- Volume 87, Issue 5, 2006

The human γ 1-herpesvirus Epstein–Barr virus (EBV) and the γ 2-herpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV), rhesus rhadinovirus (RRV), herpesvirus saimiri (HVS) and herpesvirus ateles (HVA) all contain genes located adjacent to the terminal-repeat region of their genomes, encoding membrane proteins involved in signal transduction. Designated ‘terminal membrane proteins' (TMPs) because of their localization in the viral genome, they interact with a variety of cellular signalling molecules, such as non-receptor protein tyrosine kinases, tumour-necrosis factor receptor-associated factors, Ras and Janus kinase (JAK), thereby initiating further downstream signalling cascades, such as the MAPK, PI3K/Akt, NF-κB and JAK/STAT pathways. In the case of TMPs expressed during latent persistence of EBV and HVS (LMP1, LMP2A, Stp and Tip), their modulation of intracellular signalling pathways has been linked to the provision of survival signals to latently infected cells and, hence, a contribution to occasional cellular transformation. In contrast, activation of similar pathways by TMPs of KSHV (K1 and K15) and RRV (R1), expressed during lytic replication, may extend the lifespan of virus-producing cells, alter their migration and/or modulate antiviral immune responses. Whether R1 and K1 contribute to the oncogenic properties of KSHV and RRV has not been established satisfactorily, despite their transforming qualities in experimental settings.

Several cellular molecules have been identified as putative receptors for Hepatitis C virus (HCV): CD81 tetraspanin, scavenger receptor class B type I (SR-BI), mannose-binding lectins DC-SIGN and L-SIGN, low-density lipoprotein receptor, heparan sulphate proteoglycans and the asialoglycoprotein receptor. Due to difficulties in propagating HCV in cell culture, most of these molecules have been identified by analysing their interaction with a soluble, truncated form of HCV glycoprotein E2. A recent major step in investigating HCV entry was the development of pseudoparticles (HCVpp), consisting of unmodified HCV envelope glycoproteins assembled onto retroviral core particles. This system has allowed the investigation of the role of candidate receptors in the early steps of the HCV life cycle and the data obtained can now be confirmed with the help of a newly developed cell-culture system that allows efficient amplification of HCV (HCVcc). Interestingly, CD81 and SR-BI have been shown to play direct roles in HCVpp and/or HCVcc entry. However, co-expression of CD81 and SR-BI in non-hepatic cell lines does not lead to HCVpp entry, indicating that other molecule(s), expressed only in hepatic cells, are necessary for HCV entry. In this review, the molecules that have been proposed as potential HCV receptors are described and the experimental data indicating that CD81 and SR-BI are potentially involved in HCV entry are presented.

Micromonas pusilla reovirus (MpRV) is an 11-segmented, double-stranded RNA virus isolated from the marine protist Micromonas pusilla. Sequence analysis (including conserved termini and presence of core motifs of reovirus polymerase), morphology and physicochemical properties confirmed the status of MpRV as a member of the family Reoviridae. Electron microscopy showed that intact virus particles are unusually larger (90–95 nm) than the known size of particles of viruses belonging to the family Reoviridae. Particles that were purified on caesium chloride gradients had a mean size of 75 nm (a size similar to the size of intact particles of members of the family Reoviridae), indicating that they lost outer-coat components. The subcore particles had a mean size of 50 nm and a smooth surface, indicating that MpRV belongs to the non-turreted Reoviridae. The maximum amino acid identity with other reovirus proteins was 21 %, which is compatible with values existing between distinct genera. Based on morphological and sequence findings, this virus should be classified as the representative of a novel genus within the family Reoviridae, designated Mimoreovirus (from Micromonas pusilla reovirus). The topology of the phylogenetic tree built with putative polymerase sequences of the family Reoviridae suggested that the branch of MpRV could be ancestral. Further analysis showed that segment 1 of MpRV was much longer (5792 bp) than any other reovirus segment and encoded a protein of 200 kDa (VP1). This protein exhibited significant similarities to O-glycosylated proteins, including viral envelope proteins, and is likely to represent the additional outer coat of MpRV.

Historically, coronaviruses have been recognized as a cause of minor respiratory infections in humans. However, the recent identification of three novel human coronaviruses, one causing severe acute respiratory syndrome (SARS), has prompted further examination of these viruses. Previous studies of geographically and chronologically distinct Human coronavirus 229E (HCoV-229E) isolates have found only limited variation within S gene nucleotide sequences. In contrast, analysis of the S genes of contemporary Human coronavirus OC43 variants identified in Belgium revealed two distinct viruses circulating during 2003 and 2004. Here, the S and N gene sequences of 25 HCoV-229E variants identified in Victoria, Australia, between 1979 and 2004 in patients with symptomatic infections were determined. Phylogenetic analysis showed clustering of the isolates into four groups, with evidence of increasing divergence with time. Evidence of positive selection in the S gene was also established.

The importance of sialic acid for infection by avian Infectious bronchitis virus (IBV) has been analysed. Neuraminidase treatment rendered Vero, baby hamster kidney and primary chicken kidney cells resistant to infection by the IBV-Beaudette strain. Sialic acid-dependent infection was also observed with strain M41 of IBV, which infects primary chicken kidney cells but not cells from other species. In comparison with Influenza A virus and Sendai virus, IBV was most sensitive to pre-treatment of cells with neuraminidase. This finding suggests that IBV requires a greater amount of sialic acid on the cell surface to initiate an infection compared with the other two viruses. In previous studies, with respect to the haemagglutinating activity of IBV, it has been shown that the virus preferentially recognizes α2,3-linked sialic acid. In agreement with this finding, susceptibility to infection by IBV was connected to the expression of α2,3-linked sialic acid as indicated by the reactivity with the lectin Maackia amurensis agglutinin. Here, it is discussed that binding to sialic acid may be used by IBV for primary attachment to the cell surface; tighter binding and subsequent fusion between the viral and the cellular membrane may require interaction with a second receptor.

Several disulfide-based and azoic compounds have shown antiviral and virucidal properties against arenaviruses in virus yield-inhibition and inactivation assays, respectively. The most effective virucidal agent, the aromatic disulfide NSC20625, was able to inactivate two strains of the prototype arenavirus species Lymphocytic choriomeningitis virus (LCMV). Inactivated viral particles retained the biological functions of the virion envelope glycoproteins in virus binding and uptake, but were unable to perform viral RNA replication. Furthermore, in inactivated virions, the electrophoretic profile of the Z protein was altered when analysed under non-reducing conditions, whereas the patterns of the proteins NP and GP1 remained unaffected. Treatment of a recombinant LCMV Z protein with the virucidal agents induced unfolding and oligomerization of Z to high-molecular-mass aggregates, probably due to metal-ion ejection and the formation of intermolecular disulfide bonds through the cysteine residues of the Z RING finger. NSC20625 also exhibited antiviral properties in LCMV-infected cells without affecting other cellular RING-motif proteins, such as the promyelocytic leukaemia protein PML. Altogether, the investigations described here illustrate the potential of the Z protein as a promising target for therapy and the prospects of the Z-reactive compounds to prevent arenavirus dissemination.

Caliciviruses infect a wide range of mammalian hosts and include the genus Norovirus, the major cause of food-borne viral gastroenteritis in humans. Using publicly available sequence data and phylogenetic analysis tools, the origins and virus–host co-phylogeny of these viruses were investigated. Here, evidence is presented in support of host switching by caliciviruses, but showing that zoonotic transfer does not appear to have occurred in the history of these viruses. The age or demography of the caliciviruses cannot yet be estimated with any firm degree of support, but further studies of this family, as new dated sequences become available, could provide key information of importance to human health and in understanding the emergence of food-borne disease.

The leader (L) proteins encoded by picornaviruses of the genus Cardiovirus [Theiler's murine encephalomyelitis virus (TMEV) and Encephalomyocarditis virus (EMCV)] are small proteins thought to exert important functions in virus–host interactions. The L protein of persistent TMEV strains was shown to be dispensable for virus replication in vitro, but crucial for long-term persistence of the virus in the central nervous system of the mouse. The phenotype of chimeric viruses generated by exchanging the L-coding regions was analysed and it was shown that the L proteins of neurovirulent and persistent TMEV strains are functionally interchangeable in vitro and in vivo, despite the fact that L is the second most divergent protein encoded by these viruses after the L* protein. The L protein encoded by EMCV and Mengo virus (an EMCV strain) shares about 35 % amino acid identity with that of TMEV. It differs from the latter by lacking a serine/threonine-rich C-terminal domain and by carrying phosphorylated residues not conserved in the TMEV L protein. Our data show that, in spite of these differences, the L protein of Mengo virus shares, with that of TMEV, the ability to inhibit the transcription of type I interferon, cytokine and chemokine genes and to interfere with nucleocytoplasmic trafficking of host-cell proteins. Interestingly, analysis of viral RNA replication of the recombinant viruses raised the hypothesis that L proteins of TMEV and EMCV diverged during evolution to adapt to the different replication fitness of these viruses.

Transient expression of Ebola virus (EBOV) glycoprotein GP causes downregulation of surface proteins, cell rounding and detachment, a phenomenon believed to play a central role in the pathogenicity of the virus. In this study, evidence that moderate expression of GP does not result in such morphological changes was provided. It was shown that GP continuously produced in 293T cells from the Kunjin virus replicon was correctly processed and transported to the plasma membrane without affecting the surface expression of β1 and α5 integrins and major histocompatibility complex I molecules. The level of GP expression in Kunjin replicon GP-expressing cells was similar to that observed in cells infected with EBOV early in infection and lower than that produced in cells transfected with plasmid DNA, phCMV-GP, expressing GP from a strong promoter. Importantly, transient transfection of Kunjin replicon GP-expressing cells with GP-coding plasmid DNA resulted in overexpression of GP, which lead to the downregulation of surface molecules and massive rounding and detachment of transfected cells. Here, it was also demonstrated that cell rounding and downregulation of the surface markers are the late events in EBOV infection, whereas synthesis and massive release of virus particles occur at early steps and do not cause significant cytotoxic effects. These findings indicate that the synthesis of EBOV GP in virus-infected cells is controlled well by several mechanisms that do not allow GP overexpression and hence the early appearance of its cytotoxic properties.

A defective interfering (DI) virus differs from the infectious virus from which it originated in having at least one major deletion in its genome. Such DI genomes are replicated only in cells infected in trans with homologous infectious virus and, as their name implies, they interfere with infectious virus replication and reduce the yield of progeny virus. This potent antiviral activity has been abundantly demonstrated in cell culture with many different DI animal viruses, but few in vivo examples have been reported, with the notable exception of DI Influenza A virus. A clue to this general lack of success arose recently when an anomaly was discovered in which DI Influenza A virus solidly protected mice from lethal disease caused by A/PR/8/34 (H1N1) and A/WSN/40 (H1N1) viruses, but protected only marginally from disease caused by A/Japan/305/57 (A/Jap, H2N2). The problem was not any incompatibility between the DI and infectious genomes, as A/Jap replicated the DI RNA in vivo. However, A/Jap required 300-fold more mouse infectious units to cause clinical disease than A/PR8 and it was hypothesized that it was this excess of infectivity that abrogated the protective activity of the DI virus. This conclusion was verified by varying the proportions of DI and challenge virus and showing that increasing the DI virus : infectious virus ratio in infected mice resulted in interference. Thus, counter-intuitively, DI virus is most effective against viruses that cause disease with low numbers of particles, i.e. virulent viruses.

Monoclonal antibodies (mAbs) to conserved epitopes on the G glycoprotein of human respiratory syncytial virus (HRSV) subgroup A fail to neutralize the virus in cell culture in the absence of complement, but are protective in rodent models of infection. They may have potential as prophylactic agents in human infants. In order to investigate the role of Fc-dependent pathways in protection by one such antibody, 1C2, the VH and VL genes were isolated by RT-PCR and assembled with human κ light-chain and human γ1 heavy-chain constant-region genes to form two mouse–human chimaeras, which were expressed in NS0 cells. One of the chimaeras carried a wild-type γ1 chain, whilst the other had an aglycosyl mutation in the CH2 domain rendering the antibody defective in complement activation and FcγR binding. Whilst both chimaeric antibodies exhibited similar avidity for HRSV in ELISA, only the fully glycosylated wild type was capable of neutralizing the virus in the presence of complement. In mice passively immunized with either murine or wild-type γ1 chimaeric antibody, no virus could be recovered from the lungs 4 days after intranasal inoculation of HRSV. In mice immunized with the aglycosyl γ1 chimaera, however, virus was present in the lungs following challenge, although virus titres were significantly reduced compared with controls (P<0·005). These results indicate that the protective effect of this antibody is mediated by both Fc-dependent and Fc-independent pathways.

Rotaviruses recognize several cell-surface molecules, including the α2β1 integrin, and the processes of rotavirus cell attachment and entry appear to be multifactorial. The VP5* subunit of the rotavirus spike protein VP4 contains the α2β1 ligand sequence Asp–Gly–Glu at residues 308–310. Binding to α2β1 and infectivity of monkey rotavirus strain RRV and human rotavirus strain Wa, but not porcine rotavirus strain CRW-8, are inhibited by peptides containing Asp–Gly–Glu. Asp308 and Gly309 are necessary for the binding of RRV VP5* (aa 248–474) to expressed I domain of the α2 integrin subunit. Here, the ability of RRV VP5* to bind cells and affect rotavirus–integrin interactions was determined. Interestingly, VP5* bound to cells at 4 and 37 °C, both via α2β1 and independently of this integrin. Prior VP5* binding at 37 °C eliminated RRV binding to cellular α2β1 and reduced RRV and Wa infectivity in MA104 cells by 38–46 %. VP5* binding did not affect the infectivity of CRW-8. VP5* binding at 4 °C did not affect permissive-cell infection by RRV, indicating an energy requirement for VP5* competition with virus for infectivity. Mutagenesis of VP5* Asp308 and Gly309 eliminated VP5* binding to α2β1 and the VP5* inhibition of rotavirus cell binding and infection, but not α2β1-independent cell binding by VP5*. These studies show for the first time that expressed VP5* binds cell-surface α2β1 using Asp308 and Gly309 and inhibits the infection of homologous and heterologous rotaviruses that use α2β1 as a receptor.

Human immunodeficiency virus type 1 (HIV-1) infection is established by virus variants that use the CCR5 co-receptor for entry (CCR5-tropic or R5 variants), whereas viruses that use CXCR4 as co-receptor (CXCR4-tropic or X4 variants) emerge during disease progression in approximately 50 % of infected subjects. X4 variants may have a higher fitness ex vivo and their detection is usually accompanied by faster T-cell depletion and the onset of AIDS in HIV-1-positive individuals. Here, the relationship between the sequence variation of the HIV-1 env V3–V5 region and positive selective pressure on R5 and X4 variants from infected subjects with CD4 T cell counts below 200 cells μl−1 was studied. A correlation was found between genetic distance and CD4+ cell count at late stages of the disease. R5 variants that co-existed with X4 variants were significantly less heterogeneous than R5 variants from subjects without X4 variants (P<0·0001). Similarly, X4 variants had a significantly higher diversity than R5 variants (P<0·0001), although residues under positive selection had a similar distribution pattern in both variants. Therefore, both X4 and R5 variants were subjected to high selective pressures from the host. Furthermore, the interaction between X4 and R5 variants within the same subject resulted in a purifying selection on R5 variants, which only survived as a homogeneous virus population. These results indicate that R5 variants from X4 phenotype samples were highly homogeneous and under weakly positive selective pressures. In contrast, R5 variants from R5 phenotype samples were highly heterogeneous and subject to positive selective pressures.

Concurrent uncontrolled development of human immunodeficiency virus type 1 (HIV-1) and Leishmania spp. is regarded as an emerging pathogenic combination in countries where human beings are exposed to these two micro-organisms. The present study was aimed at exploring whether HIV-1 development within a culture of human monocyte-derived macrophages (MDMs) affected the further development of luciferase-encoding Leishmania infantum using the luciferase activity as a readout assay. It was demonstrated that, in cultures of HIV-1-loaded MDMs exposed to axenic amastigotes, the luciferase activity was higher than in HIV-1-free MDMs. As a preliminary approach to deciphering the possible mechanism through which HIV-1 can affect Leishmania infantum, attention was focused on the very early processes that could underlie this increased luciferase activity. Using GFP-labelled parasites, it was possible to establish that, in HIV-1-infected MDMs, the percentage of GFP-expressing MDMs was higher (10–20 %) than in cell cultures not exposed to HIV-1 (5 %). Two-colour immunofluorescence staining suggested that HIV-1 indirectly affects the uptake of parasites inside MDMs. Thus, the observed phenomenon seems to be linked with a higher uptake of parasites within MDMs. Taken together, the data reported here may contribute to our understanding of disseminated Leishmania infection in HIV-1-infected individuals.

Human immunodeficiency virus type 1 subtype C isolates belong to one of the most prevalent strains circulating worldwide and are responsible for the majority of new infections in the sub-Saharan region and other highly populated areas of the globe. In this work, the impact of drug-resistance mutations in the protease gene of subtype C viruses was analysed and compared with that of subtype B counterparts. A series of recombinant subtype C and B viruses was constructed carrying indinavir (IDV)-resistance mutations (M46V, I54V, V82A and L90M) and their susceptibility to six FDA-approved protease inhibitor compounds (amprenavir, indinavir, lopinavir, ritonavir, saquinavir and nelfinavir) was determined. A different impact of these mutations was found when nelfinavir and lopinavir were tested. The IDV drug-resistance mutations in the subtype C protease backbone were retained for a long period in culture without selective pressure when compared with those in subtype B counterparts in washout experiments.

A better understanding of virological events during the early phase of human immunodeficiency virus 1 (HIV-1) infection is important for development of effective antiviral vaccines. In this study, by using quantitative PCR and an infectious plaque assay, virus distribution and replication were examined in various internal organs of rhesus macaques for almost 1 month after intrarectal inoculation of a pathogenic simian immunodeficiency virus/HIV chimeric virus (SHIV-C2/1-KS661c). At 3 days post-inoculation (p.i.), proviral DNA was detected in the rectum, thymus and axillary lymph node. In lymphoid tissues, infectious virus was first detected at 6 days p.i. and a high level of proviral DNA and infectious virus were both detected at 13 days p.i. By 27 days p.i., levels of infectious virus decreased dramatically, although proviral DNA load remained unaltered. In the intestinal tract, levels of infectious virus detected were much lower than in lymphoid tissues, whereas proviral DNA was detected at the same level as in lymphoid tissues throughout the infection. In the thymus and jejunum, CD4CD8 double-positive T cells were depleted earlier than CD4 single-positive cells. These results show that the virus spread quickly to systemic tissues after mucosal transmission. Thereafter, infectious virus was actively produced in the lymphoid tissues, but levels decreased significantly after the peak of viraemia. In contrast, in the intestinal tract, infectious virus was produced at low levels from the beginning of infection. Moreover, virus pathogenesis differed in CD4 single-positive and CD4CD8 double-positive T cells.

The protease (PR) of Murine leukemia virus (MLV) was expressed in Escherichia coli, purified to homogeneity and characterized by using various assay methods, including HPLC-based, photometric and fluorometric activity measurements. The specificity of the bacterially expressed PR was similar to that of virion-extracted PR. Compared with human immunodeficiency virus type 1 (HIV-1) PR, the pH optimum of the MLV enzyme was higher. The specificity of the MLV PR was further compared with that of HIV-1 PR by using various oligopeptides representing naturally occurring cleavage sites in MLV and HIV-1, as well as by using bacterially expressed proteins having part of the MLV Gag. Inhibitors designed against HIV-1 PR were also active on MLV PR, although all of the tested ones were substantially less potent on this enzyme than on HIV-1 PR. Nevertheless, amprenavir, the most potent inhibitor against MLV PR, was also able to block Gag processing in MLV-infected cells. These results indicate that, in spite of the similar function in the life cycle of virus infection, the two PRs are only distantly related in their specificity.

A squirrel monkey model of human T-cell leukemia virus type 1 (HTLV-1) infection was used to evaluate the immunogenicity and protective efficacy of a chimeric peptide vaccine composed of a B-cell epitope from the envelope region (aa 175–218) and three HLA-A*0201-restricted cytotoxic T-lymphocyte epitopes derived from Tax protein (Tri-Tax). These selected Tax peptides induced secretion of gamma interferon (IFN-γ) in peripheral blood mononuclear cells obtained from monkeys chronically infected with HTLV-1. After immunization, a high titre of antibodies and a high frequency of IFN-γ-producing cells were detected against the Env and the Tri-Tax immunogens, but not against the individual Tax peptides. This might indicate that epitope(s) distinct from those recognized by humans are recognized by responder monkeys. After challenge, it was shown by competitive PCR that partial protection against HTLV-1 infection could be raised in immunized animals. Further studies should be developed to determine the duration of this protection.

Integration-site selection by retroviruses and retroviral vectors has gained increased scientific interest. Foamy viruses (FVs) constitute a unique subfamily (Spumavirinae) of the family Retroviridae, for which the integration pattern into the human genome has not yet been determined. To accomplish this, 293 cells were transduced with FV vectors and the integration sites into the cellular genome were determined by a high-throughput method based on inverse PCR. For comparison, a limited number of murine leukemia virus (MLV) and human immunodeficiency virus (HIV) integration sites were analysed in parallel. Altogether, 628 FV, 87 HIV and 141 MLV distinct integration sites were mapped to the human genome. The sequences were analysed for RefSeq genes, promoter regions, CpG islands and insertions into cellular oncogenes. Compared with the integration-site preferences of HIV, which strongly favours active genes, and MLV, which favours integration near transcription-start regions, our results indicate that FV integration has neither of these preferences. However, once integration has occurred into a transcribed region of the genome, FVs tend to target promoter-close regions, albeit with less preference than MLV. Furthermore, our study revealed a palindromic consensus sequence for integration, which was centred on the virus-specific, four-base-duplicated target site. In summary, it is shown that the integration pattern of FVs appears to be unique compared with those of other retroviral genera.

The present work studies the physico-chemical properties of retroviral vector membrane, in order to provide some explanation for the inactivation kinetics of these vectors and to devise new ways of improving transduction efficiency. For this purpose, vectors with an amphotropic envelope produced by TE Fly A7 cells at two culture temperatures (37 and 32 °C) were characterized by different techniques. Electron paramagnetic resonance (EPR) results showed that vectors produced at 32 °C are more rigid than those produced at 37 °C. Further characterization of vector membrane composition allowed us to conclude that the vector inactivation rate increases with elevated cholesterol to phospholipid ratio. Differential scanning calorimetry (DSC) showed that production temperature also affects the conformation of the membrane proteins. Transduction studies using HCT116 cells and tri-dimensional organ cultures of mouse skin showed that vectors produced at 37 °C have higher stability and thus higher transduction efficiency in gene therapy relevant cells as compared with vectors produced at 32 °C. Overall, vectors produced at 37 °C show an increased stability at temperatures below 4 °C. Since vector membrane physico-chemical properties are affected in response to changes in culture temperature, such changes, along with alterations in medium composition, can be used prospectively to improve the stability and the transduction efficiency of retroviral vectors for therapeutic purposes.