- Volume 85, Issue 5, 2004

This review summarizes the combined insights from recent structural and functional studies of viral RNA-dependent RNA polymerases (RdRPs) with the primary focus on the mechanisms of initiation of RNA synthesis. Replication of RNA viruses has traditionally been approached using a combination of biochemical and genetic methods. Recently, high-resolution structures of six viral RdRPs have been determined. For three RdRPs, enzyme complexes with metal ions, single-stranded RNA and/or nucleoside triphosphates have also been solved. These advances have expanded our understanding of the molecular mechanisms of viral RNA synthesis and facilitated further RdRP studies by informed site-directed mutagenesis. What transpires is that the basic polymerase right hand shape provides the correct geometrical arrangement of substrate molecules and metal ions at the active site for the nucleotidyl transfer catalysis, while distinct structural elements have evolved in the different systems to ensure efficient initiation of RNA synthesis. These elements feed the template, NTPs and ions into the catalytic cavity, correctly position the template 3′ terminus, transfer the products out of the catalytic site and orchestrate the transition from initiation to elongation.

The intracellular transport of the surface glycoprotein E2 of bovine viral diarrhoea virus was analysed by expressing the cloned gene in the absence of other viral proteins. Immunofluorescence analysis and surface biotinylation indicated that E2 is located in an early compartment of the secretory pathway and not transported to the cell surface. In agreement with this result, E2 was found to contain only high-mannose oligosaccharide side-chains but no N-glycans of the complex type. To define the intracellular localization signal of the E2 protein, chimeric proteins were generated. E2 chimeras containing the MT (membrane anchor plus carboxy-terminal domain) of the G protein of vesicular stomatitis virus (VSV) or of the F protein of bovine respiratory syncytial virus (BRSV) were transported to the cell surface. On the other hand, VSV G protein containing the MT domain of E2 was detected only in the ER, indicating that this domain contains an ER localization signal. A chimeric E2 protein, in which not the membrane anchor but only the carboxy-terminal end was replaced by the corresponding domain of the BRSV F protein, was also localized in the ER. Therefore, it was concluded that the membrane anchor contains the ER localization signal of E2. Interestingly, the ER export signal within the VSV G protein cytoplasmic tail was found to overrule the ER localization signal in the E2 protein membrane anchor.

Presented here is a comprehensive computational survey of evolutionarily conserved secondary structure motifs in the genomic RNAs of the family Flaviviridae. This virus family consists of the three genera Flavivirus, Pestivirus and Hepacivirus and the group of GB virus C/hepatitis G virus with a currently uncertain taxonomic classification. Based on the control of replication and translation, two subgroups were considered separately: the genus Flavivirus, with its type I cap structure at the 5′ untranslated region (UTR) and a highly structured 3′ UTR, and the remaining three groups, which exhibit translation control by means of an internal ribosomal entry site (IRES) in the 5′ UTR and a much shorter less-structured 3′ UTR. The main findings of this survey are strong hints for the possibility of genome cyclization in hepatitis C virus and GB virus C/hepatitis G virus in addition to the flaviviruses; a surprisingly large number of conserved RNA motifs in the coding regions; and a lower level of detailed structural conservation in the IRES and 3′ UTR motifs than reported in the literature. An electronic atlas organizes the information on the more than 150 conserved, and therefore putatively functional, RNA secondary structure elements.

Caliciviruses are small, non-enveloped, positive-stranded RNA viruses that are pathogenic for both animals and man. Although their capsid structure and genomic organization are distinct from picornaviruses, they have similarities to these viruses in their non-structural proteins. Picornaviruses induce a rapid inhibition of host-cell cap-dependent protein synthesis and this is mainly achieved through cleavage of eIF4G and/or dephosphorylation of 4E-BP1. In this study, the effect of calicivirus infection was examined on host-cell protein synthesis in order to determine whether they also induce host shut-off. We report that infection of cells with feline calicivirus (FCV) leads to the inhibition of cellular protein synthesis. This is accompanied by the cleavage of the eukaryotic translation initiation factors eIF4GI and eIF4GII in a manner reminiscent of that induced by picornaviruses. However, the cleavages occur at different sites. The potential mechanisms of these cleavage events and the implications for the translation of calicivirus mRNA are discussed.

Our earlier identification of the non-structural protein 3 (NS3) of Japanese encephalitis virus (JEV) as a dominant CD4+ as well as CD8+ T cell-eliciting antigen in a healthy JEV-endemic cohort with a wide HLA distribution implied the presence of several epitopes dispersed over the length of the protein. Use of various truncated versions of NS3 in lymphocyte stimulation and interferon (IFN)-γ secretion assays revealed that amino acids (aa) 193–324 of NS3 were comparable with, if not superior to, the full-length protein in evoking Th1 responses. The potential of this 14·4 kDa stretch to stimulate IFN-γ production from both subtypes of T cells in a manner qualitatively and quantitatively similar to the 68 kDa parent protein suggested the presence within it of both class I and II epitopes and demonstrated that the entire immunogenicity of NS3 was focused on aa 193–324. Interestingly, this segment contained five of the eight helicase motifs of NS3. Analysis of variability of the NS3 protein sequence across 16 JEV isolates revealed complete identity of aa 219–318, which is contained within the above segment, suggesting that NS3-specific epitopes tend to cluster in relatively conserved regions that harbour functionally critical domains of the protein.

The complete nucleotide sequence of a new insect picorna-like virus, Ectropis obliqua picorna-like virus (EoPV), which causes a fatal infection of Ectropis obliqua larvae, has been determined. The genomic RNA of EoPV is 9394 nt in length and contains a single, large open reading frame (nt 391–9351) encoding a polyprotein of 2987 aa. Sequence comparisons with other viral polyproteins revealed that the consensus sequences for picornavirus RNA helicase, protease and RNA-dependent RNA polymerase proteins are found on the genome in order in the 5′→3′ direction. All structural genes were located at the 5′ terminus. In terms of sequence similarity, identity and genome organization, EoPV resembles mammalian picornaviruses and three other insect picorna-like viruses: Infectious flacherie virus of silkworm, Sacbrood virus of honeybee and Perina nuda picorna-like virus (PnPV). Phylogenetic analysis showed that EoPV is most closely related to PnPV and suggests that these four insect picorna-like viruses might constitute a new group of insect-infectious RNA viruses.

The cellular distribution of the small hydrophobic (SH) protein in respiratory syncytial virus (RSV)-infected cells was examined. Although the SH protein was distributed throughout the cytoplasm, it appeared to accumulate in the Golgi complex within membrane structures that were enriched in the raft lipid, GM1. The ability of the SH protein to interact with lipid-raft membranes was further confirmed by examining its detergent-solubility properties in Triton X-100 at 4 °C. This analysis showed that a large proportion of the SH protein exhibited detergent-solubility characteristics that were consistent with an association with lipid-raft membranes. Analysis of virus-infected cells by immuno-transmission electron microscopy revealed SH protein clusters on the cell surface, but only very low levels of the protein appeared to be associated with mature virus filaments and inclusion bodies. These data suggest that during virus infection, the compartments in the secretory pathway, such as the endoplasmic reticulum (ER) and Golgi complex, are major sites of accumulation of the SH protein. Furthermore, although a significant amount of this protein interacts with lipid-raft membranes within the Golgi complex, its presence within mature virus filaments is minimal.

Viral haemorrhagic septicaemia (VHS) caused by the rhabdovirus VHSV is economically the most important viral disease in European rainbow trout farming. Until 1989, this virus was mainly isolated from freshwater salmonids but in the last decade, it has also been isolated from an increasing number of free-living marine fish species. To study the genetic evolution of VHSV, the entire G gene from 74 isolates was analysed. VHSV from wild marine species caught in the Baltic Sea, Skagerrak, Kattegat, North Sea, and English Channel and European freshwater isolates, appeared to share a recent common ancestor. Based on the estimated nucleotide substitution rate, the ancestor of the European fresh water isolates was dated some 50 years ago. This finding fits with the initial reports in the 1950s on clinical observations of VHS in Danish freshwater rainbow trout farms. The study also indicates that European marine VHSV and the North American marine line separated approx. 500 years ago. The codon substitution rate among the freshwater VHSV isolates was found to be 2·5 times faster than among marine isolates. The data support the hypothesis of the marine environment being the original reservoir of VHSV and that the change in host range (to include rainbow trout) may have occurred several times. Virus from the marine environment will therefore continue to represent a threat to the trout aquaculture industry.

The complete open reading frame of Tula hantavirus (TULV) L RNA was cloned in three parts. The middle third (nt 2191–4344) could be expressed in E. coli and was used to immunize rabbits. The resultant antiserum was then used to immunoblot concentrated TULV and infected Vero E6 cells. The L protein of a hantavirus was detected, for the first time, in infected cells and was found to be expressed as a single protein with an apparent molecular mass of 250 kDa in both virions and infected cells. Using the antiserum, the expression level of the L protein was followed and image analysis of immunoblots indicated that there were 104 copies per cell at the peak level of expression. The antiserum was also used to detect the L protein in cell fractionation studies. In cells infected with TULV and cells expressing recombinant L, the protein pelleted with the microsomal membrane fraction. The membrane association was confirmed with membrane flotation assays. To visualize L protein localization in cells, a fusion protein of L and enhanced green fluorescent protein, L–EGFP, was expressed in Vero E6 cells with a plasmid-driven T7 expression system. L–EGFP localized in the perinuclear region where it had partial co-localization with the Golgi matrix protein GM130 and the TULV nucleocapsid protein.

We investigated the protective efficacy of a systemic triple vector (DNA/rSFV/rMVA)-based vaccine against mucosal challenge with pathogenic simian immunodeficiency virus (SIV) in cynomolgus monkeys. Animals were immunized at week 0 with DNA (intradermally), at weeks 8 and 16 with recombinant Semliki Forest virus (rSFV, subcutaneously) and finally, at week 24, with recombinant modified vaccinia virus Ankara strain (rMVA, intramuscularly). Both DNA and recombinant viral vectors expressed a wide range of SIV proteins (Gag, Pol, Tat, Rev, Env and Nef). This immunization strategy elicited cell-mediated rather than humoral responses that were especially increased following the last boost. Upon intrarectal challenge with pathogenic SIVmac251, three of the four vaccinated monkeys dramatically abrogated virus load to undetectable levels up to 41 weeks after challenge. A major contribution to this vaccine effect appeared to be the T-cell-mediated immune response to vaccine antigens (Gag, Rev, Tat, Nef) seen in the early phase of infection in three of the four vaccinated monkeys. Indeed, the frequency of T-cells producing antigen-induced IFN-γ mirrored virus clearance in the vaccinated and protected monkeys. These results, reminiscent of the efficacy of live attenuated virus vaccines, suggest that vaccination with a combination of many viral antigens can induce a robust and stable vaccine-induced immunity able to abrogate virus replication.

We examined the structural genes (gag, pol and env) of the human endogenous retrovirus (HERV-W) family from 12 normal human tissues and 18 human cancer cell lines using RT-PCR. For the gag and pol genes, their expression patterns showed tissue or cell specificity, depending on the samples, whereas the env gene was expressed in all tissues and cancer cells examined. To identify active HERV-W elements in tissues and cancer cells, the RT-PCR products were cloned and sequenced. Ninety-five clones for the env gene, 55 clones for the pol gene and 17 clones for the gag gene of the HERV-W family were isolated from human tissues and sequenced, while 85 clones for the env gene, 61 clones for the pol gene and no clones for the gag gene of the HERV-W family were isolated and sequenced from cancer cells. Among these clones, 50 clones from tissues and 44 from cancer cells showed putative amino acids of the HERV-W env gene, while only two clones from cancer cells showed putative amino acids of the HERV-W pol gene. Phylogenetic analysis indicated that several clones identified previously from human monochromosomes had sister relationships with the clones from the different tissues and cancer cells. These data suggest that HERV-W elements are actively expressed in human tissues and cancer cells. These active HERV-W elements deserve further investigation as potential causative agents of various human diseases including cancers.

Phylogenetic analysis of hepatitis B virus (HBV) has led to its classification into eight genotypes, A to H. The dominant genotype in South Africa is genotype A, which consists of two subgenotypes, A1 and A2. Subgenotype A1 (previously subgroup A′) predominates over subgenotype A2 (previously subgroup A minus A′). The complete genome of HBV isolated from 18 asymptomatic carriers of the virus and five acute hepatitis B patients was amplified; the resulting amplicons were cloned and sequenced. All acute hepatitis isolates belonged to subgenotype A1 and had no distinguishing mutations relative to the isolates from asymptomatic carriers, which had a distribution of ten subgenotype A1, two subgenotype A2 and six genotype D. The presence of the previously described amino acid residues that distinguish subgenotype A1 (subgroup A′) from the remainder of genotype A in the S and polymerase genes was confirmed. Moreover, the large number of subgenotype A1 isolates sequenced allowed identification in the other open reading frames of additional nucleotide and amino acid changes that are characteristic of subgenotype A1. In particular, nucleotide mutations at positions 1809–1812 that alter the Kozak sequence of the precore/core open reading frame, and A1888 in the precore region, were found exclusively in subgenotype A1 isolates. Unique sequence alterations of the transcriptional regulatory elements were also found in subgenotype A1 isolates. The mean nucleotide divergence of subgenotype A1 was greater than that of subgenotype A2, suggesting that this subgenotype has been endemic for a longer time in the South African black population than had subgenotype A2.

For functional diversity, the large (L) envelope protein of hepatitis B virus (HBV) acquires a dual transmembrane topology via co-translational membrane integration of the S region and partial post-translational translocation of the preS subdomain. Because each process requires the second transmembrane segment (TM2), we explored the action of this determinant by using protease protection analysis of mutant L proteins. We demonstrated that neither the disruption of a leucine zipper-like motif by multiple alanine substitutions nor the flanking charges of TM2 affected the topological reorientation of L. The dispensability of both putative subunit interaction modules argues against a link between preS post-translocation and envelope assembly. Phenotypic mixing experiments revealed that the preS and S protein domains of the related duck HBV L polypeptide failed to substitute functionally for the topogenic elements of HBV in directing the correct L topogenesis, implicating different translocation mechanisms used by the two hepadnavirus genera.

Gene transfer with recombinant adenoviruses (rAds) is a powerful means of inducing an immune response against a transgene product. However, little is known about the mechanisms that underlie the induction of the immune response after intramuscular inoculation of adenovirus and, in particular, the relative role of the different cell types transduced. Several studies have suggested that CD8+ cytotoxic T lymphocyte responses elicited after inoculation of adenoviruses (Ads) are induced both by direct transduction of antigen presenting cells (APCs) and by cross-priming. In the present study, a library of fibre-chimeric rAds was screened in order to identify rAds with distinct capacities to express transgene product in murine cell types naturally found in muscle, i.e. myoblasts, endothelial cells (both representing non-APCs) and dendritic cells (representing APCs). Four selected pseudotypes, differing in their ability to infect muscular cells were used to immunize C57BL/6 mice. The relationship between the capacity to transduce non-APC or APC in vitro and the ability to induce humoral and cellular responses against the β-galactosidase antigen after intramuscular inoculation were studied. Results indicate that CD8+ T cell responses against the β-galactosidase antigen were similar after inoculation of the four viruses, thus revealing no direct relationship with their ability to transduce myoblasts, endothelial cells or dendritic cells in vitro.

The prevalence of human papillomavirus type 16 E6 variant lineages was characterized in a cross-sectional study of 24 human immunodeficiency virus type 1 (HIV)-positive and 33 HIV-negative women in New Orleans. The European prototype was the predominant variant in the HIV-negative women (39·4 %), while in the HIV-positive women the European 350G variant was predominant (29·1 %). In exact logistic regression models, HIV-positive women were significantly more likely to harbour any variant with a nucleotide G-350 mutation compared with HIV-negative women [58·3 % vs 21·1 %; adjusted odds ratio (AOR)=6·28, 95 % confidence interval (CI)=1·19–46·54]. Models also revealed a trend towards increased prevalence of Asian–American lineage in HIV-positive women compared with HIV-negative women (25·0 % vs 6·0 %; AOR=6·35, 95 % CI=0·77–84·97). No association was observed between any variant and cytology or CD4 cell counts or HIV-1 viral loads. These observations reflect a difference in the distribution of HPV-16 variants among HIV-positive and -negative women, indicating that HIV-positive status may lead to increased prevalence of a subset of variants.

The L-X-C-X-E pRB-binding motif of papillomavirus (PV) E7 proteins has been implicated in the immortalization and transformation of the host cell. However, sequencing of the complete genomes of bovine papillomavirus type 3 (BPV-3), bovine papillomavirus type 5 (BPV-5), equine papillomavirus (EQPV) and reindeer (Rangifer tarandus) papillomavirus (RPV) supports the notion that the pRB-binding motif is not ubiquitous among E7 proteins in the PV proteome. Key among the animal groups that lack the pRB-binding domain are the artiodactyl PVs, including European elk PV (EEPV), deer PV (DPV), reindeer PV (RPV), ovine PVs types 1 and 2 (OvPV-1 and -2) and bovine PVs 1, 2 and 5 (BPV-1, -2 and -5). Whereas the presence of the pRB-binding domain is normally associated with papillomas, the artiodactyl PVs are marked by the development of fibropapillomas on infection. Previous studies emphasized the role of E5 in the pathogenic mechanism of fibropapilloma development, but correlation between the lack of an E7 pRB-binding domain and the unique pathology of the artiodactyl PVs suggests a more complicated mechanism and an early evolutionary divergence from a pRB-binding ancestor.

Human cell lines are permissive for LuIII, a member of the rodent group of autonomous parvoviruses. However, LuIII vectors pseudotyped with feline panleukopaenia virus (FPV) capsid proteins can transduce feline cells but not human cells. Feline transferrin receptor (FelTfR) functions as a receptor for FPV. Transfection of Rh18A, a human rhabdomyosarcoma cell line, with FelTfR enabled transduction by vector with FPV capsid. This was not true of other human lines, suggesting restriction at some additional, post-entry, level(s) in human cells other than Rh18A. It seemed a reasonable hypothesis that a second blockage might be in nuclear delivery mediated by the N-terminal region of the minor capsid protein, VP1. We therefore generated virions containing an LuIII–luciferase genome, packaged using chimaeric VP1 molecules (N-terminal region of LuIII VP1, fused with body of FPV, and vice versa) together with the major capsid protein, VP2, of FPV or LuIII. The virions were tested for ability to transduce feline and human cells. Our hypothesis predicted that the N-terminal region of LuIII VP1 should allow transduction of human cells expressing FelTfR, while the FPV N-terminal region should not allow transduction of human cells (except for Rh18A). The experimental results did not bear out either of these predictions. Therefore, the VP1 N-terminal region appears not to be a major determinant of permissiveness for LuIII, versus FPV, capsid in human cells.

The aim of this study was to describe the generation of a PCV2 (porcine circovirus type 2) infectious clone (pIC-PCV2) and its infectivity under in vitro and in vivo conditions. The constructed pIC-PCV2 contained the whole PCV2 genome from a German isolate together with a partial duplication of 467 bp. PK-15 cells were transfected with pIC-PCV2 and an indirect immune fluorescence assay (IFA) was performed 7 days post-transfection. The PCV2 Cap gene was expressed in approximately 20 % of the cultured cells, and only the recombination product, and not pIC-PCV2, was subsequently detected by PCR and Southern blot. This result indicated that infection by pIC-PCV2 delivered genomic PCV2 DNA specifically into susceptible cells and led to the expression of a functional virus genome. Eighteen 30- to 40-day-old conventional pigs were distributed into three groups. Group 1 pigs (n=6) were inoculated intranasally (i.n.) with a Spanish isolate of PCV2 propagated in cell culture; pigs from group 2 (n=6) were inoculated with pIC-PCV2 intramuscularly (i.m.), and the last group of pigs (n=6) was inoculated with pIC-PCV2 intraperitoneally (i.p.). All pigs remained clinically healthy during the whole experimental period (35 days). Pigs that received pIC-PCV2 i.p. and i.m., as well as those PCV2 i.n. inoculated, became infected based on an in situ hybridization (ISH), PCR, TaqMan PCR and serological results. The results of this study confirm that cloned PCV2 genomic DNA is infectious both in vitro and in vivo, and is able to cause PMWS-like lesions in i.p. and i.m. experimentally inoculated pigs.