors for enhanced gene delivery, multiple modifications to a prototype moloney murine leukemia virus (MoMLV)-derived vector system were made including adaptation of the vector system to simian virus 40 ori/T antigen-mediated episomal replication in packaging cells, replacement of the MoMLV 5′ U3 promoter with a series of stronger composite promoters and addition of an extra polyadenylation signal downstream of the 3′ long terminal repeat. These modifications enhanced vector production by 2–3 logs. High-titre vector stocks were tested for their ability to infect a variety of cells derived from humans and mice, including primary monocyte-derived macrophage cultures. Whilst the lentiviral vector was significantly restricted at the integration level, the MoMLV-based vector showed effective gene transduction of mouse cells. This high-titre retroviral vector system represents a useful tool for efficient gene delivery into human and mouse haematopoietic and lymphocytic cells, with particular application in mice as a small animal model for novel gene therapy tests.

Cell entry and membrane fusion of the hepatitis C virus (HCV) depend on its envelope glycoproteins E1 and E2. HCV pseudotyped particles (HCVpps) are relevant and popular models to study the early steps of the HCV life cycle. However, no structural characterization of HCVpp has been available so far. Using cryo-transmission electron microscopy (cryo-TEM), providing structural information at nanometric resolution, the molecular details of HCVpps and their fusion with liposomes were studied. Cryo-TEM revealed HCVpps as regular 100 nm spherical structures containing the dense retroviral nucleocapsid surrounded by a lipid bilayer. E1–E2 glycoproteins were not readily visible on the membrane surface. Pseudoparticles bearing the E1–E2 glycoproteins of Semliki forest virus looked similar, whereas avian influenza A virus (fowl plague virus) haemagglutinin/neuraminidase-pseudotyped particles exhibited surface spikes. To further characterize HCVpp structurally, a novel method was designed based on magnetic beads covered with anti-HCV antibodies to enrich the samples with particles containing E1–E2. This strategy efficiently sorted HCVpps, which were then directly observed by cryo-TEM in the presence or absence of liposomes at low or neutral pH. After acidification, HCVpps looked the same as at neutral pH and closely contacted the liposomes. These are the first visualizations of early HCV membrane fusion events at the nanometer scale. Furthermore, fluorimetry analysis revealed a relative resistance of HCVpps regarding their fusion capacity when exposed to low pH. This study therefore brings several new molecular details to HCVpp characterization and this efficient strategy of virion immunosorting with magnetic nanobeads is direct, efficient and adaptable to extensive characterization of any virus at a nanometric resolution.

Hepatitis C virus (HCV) infection is prevalent throughout the world and interferon (IFN)-based treatments are currently the only therapeutic option. However, depending upon variations in their human leukocyte antigen (HLA), some patients do not respond well to IFN therapy. The current study evaluated the HLA allele and haplotype distribution of 204 HCV-seropositive individuals from Islamabad, Pakistan, who were receiving standard IFN therapy. In this cohort, 150 patients (74 %) showed a sustained virological response to IFN therapy, whereas 54 (26 %) did not. In addition to the HCV patients, 102 unrelated healthy volunteers were used as controls. DNA was isolated from the blood of the patients and controls for HLA-DRB1 and HLA-DQB1 allele typing, whilst plasma was used for HCV detection and genotyping. HLA-DRB1*04 was found to impart a significant protective advantage [Bonferroni-corrected P value (pc)=0.047] against HCV infection. In patients on IFN therapy, HLA-DRB1*11 and -DQB1*0301 (pc=0.044) were found to be associated with viral clearance. In contrast, HLA-DRB1*07 (pc=0.008) individually or in combination with HLA-DQB1*02 was found to be associated with viral persistence. These associations of HLA with HCV persistence or clearance will be beneficial in deciding the therapeutic regimen for Pakistani patients infected with HCV genotype 3a.

Bovine viral diarrhea virus (BVDV) is a positive-sense, single-stranded RNA virus that causes an economically important livestock disease worldwide. Previous studies have suggested that non-structural protein 5A (NS5A) from hepatitis C virus (HCV) and BVDV plays a similar role during virus infection. Extensive reports are available on HCV NS5A and its interactions with the host cellular proteins; however, the role of NS5A during BVDV infection remains largely unclear. To identify the cellular proteins that interact with the N terminus of NS5A and could be involved in its function, we conducted a yeast two-hybrid screening. As a result, we identified a cellular protein termed bovine NIK- and IKKβ-binding protein (NIBP), which is involved in protein trafficking and nuclear factor kappa B (NF-κB) signalling in cells. The interaction of NS5A with NIBP was confirmed both in vitro and in vivo. Complementing our glutathione S-transferase pull-down and immunoprecipitation data are the confocal immunofluorescence results, which indicate that NS5A colocalized with NIBP on the endoplasmic reticulum in the cytoplasm of BVDV-infected cells. Moreover, the minimal residues of NIBP that interact with NS5A were mapped as aa 597–623. In addition, overexpression of NS5A inhibited NF-κB activation in HEK293 and LB9.K cells as determined by luciferase reporter-gene assay. We further showed that inhibition of endogenous NIBP by small interfering RNA molecules enhanced virus replication, indicating the importance of NIBP implications in BVDV pathogenesis. Being the first reported interaction between NIBP and a viral protein, this finding suggests a novel mechanism whereby viruses may subvert host-cell machinery for mediating trafficking as well as NF-κB signalling.

From 1963 to 1986, human enterovirus 71 (HEV71) infections in the Netherlands were successively caused by viruses of subgenogroups B0, B1 and B2. A genogroup shift occurred in 1987, after which viruses of subgenogroups C1 and C2 were detected exclusively. This is in line with HEV71 typing in Australia, Europe and the USA, but is distinct from that in the Asian Pacific region, where HEV71 subgenogroups B3–B5 and C4–C5 have caused large outbreaks since 1997. To understand these observations in HEV71 epidemiology, the VP1-encoding regions of 199 HEV71 strains isolated in the Netherlands between 1963 and 2008 were used to study the detailed evolutionary trajectory and population dynamics of HEV71. Genogroup B viruses showed an epochal evolution, whereas genogroup C viruses evolved independently, which is in line with the co-circulation of C1 and C2 viruses in the Netherlands since 1997. Considering that strains from the Netherlands are interspersed phylogenetically with GenBank reference strains, the evolution of B1–B2, C1–C2 viruses has a global nature. Phylodynamic analysis confirmed that increased reporting of HEV71 infections in 1986 and 2007 reflected true epidemics of B2 and C2 viruses, respectively. Sequence analysis of the complete capsid region of a subset of isolates revealed several (sub)genogroup-specific residues. Subgenogroup B2-specific rabbit antiserum showed cross-neutralization of B0, B1 and B2 viruses, but not of subgenogroup C1 or C2 viruses, probably explaining the global shift to genogroup C in 1987 following a B2 epidemic. Anti-C1 rabbit serum neutralized both genogroup B and C viruses. Global herd immunity against C1 and C2 viruses possibly explains why epidemics with subgenogroups B4 and C4 are restricted to the Asian Pacific region.

In viral myocarditis, adeno- and enteroviruses have most commonly been implicated as causes of infection. Both viruses require the human coxsackie-adenovirus receptor (CAR) to infect the myocardium. Due to its crucial role for viral entry, CAR-downregulation may lead to novel approaches for treatment for viral myocarditis. In this study, we report on pharmaceutical drug influences on CAR levels in human umbilical vein endothelial cells (HUVEC) and cervical carcinoma cells (HeLa) detected by immunoblotting, quantitative real time-PCR and cellular susceptibility to the cardiotropic coxsackie-B3 virus strain Nancy (CVB3). Our results indicate, for the first time, a dose-dependent CAR mRNA and protein downregulation upon Valsartan and Bosentan treatment. Most interestingly, drug-induced CAR diminution significantly reduced the viral load in CVB3-infected HUVEC. In order to assess the regulatory effects of both drugs in detail, we knocked down their protein targets, the G-protein coupled receptors angiotensin-II type-1 receptor (AT1R) and endothelin-1 type-A and -B receptors (ETAR/ETBR) in HUVEC. Receptor-specific gene silencing indicates that CAR gene expression is regulated by agonistic and antagonistic binding to ETBR, but not ETAR. In addition, neither stimulation nor inhibition of AT1R seemed to be involved in CAR gene regulatory processes. Our study indicates that Valsartan and Bosentan protected human endothelial cells from CVB3-infection. Therefore, besides their well-known anti-hypertensive effects these drugs may also protect the myocardium and other tissues from coxsackie- and adenoviral infection.

Equine rhinitis A virus (ERAV) shares many features with foot-and-mouth disease virus (FMDV) and both are classified within the genus Aphthovirus of the family Picornaviridae. ERAV is used as a surrogate for FMDV research as it does not require high-level biosecurity. In contrast to FMDV, which uses integrins as cellular receptors, the receptor for ERAV has been reported to involve the sugar moiety sialic acid. This study confirmed the importance of sialic acid for cell entry by ERAV and reports the crystal structure of ERAV particles complexed with the receptor analogue 3′-sialyllactose. The receptor is attached to the rim of a capsid pit adjacent to the major immunogenic site and distinct from the sialic acid binding site used by a related picornavirus, the cardiovirus Theiler's murine encephalitis virus. The structure of the major antigenic determinant of the virus, previously identified from antibody escape mutations, is also described as the EF loop of VP1, which forms a hairpin stretching across the capsid surface close to the icosahedral fivefold axis, neighbouring the receptor-binding site, and spanning two protomeric units.

We characterized low pathogenic avian influenza (LPAI) H5N2 and H9N2 viruses isolated in South Korea from 2008 to 2009. Genetic analysis of the H5N2 viruses isolated from wild birds and domestic ducks demonstrated that they were related to the recently isolated southern Chinese LPAI H5 viruses and various influenza viruses circulating in Eurasia. Three H9N2 viruses obtained at live bird markets and duck farms were reassortant viruses generated from the H5N2 viruses of domestic ducks and the H9N2 virus endemic in Korean chickens. The H5N2 viruses did not replicate well in experimentally infected chickens and mice, but novel H9N2 viruses, without pre-adaptation, were recovered at high titres in chickens. Our results show that reassortment between H5N2 and H9N2 viruses must have occurred in domestic ducks and may have contributed to the diversity expansion of the gene pool, which has potential to alter the pathogenicity and host range of the influenza virus.

Highly pathogenic avian H5N1 viruses have circulated in South-east Asia for more than a decade and have now spread to more than 60 countries. The evolution of these viruses is characterized by frequent reassortment of the so-called ‘internal’ genes, creating novel genotypes. Additionally, over time, the surface glycoprotein, haemagglutinin (HA), which is the primary target of the adaptive immune response, has evolved by point mutation into 20 genetically and potentially antigenically distinct clades. To investigate the evolution of avian H5N1 influenza viruses, we undertook a high-resolution analysis of the reassortment of internal genes and evolution of HA of 651 avian H5N1 viruses from 2000 to 2008. Our analysis suggested: (i) all current H5N1 genotypes were derived from a single, clearly defined sequence of initial reassortment events; (ii) reassortment of just three of the internal genes had the most importance in avian H5N1 virus evolution; (iii) HA and the constellation of internal genes may be jointly important in the emergence of dominant variants. Further, our analysis led to the identification of evolutionarily significant molecular changes in the internal genes that may be significant for the emergence of these dominant variants.

The NS1 protein has been associated with the virulence of influenza A viruses. To evaluate the role of the NS1 protein in pathogenicity of pandemic H5N1 avian influenza and H1N1 2009 influenza viruses, recombinant Newcastle disease viruses (rNDVs) expressing NS1 proteins were generated. Expression of the NS1 proteins resulted in inhibition of host innate immune responses (beta interferon and protein kinase R production). In addition, the NS1 proteins were localized predominantly in the nucleus of virus-infected cells. Consequently, expression of the NS1 protein contributed to an increase in pathogenicity of rNDV in chickens. In particular, mutational analysis of H5N1 NS1 protein indicated that both the RNA-binding and effector domains affect virus pathogenicity synergistically. Our study also demonstrated that expression of H1N1/09 NS1 resulted in enhanced replication of rNDV in human cells, indicating that function of the NS1 proteins can be host-species-specific.

Bunyamwera virus NSs protein is involved in the inhibition of cellular transcription and the interferon (IFN) response, and it interacts with the Med8 component of Mediator. A spontaneous mutant of a recombinant NSs-deleted Bunyamwera virus (rBUNdelNSs2) was identified and characterized. This mutant virus, termed mBUNNSs22, expresses a 21 aa N-terminally truncated form of NSs. Like rBUNdelNSs2, mBUNNSs22 is attenuated in IFN-deficient cells, and to a greater extent in IFN-competent cells. Both rBUNdelNSs2 and mBUNNSs22 are potent IFN inducers and their growth can be rescued by depleting cellular IRF3. Strikingly, despite encoding an NSs protein that contains the Med8 interaction domain, mBUNNSs22 fails to block RNA polymerase II activity during infection. Overall, our data suggest that both the interaction of NSs with Med8 and a novel unidentified function of the NSs N-terminus, seem necessary for Bunyamwera virus to counteract host antiviral responses.

Intestinal epithelial cell death following rotavirus infection is associated with villus atrophy and gastroenteritis. Roles for both apoptosis and necrosis in cytocidal activity within rotavirus-infected epithelial cells have been proposed. Additionally, inactivated rotavirus has been reported to induce diarrhoea in infant mice. We further examined the death mechanisms induced in epithelial cell lines following rotavirus infection or inactivated rotavirus exposure. Monolayer integrity changes in MA104, HT-29 and partially differentiated Caco-2 cells following inactivated rotavirus exposure or RRV or CRW-8 rotavirus infection paralleled cell metabolic activity and viability reductions. MA104 cell exposure to rotavirus dsRNA also altered monolayer integrity. Inactivated rotaviruses induced delayed cell function losses that were unrelated to apoptosis. Phosphatidylserine externalization, indicating early apoptosis, occurred in MA104 and HT-29 but not in partially differentiated Caco-2 cells by 11 h after infection. Rotavirus activation of phosphatidylinositol 3-kinase partially protected MA104 and HT-29 cells from early apoptosis. In contrast, activation of the stress-activated protein kinase JNK by rotavirus did not influence apoptosis induction in these cells. RRV infection produced DNA fragmentation, indicating late-stage apoptosis, in fully differentiated Caco-2 cells only. These studies show that the apoptosis initiation and cell death mechanism induced by rotavirus infection depend on cell type and degree of differentiation. Early stage apoptosis resulting from rotavirus infection is probably counter-balanced by virus-induced phosphatidylinositol 3-kinase activation. The ability of inactivated rotaviruses and rotavirus dsRNA to perturb monolayer integrity supports a potential role for these rotavirus components in disease pathogenesis.

Equine herpesvirus type 1 (EHV-1) is the causative agent of equine herpes myeloencephalopathy, of which outbreaks are reported with increasing frequency throughout North America and Europe. This has resulted in its classification as a potentially emerging disease by the US Department of Agriculture. Recently, it was found that a single nucleotide polymorphism (SNP) in the viral DNA polymerase gene (ORF30) at aa 752 (N→D) is associated with the neurovirulent potential of EHV-1. In the present study, equine respiratory mucosal explants were inoculated with several Belgian isolates typed in their ORF30 as D752 or N752, to evaluate a possible difference in replication in the upper respiratory tract. In addition, to evaluate whether any observed differences could be attributed to the SNP associated with neurovirulence, the experiments were repeated with parental Ab4 (reference neurovirulent strain), parental NY03 (reference non-neurovirulent strain) and their N/D revertant recombinant viruses. The salient findings were that EHV-1 spreads plaquewise in the epithelium, but plaques never cross the basement membrane (BM). However, single EHV-1-infected cells could be observed below the BM at 36 h post-inoculation (p.i.) for all N752 isolates and at 24 h p.i. for all D752 isolates, and were identified as monocytic cells and T lymphocytes. Interestingly, the number of infected cells was two to five times higher for D752 isolates compared with N752 isolates at every time point analysed. Finally, this study showed that equine respiratory explants are a valuable and reproducible model to study EHV-1 neurovirulence in vitro, thereby reducing the need for horses as experimental animals.

Interaction between human cytomegalovirus uracil DNA glycosylase (UL114) and the viral DNA polymerase accessory subunit (UL44) has been reported; however, no such association was found in proteomic studies of UL44-interacting proteins. Utilizing virus expressing FLAG-tagged UL114, nuclease-resistant association of UL44 and the DNA polymerase catalytic subunit UL54 with UL114 was observed by co-immunoprecipitation. Contrary to a previous report, we observed that UL114 was much less abundant than UL44. Interaction of UL114 with UL54, independent of the UL54 carboxyl terminus, but not with UL44 was detected in vitro. Our data are consistent with a direct UL114–UL54 interaction, and suggest that UL114 and UL54 act in concert during base excision repair of the viral genome.

Human cytomegalovirus (HCMV) UL141 induces protection against natural killer cell-mediated cytolysis by downregulating cell surface expression of CD155 (nectin-like molecule 5; poliovirus receptor), a ligand for the activating receptor DNAM-1 (CD226). However, DNAM-1 is also recognized to bind a second ligand, CD112 (nectin-2). We now show that HCMV targets CD112 for proteasome-mediated degradation by 48 h post-infection, thus removing both activating ligands for DNAM-1 from the cell surface during productive infection. Significantly, cell surface expression of both CD112 and CD155 was restored when UL141 was deleted from the HCMV genome. While gpUL141 alone is sufficient to mediate retention of CD155 in the endoplasmic reticulum, UL141 requires assistance from additional HCMV-encoded functions to suppress expression of CD112.

Recent studies have shown that long-term persistence of human cytomegalovirus (HCMV) in mononuclear cells of myeloid lineage is dependent on the UL138 open reading frame, which promotes latent infection. Although T-cell recognition of protein antigens from all stages of lytic HCMV infection is well established, it is not clear whether proteins expressed during latent HCMV infection can also be recognized. This study conducted an analysis of T-cell response towards proteins associated with HCMV latency. Ex vivo analysis of T cells from healthy virus carriers revealed a dominant CD8+ T-cell response to the latency-associated pUL138 protein, which recognized a non-canonical 13 aa epitope in association with HLA-B*3501. These pUL138-specific T cells displayed a range of memory phenotypes that were in general less differentiated than that previously described in T cells specific for HCMV lytic antigens. Antigen-presentation assays revealed that endogenous pUL138 could be presented efficiently by HCMV-infected cells. However, T-cell recognition of pUL138 was dependent on newly synthesized protein, with little presentation from stable, long-lived protein. These data demonstrate that T cells targeting latency-associated protein products exist, although HCMV may limit the presentation of latent proteins, thereby restricting T-cell recognition of latently infected cells.

Nine male chimpanzees originally reared in solitary cages were set up to form a group. Plasma viral load of the lymphocryptovirus (LCV) of chimpanzee [Epstein–Barr virus chimpanzee (EBVcmp)] was measured by real-time PCR. In the group formation (Form) period, the first-ranking male showed an imminent increase in plasma EBVcmp load compared with 1 week before (pre-Form) and 3 months after (post-Form) group formation. Other upper-ranking males such as the second-, third- and fourth-male also showed the highest level of viral load in the Form period. The kinetics of EBVcmp load in the Form period were statistically different from other periods (against pre-Form, t=−4.878, P<0.001; against post-Form, t=6.434, P<0.001). The effect of the male dominance rank did not differ between the pre-Form and post-Form periods (t=−1.557, P=0.12). Reactivation of LCV (EBV) as an immunological stress marker for humans might also be applied to chimpanzees.

Epstein–Barr virus (EBV) is ubiquitous in the human population and seroepidemiological studies have revealed that more than 90 % of adults are infected with the virus in Thailand. It has been suggested that latent membrane protein 1 (LMP1) variants may differ in their tumorigenicity and geographical localization. The distribution of LMP1 variants of EBV in the Thai population was studied. A total of 259 LMP1 sequences from ten Thai ethnic groups (Lahu, Lisu, Shan, Red Karen, White Karen, Hmong, Akha, Mlabri, Moken and Urak Lawoi) were studied using direct PCR sequencing. Nucleotide sequences corresponding to the C terminus of the LMP1, including previously published sequences from central and southern Thais, were used in the phylogenetic analysis. Five strains – the B95-8 prototype, China 1, China 2, Mediterranean (Med) and SEA 2 – were identified in ethnic groups in Thailand. The major strain and the distribution pattern differed by group and location. When the ethnic groups were classified by linguistic group, the prevalence of the SEA 2 strain was significantly different between Austro-Thais and other linguistic groups (P=0.0001), whereas, among Tibeto-Burman linguistic groups, the prevalence of the Med strain was different between matrilocal and patrilocal groups (P=0.0245). The distribution of LMP1 strains in ethnic minorities in Thailand is associated with ethnogeographical factors and the social/marriage system. This study thus provides evidence for the importance of interactions between populations in virus diversity.