- Volume 89, Issue 1, 2008

Computer analysis of 158 hepatitis C virus (HCV) 5′ untranslated region (5′ UTR) sequences from the six genotypes showed that the 5′ UTR from genotype 3 displays seven specific non-contiguous nucleotide changes, at positions 8, 13, 14, 70, 97, 203 and 224. The purpose of this study was to investigate the impact of these changes on translation and replication activities. Indeed, these modifications could alter both the internal ribosome entry site (IRES) present in the 5′ UTR of the plus-strand RNA and the 3′ end of the minus strand involved in the initiation of plus-strand RNA synthesis. We found that the genotype 3-specific nucleotide changes do not modify the in vitro or ex vivo translation activity of the corresponding IRES, in comparison with that of genotype 1. In contrast, in vitro replication from the minus-strand RNA is eight times less efficient for genotype 3 than for genotype 1 RNA, suggesting the involvement of some nucleotide changes in the reduction of RNA synthesis. Nucleotides 13, 14 and 224 were found to be responsible for this effect. Moreover, a reduced replicative activity was confirmed ex vivo for genotype 3, but to a lesser extent than that observed in vitro, using an RNA minigenome.

The hepatitis C virus (HCV) genome possesses an open reading frame (ORF) overlapping the core gene at +1 nucleotide (core+1 ORF). Initial in vitro studies suggested that the core+1 ORF is translated by a ribosomal −2/+1 frameshift mechanism during elongation of the viral polyprotein. Recent studies, however, based on transfection of mammalian cells with reporter constructs have shown that translation of the core+1 ORF is mediated from internal core+1 codons. To resolve the apparent discrepancies associated with the mechanism of core+1 translation, we examined the expression of the HCV-1 and HCV-1a (H) core+1 ORF in a cytoplasmic transcription system based on Huh-7/T7 cells that constitutively synthesize the T7 RNA polymerase in comparison to that in Huh-7 cells. We showed that the efficiency of both the −2/+1 and −1/+2 frameshift events operating at the HCV-1 core codons 8–11 is significantly enhanced in the Huh-7/T7 cytoplasmic transcription system and is dependent on the presence of the consecutive adenine (A) residues within core codons 8–11. In contrast, internal translation initiation at core+1 codons 85/87 occurs in both the nuclear and cytoplasmic transcription systems and is not repressed by the ribosomal frameshifting event. Finally, although core+1 codons 85/87 is the most efficient site for internal initiation of core+1 translation, it may not be unique, as additional internal core+1 codon(s) appear to drive translation at low levels.

If we could rewind the tape of evolution and play it again, would it turn out to be similar to or different from what we know? Obviously, this key question can only be addressed by fragmentary experimental approaches. Twenty-two years ago, we described the establishment of BHK-21 cells persistently infected with foot-and-mouth disease virus (FMDV), a system that displayed as its major biological feature a coevolution of the cells and the resident virus in the course of persistence. Now we report the establishment of two persistently infected cell lines in parallel, starting with the same clones of FMDV and BHK-21 cells used 22 years ago. We have asked whether the evolution of the two newly established cell lines and of the earlier cell line would be similar or different. The main conclusions of the study are: (i) the basic behaviour characterized by virus–cell coevolution is similar in the three carrier cell lines, despite differences in some genetic alterations of FMDV; (ii) a strikingly parallel behaviour has been observed with the two newly established cell lines passaged in parallel, unveiling a deterministic virus behaviour during persistence; and (iii) selective RT-PCR amplifications have detected imbalances in the proportion of positive- versus negative-strand viral RNA, mediated by both viral and cellular factors. The results confirm coevolution of cells and virus as a major and reproducible feature of FMDV persistence in cell culture, and suggest that rapidly evolving viruses may constitute adequate test systems to probe the influence of historical contingency on evolutionary events.

A truncated structural protein of hepatitis E virus (HEV), p239, occurs as 23 nm particles consisting of partial homodimers. As the latter resemble the HEV capsomere structurally and antigenically, it was postulated that the recombinant protein may serve as a probe for the HEV receptor. This hypothesis was supported by findings that purified p239 bound and penetrated different cell lines that are susceptible to HEV, and inhibited HEV infection of these cells. The binding was blocked by four of six monoclonal antibodies (mAbs) reactive against the dimeric domain of p239, and by two of three mAbs reactive against its monomeric domain, suggesting that binding may involve a portion of each domain. Mutation affecting the monomeric domain had no effect on binding or capacity to block HEV infection, whereas that affecting the dimeric domain diminished binding of the mutant peptide markedly and abrogated its capacity to block HEV infection. These results suggest that HEV infection might involve distinct receptor-binding sites.

As respiratory syncytial virus (RSV) targets the mucosal surfaces of the respiratory tract, induction of both systemic and mucosal immunity will be critical for optimal protection. In this study, the ability of an intranasally delivered, formalin-inactivated bovine RSV (FI-BRSV) vaccine co-formulated with CpG oligodeoxynucleotides (ODN) and polyphosphazenes (PP) to induce systemic and mucosal immunity, as well as protection from BRSV challenge, was evaluated. Intranasal immunization of mice with FI-BRSV formulated with CpG ODN and PP resulted in both humoral and cell-mediated immunity, characterized by enhanced production of BRSV-specific serum IgG, as well as increased gamma interferon and decreased interleukin-5 production by in vitro-restimulated splenocytes. These mice also developed mucosal immune responses, as was evident from increased production of BRSV-specific IgG and IgA in lung-fragment cultures. Indeed, the increases in serum and mucosal IgG, and in particular mucosal IgA and virus-neutralizing antibodies, were the most critical differences observed between FI-BRSV formulated with both CpG ODN and PP in comparison to formulations with CpG ODN, non-CpG ODN or PP individually. Finally, FI-BRSV/CpG/PP was the only formulation that resulted in a significant reduction in viral replication upon BRSV challenge. Co-formulation of CpG ODN and PP is a promising new vaccine platform technology that may have applications in mucosal immunization in humans.

Respiratory syncytial virus (RSV) is the primary cause of bronchiolitis in young children. In general, RSV is considered to be a poor inducer of type I (alpha/beta) interferons (IFNs). Measurement of active type I IFN production during infection in vivo is demanding, as multiple IFN subtypes with overlapping activities are produced. In contrast, Mx gene expression, which is tightly regulated by type I IFN expression, is easily determined. This study therefore measured Mx expression as a reliable surrogate marker of type I IFN activity during RSV infection in vivo in a cotton rat model. It was shown that expression of Mx genes was dramatically augmented in the lungs of infected animals in a dose- and virus strain-dependent manner. The expression of Mx genes in the lungs was paralleled by their induction in the nose and spleen, although in spleen no simultaneous virus gene expression was detected. Reinfection of RSV-immune animals leads to abortive virus replication in the lungs. Thus, type I IFN and Mx gene expression was triggered in reinfected animals, even though virus could not be isolated from their lungs. Furthermore, it was demonstrated that immunity to RSV wanes with time. Virus replication and Mx gene expression became more prominent with increasing intervals between primary infection and reinfection. These results highlight the role of type I IFN in modulation of the immune response to RSV.

European porcine H1N2 influenza viruses arose after multiple reassortment steps involving a porcine influenza virus with avian-influenza-like internal segments and human H1N1 and H3N2 viruses in 1994. In Germany, H1N2 swine influenza viruses first appeared in 2000. Two German H1N2 swine influenza virus strains isolated from pigs with clinical symptoms of influenza are described. They were characterized by the neutralization test, haemagglutination inhibition (HI) test and complete sequencing of the viral genomes. The data demonstrate that these viruses represent a novel H1N2 reassortant. The viruses showed limited neutralization by sera raised against heterologous A/sw/Bakum/1832/00-like H1N2 viruses. Sera pools from recovered pigs showed a considerably lower HI reaction, indicative of diagnostic difficulties in using the HI test to detect these viruses with A/sw/Bakum/1832/00-like H1N2 antigens. Genome sequencing revealed the novel combination of the human-like HAH1 gene of European porcine H1N2 influenza viruses and the NAN2 gene of European porcine H3N2 viruses.

The lymphotropic lentiviruses feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV) enter cells by sequential interaction with primary receptors CD134 or CD4, respectively, and subsequently with chemokine receptors. The host-cell range for FIV is broader than that for HIV, but whether this is a function of receptor expression is unknown. Lack of reagents specific to feline molecules has limited detection and analysis of receptors and their interaction with viral components. Here, the expression of CD134 and CXCR4 on feline T and B lymphocytes, dendritic cells (DCs) and macrophages was examined and the kinetics of FIV replication were assessed. Quantification of CD134 mRNA by real-time PCR indicated expression in all leukocytes, with significantly more transcripts in CD4+ lymphocytes than in other leukocytes. Antibodies against human CD134 bound inconsistently to feline leukocytes. CXCR4 was detected with antibody clone 12G5 on the surface of monocyte-derived cells only, but gene transcripts were present in all cells, with the highest copy number in lymphocytes. CXCR4 expression decreased and CD134 expression increased with cell activation in lymphocytes. A subtype B biological isolate of FIV infected DCs, macrophages and lymphocytes, with the highest replication in CD4+ lymphocytes, whilst cloned FIV P14 infected all cells, but replicated less efficiently. Although viral replication was lower in DCs and macrophages than in lymphocytes, DCs expressed specific receptors and were infected productively with FIV, as indicated by viral ultrastructure and DNA detection. These results may implicate altered function of DCs in the induction of specific immunity against FIV.

Myristoylation of the human immunodeficiency virus type 1 (HIV-1) proteins Gag and Nef by N-myristoyltransferase (NMT) is a key process in retroviral replication and virulence, yet remains incompletely characterized. Therefore, the roles of the two isozymes, NMT1 and NMT2, in myristoylating Gag and Nef were examined using biochemical and molecular approaches. Fluorescently labelled peptides corresponding to the N terminus of HIV-1 Gag or Nef were myristoylated by recombinant human NMT1 and NMT2. Kinetic analyses indicated that NMT1 and NMT2 had 30- and 130-fold lower K m values for Nef than Gag, respectively. Values for K cat indicated that, once Gag or Nef binds to the enzyme, myristoylation by NMT1 and NMT2 proceeds at comparable rates. Furthermore, the catalytic efficiencies for the processing of Gag by NMT1 and NMT2 were equivalent. In contrast, NMT2 had approximately 5-fold higher catalytic efficiency for the myristoylation of Nef than NMT1. Competition experiments confirmed that the Nef peptide acts as a competitive inhibitor for the myristoylation of Gag. Experiments using full-length recombinant Nef protein also indicated a lower K m for Nef myristoylation by NMT2 than NMT1. Small interfering RNAs were used to selectively deplete NMT1 and/or NMT2 from HEK293T cells expressing a recombinant Nef–sgGFP fusion protein. Depletion of NMT1 had minimal effect on the intracellular distribution of Nef–sgGFP, whereas depletion of NMT2 altered distribution to a diffuse, widespread pattern, mimicking that of a myristoylation-deficient mutant of Nef–sgGFP. Together, these findings indicate that Nef is preferentially myristoylated by NMT2, suggesting that selective inhibition of NMT2 may provide a novel means of blocking HIV virulence.

A Mus dunni tail fibroblast (MDTF) cell line is highly resistant to infection by ecotropic Moloney murine leukemia virus (Mo-MLV). The cationic amino acid transporter type 1 (CAT1) paralogues of murine NIH 3T3 and MDTF cells (mCAT1 and dCAT1, respectively) contain two conserved N-linked glycosylation sites in the third extracellular loop (ECL3, the putative Mo-MLV binding site). Glycosylation of dCAT1 inhibits Mo-MLV infection, but that of mCAT1 does not. Compared with mCAT1, dCAT1 possesses an Ile-to-Val substitution at position 214 and a Gly insertion at position 236 in the ECL3. To determine the residues responsible for the loss of dCAT1 receptor function, mutants of mCAT1 were constructed. The mCAT1/insG receptor (with a Gly residue inserted at mCAT1 position 236) had greatly reduced Mo-MLV receptor function compared with mCAT1. Treatment of mCAT1/insG-expressing cells with tunicamycin, an N-linked glycosylation inhibitor, increased the transduction titre. In addition, the reduced susceptibility to Mo-MLV observed with mCAT1/insG-expressing cells correlated with impaired binding of Mo-MLV. These results show that a single amino acid insertion confers mCAT1 receptor properties on dCAT1 and provide an important insight into the co-evolution of virus–host interactions.

In the current study, it was shown that repressed virus genomes in quiescently infected MRC5 cells adopt a repressed histone-associated structure marked by the enrichment of deacetylated histones at a wide variety of herpes simplex virus type 1 (HSV-1) promoters. In addition, it was shown that genome de-repression, mediated by HSV-2 superinfection or delivery of ICP0 using a recombinant adenovirus vector, resulted in the enrichment of acetylated histones on HSV DNA. These data indicate that ICP0-mediated genome de-repression is intimately linked to enrichment of acetylated histones at virus promoters. The fold change in association of pan-acetylated histone H3 following Ad.TRE.ICP0-mediated de-repression consistently revealed promoter-specific variation, with the highest fold changes (>50-fold) being observed at the latency-associated transcript promoter and enhancer regions. Chromatin immunoprecipitation analyses using an antibody specific to the C terminus of histone H3 as a surrogate measure of nucleosome occupancy revealed little variability in the total loading of histone H3 at the various HSV promoters. This observation suggests that acetylation of histone H3 in response to ICP0 expression is not uniformly targeted across the HSV-1 genome during ICP0-mediated de-repression.

An efficient host response to human cytomegalovirus (HCMV) infection may depend on rapid sensing of the infection by the innate immune response prior to deployment of viral immunosubversive functions. Control of HCMV dissemination could be ensured by apoptosis of cells immediately following infection. In the present report, it is demonstrated that changes in the ratio of c-FLIP to FLICE contributed to early sensitivity of HCMV-infected MRC5 fibroblasts to tumour necrosis factor alpha (TNF-α), providing an innate response to infection. Dendritic cells (DCs) co-cultured with HCMV-infected MRC5 cells acquired the ability to secrete TNF-α in an amount sufficient to kill infected fibroblasts. Blockage of TNF-α binding to its receptor on MRC5 cells with soluble TNF-R reduced the number of dead, HCMV-infected fibroblasts ingested by DCs, thus highlighting the impact of the apoptotic state of infected cells for efficient loading of DCs. Those DCs loaded with antigens available early in infection, such as input virion-associated pp65, could then engage antigen processing for cross-presentation to specific CD8+ T cells. Cross-presentation was impaired when MRC5 cells were treated with the pan-caspase inhibitor ZVAD before co-culture with DCs. Altogether, our data suggest that the innate killing capacity of DCs at the early stage of infection plays a role in the activation of anti-HCMV CD8+ T cells.

Here, we demonstrate that human herpesvirus 6B (HHV-6B) infection upregulates the tumour suppressor p53 and induces phosphorylation of p53 at Ser392. Interestingly, phosphorylation at the equivalent site has previously been shown to correlate with p53 tumour suppression in murine models. Although the signalling pathways leading to Ser392 phosphorylation are poorly understood, they seem to include casein kinase 2 (CK2), double-stranded RNA-activated protein kinase (PKR), p38 or cyclin-dependent kinase 9 (Cdk9). By using column chromatography and in vitro kinase assays, CK2 and p38, but not PKR or Cdk9, eluted in column fractions that phosphorylated p53 at Ser392. However, treatment of cells with neither the CK2 and Cdk9 inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) nor p38 kinase inhibitors reduced HHV-6B-induced Ser392 phosphorylation significantly. Knockdown of the CK2β subunit or p38α by small interfering RNA had no effect on HHV-6B-induced phosphorylation of p53 at Ser392. Thus, HHV-6B induces p53 Ser392 phosphorylation by an atypical pathway independent of CK2 and p38 kinases, whereas mitogen-activated protein (MAP) kinase signalling pathways are involved in viral replication.

The human cytomegalovirus (HCMV)-encoded viral G protein-coupled receptor pUS28 contributes to an array of biological effects, including cell migration and proliferation. Using FIX-BAC (bacterial artificial chromosome, derived from the HCMV clinical isolate VR1814) and lambda red recombination techniques, we generated HCMV recombinants expressing amino-terminally FLAG-tagged versions of wild-type pUS28 (FLAG–US28/WT), G-protein coupling deficient pUS28 (FLAG–US28/R129A) and chemokine-binding domain deficient pUS28 (FLAG–US28/ΔN). Infection with the FLAG–US28/R129A virus failed to induce inositol phosphate accumulation, indicating that G-protein coupling is essential for pUS28 signalling to phospholipase C-β (PLC-β) during HCMV infection. The FLAG–US28/ΔN virus induced about 80 % of the level of PLC-β signalling induced by the FLAG–US28/WT virus, demonstrating that the N-terminal chemokine-binding domain is not required for pUS28-induced PLC-β signalling in infected cells. The data presented here are the first to describe the functional analyses of several key pUS28 mutants in HCMV-infected cells. Elucidating the mechanisms by which pUS28 signals during infection will provide important insights into HCMV pathogenesis.

Human herpesvirus 6B (HHV-6B) contains an IE-B domain spanning open reading frames U16/17–U19, based on homology with human cytomegalovirus. Here, the protein product, U19, of the HHV-6B U19 gene is identified as a 47 kDa transcriptional activator. HHV-6B infection or overexpression of U19 transactivated the RANTES promoter. Mutational analysis of the promoter indicated that transactivation was not critically dependent on the promoter sites CRE, NF-κB, ISRE or NF-IL6. ND10 are nuclear substructures that are involved in several cellular regulatory pathways, including those controlling gene expression. HHV-6B infection resulted in a reduced number of ND10 structures, but with a concomitantly increased level of promyelocytic leukaemia (PML) protein expression and mRNA induction. The U19 protein co-located to ND10 with PML and heterochromatin protein 1 (HP1), but whilst PML formed a ring structure, U19 also localized to the centre of ND10. Knockdown of PML by small interfering RNA did not prevent U19 localization to ND10-like foci, but instead led to a fourfold increase in U19-induced transcription from the RANTES promoter. Generation of four truncated U19 proteins indicated that the N-terminal portion of the protein contains a sequence responsible for nuclear localization; a domain in the N-terminal half of U19 is responsible for its ND10 localization, whereas the C-terminal portion contains the transactivation domain. None of the truncated proteins retained full transactivating ability on the RANTES promoter. Thus, U19 is a transcriptional activator that co-localizes with PML and localizes to ND10-like foci independently of PML, yet is regulated negatively by PML or its associated proteins.

Most anti-human papillomavirus (HPV) capsid antibody assays are based on virus-like particles (VLP). We evaluated glutathione S-transferase (GST)–L1 fusion proteins as ELISA antigens for determining type specificity and cross-reactivity of 92 VLP-specific monoclonal antibodies (mAb) generated against nine mucosal alpha papillomavirus types of species 7, 9 and 10. The antibody panel included 25 new mAb, and 24 previously published mAb are further characterized. We determined the cross-reactivity patterns with 15 different HPV types representing 6 species (alpha1, 2, 4, 7, 9 and 10) and neutralization and cross-neutralization properties with HPV types 6, 11, 16, 18 and 45. Eighty-nine (97 %) of the antibodies including 34, 71 and 14 recognizing neutralizing, conformational and linear epitopes, respectively, reacted with the GST–L1 protein of the HPV type used as immunogen, with log titres ranging from 2.0 to 7.3. Of these 89 antibodies, 52 % were monotypic, 20 % showed intra-species and 28 % inter-species cross-reactivity. Log neutralization titres to the immunogen HPV ranged from 1.7 to 5.6. A single cross-neutralizing mAb (H6.L12) was found. ELISA titres were always higher than neutralization titres. All neutralizing epitopes were conformational and mostly type-specific. Our data show that bacterially expressed, affinity-purified GST–L1 fusion proteins display a broad variety of epitopes and thus are well suited for detection of HPV antibodies. Cross-reactivity is associated with linear as well as conformational epitopes. Distantly related mucosal and skin alpha papillomaviruses share some conformational epitopes and the phylogenetic L1-based species definition may not define a serological unit since no species-specific epitope was found.

DNA samples from a variety of New World monkeys were screened by using a broad-spectrum PCR targeting the VP1 gene of polyomaviruses. This resulted in the characterization of the first polyomavirus from a New World primate. This virus naturally infects squirrel monkeys (Saimiri sp.) and is provisionally named squirrel monkey polyomavirus (SquiPyV). The complete genome of SquiPyV is 5075 bp in length, and encodes the small T and large T antigens and the three structural proteins VP1, VP2 and VP3. Interestingly, the late region also encodes a putative agnoprotein, a feature that it shares with other polyomaviruses from humans, baboons and African green monkeys. Comparison with other polyomaviruses revealed limited sequence similarity to any other polyomavirus, and phylogenetic analysis of the VP1 gene confirmed its uniqueness.

Equine sarcoids are fibrosarcoma-like skin tumours with a prevalence of approximately 1–2 %. Strong evidence exists for a causative role of bovine papillomavirus (BPV) type 1 or type 2 in the development of sarcoids. No effective treatment of equine sarcoid is available and after surgical excision relapse of the tumours is very frequent. We developed chimeric virus-like particles (CVLPs) of BPV 1 L1–E7 for the immunotherapy of equine sarcoid. In a phase I clinical trial 12 horses suffering from equine sarcoid with an average number of more than 22 tumours per animal were vaccinated in a dose-escalation setting. The animals were followed-up for 63 days, eight of the twelve horses were followed-up for more than a year and side-effects, humoral immune responses and tumour appearance were recorded. BPV DNA was detected in tumours of 11 cases. CVLPs were well tolerated in all dose groups, a robust anti-L1 antibody response was induced in all but one of the horses. Anti-E7 antibodies were detected in five of the 12 animals at low titres. Two animals showed a clear improvement of the clinical status after treatment, i.e. the number of the tumours per horse was reduced. In another horse regression of five sarcoids was observed; three of them relapsed during the study. Two animals showed tumour regression as well as growth of new sarcoids. In two horses the clinical status remained unchanged, in another two horses growth of existing tumours or growth of additional tumours was observed. The remaining three animals showed simultaneously regression and growth of existing tumours. Neither the humoral immune responses nor the observed effects on the tumours was correlated with the dose group.