- Volume 83, Issue 12, 2002

Virus-neutralizing activity of two monoclonal antibodies (mAbs), #7-1-9 and #1-46-12, against rabies virus glycoprotein (G) was compared. Although these mAbs affected the virion’s ability to bind to host cells similarly, a big difference was found in the titres of virus neutralization (1:7132 and 1:32 for mAbs #1-46-12 and #7-1-9, respectively, at a concentration of 10 μg protein/ml). Although no big difference in virion-binding affinity between the two mAbs was found, the number of antibodies required for virus neutralization was very low, ⩽20 molecules for mAb #1-46-12 and ⩾250 molecules for mAb #7-1-9. In the latter case, the mAbs cover a major part of the virion surface and cause steric hindrance of viral receptor-binding activity. The infectivity of an epitope-preserved escape mutant virus (R-61) was not affected by the binding of high numbers of mAb #1-46-12 to the virion, which implies that mAb binding does not mask the receptor-binding site of the viral spikes. Based on these results, it is hypothesized that mAb #1-46-12 affected virus infectivity by a mechanism different from covering the virion spikes. Possible virus-neutralizing mechanisms by low numbers of mAb #1-46-12 in comparison to that of mAb #7-1-9 are discussed.

Recently, we have shown that influenza virus budding in MDCK cells is regulated by metabolic inhibitors of ATP and ATP analogues (Hui & Nayak, Virology 290, 329–341, 2001). In this report, we demonstrate that G protein signalling stimulators such as sodium fluoride, aluminium fluoride, compound 48/80 and mastoparan stimulated the budding and release of influenza virus. In contrast, G protein signalling blockers such as suramin and NF023 inhibited virus budding. Furthermore, in filter-grown lysophosphatidylcholine-permeabilized virus-infected MDCK cells, membrane-impermeable GTP analogues, such as guanosine 5’-O-(3-thiotriphosphate) or 5’-guanylylimidodiphosphate caused an increase in virus budding, which could be competitively inhibited by adding an excess of GTP. These results suggest that the G protein is involved in the regulation of influenza virus budding. We also determined the role of different protein kinases in influenza virus budding. We observed that specific inhibitors or activators of protein kinase A (H-89 and 8-bromoadenosine 3’,5’-cyclic monophosphate) or of protein kinase C (bisindolylmaleimide I and Ro-32-0432) or of phosphatidylinositol 3-kinase (LY294002 and wortmannin) did not affect influenza virus budding. However, the casein kinase 2 (CK2) inhibitor 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole decreased virus budding. We further observed an increase in the CK2 activity during the replication cycle of influenza virus, although Western blot analysis did not reveal any increase in the amount of CK2 protein in virus-infected cells. Also, in digitonin-permeabilized MDCK cells, the introduction of CK2 substrate peptides caused a down-regulation of virus budding. These results suggest that CK2 activity also regulates influenza virus budding.

The haemagglutinin (HA) protein of influenza A/H2N2 virus possesses five oligosaccharide attachment sites, two of which have overlapping glycosylation sequons at positions 20–23 (NNST) and 169–172 (NNTS). Here, the role of these two oligosaccharide attachment sites is investigated with regard to antigenic property, intracellular transport and biological activity of the HA protein. Glycosylation-site HA mutants with mutation(s) in their overlapping glycosylated sequons, each of which had one or two oligosaccharide attachment sites removed, were constructed. Comparison of electrophoretic mobility between the wt and mutant HA proteins showed that both Asn residues 20 and 21 and Asn residues 169 and 170 could be used for glycosylation. Analysis of reactivity of the mutants with anti-HA monoclonal antibodies suggested that amino acid changes at these two positions result in a conformational change of the HA molecule. Even if oligosaccharide chains linked to Asn 20 or 21 and Asn 169 or 170 are eliminated, the antigenic properties, intracellular transport and biological activities are not influenced strongly. Thus it is reasonable to conclude that the two overlapping glycosylation sequons at positions 20–23 and 169–172 are conserved among all of the HAs of influenza A/H2N2 viruses because conservation of the amino acid sequence itself rather than that of N-glycosylation is essential for the formation of the proper conformation, intracellular transport and biological activities of the H2 subtype HA.

In this study, the complete genomic sequence of chikungunya virus (CHIK; S27 African prototype) was determined and the presence of an internal polyadenylation [I-poly(A)] site was confirmed within the 3’ non-translated region (NTR) of this strain. The complete genome was 11805 nucleotides in length, excluding the 5’ cap nucleotide, an I-poly(A) tract and the 3’ poly(A) tail. It comprised two long open reading frames that encoded the non-structural (2474 amino acids) and structural polyproteins (1244 amino acids). The genetic location of the non-structural and structural proteins was predicted by comparing the deduced amino acid sequences with the known cleavage sites of other alphaviruses, located at the C-terminal region of their virus-encoded proteins. In addition, predicted secondary structures were identified within the 5’ NTR and repeated sequence elements (RSEs) within the 3’ NTR. Amino acid sequence homologies, phylogenetic analysis of non-structural and structural proteins and characteristic RSEs revealed that although CHIK is closely related to o’nyong-nyong virus, it is in fact a distinct virus. The existence of I-poly(A) fragments with different lengths (e.g. 19, 36, 43, 91, 94 and 106 adenine nucleotides) at identical initiation positions for each clone strongly suggests that the polymerase of the alphaviruses has a capacity to create poly(A) by a template-dependant mechanism such as ‘polymerase slippage’, as has been reported for vesicular stomatitis virus.

The interaction between the hepatitis C virus capsid protein and the envelope protein E1 has been demonstrated previously in vivo. To determine the binding region of the E1 protein with the capsid protein, this interaction was characterized in vitro. This study shows that the interaction between these proteins should occur in the endoplasmic reticulum membrane rather than in the cytosol and that the first hydrophobic domain of the E1 protein (aa 261–291) is important for the interaction with the capsid protein.

In dengue virus (DEN) particles, the core protein is a structural protein of the nucleocapsid. The core protein is known to be present in the nucleus of DEN-infected cells but there have been conflicting reports as to whether it is also present in the nucleolus. To clarify this, the intracellular location of the core protein was examined using a monoclonal antibody, 15B11, which was produced in this study. Immunofluorescence staining with this antibody demonstrated that the core protein first appeared in the cytoplasm and then in the nuclei and nucleoli of infected cells. Nuclear localization of the core protein was determined to be independent of other DEN proteins, since recombinant core proteins still entered the nuclei and nucleoli of cells transfected with only the core protein gene. Three putative nuclear localization signal motifs have been predicted to be present on the core protein. Deletion of the first one (KKAR), located at aa 6–9, and mutation of the second one (KKSK), located at aa 73–76, did not eliminate the nuclear localization property of the core protein. The third motif with a bipartite structure, RKeigrmlnilnRRRR, located at aa 85–100, was determined to be responsible for the nuclear localization of the core protein, since the core protein without this motif was located exclusively in the cytoplasm of DEN-infected cells and that this motif mediated nuclear localization of a normally cytoplasmic protein.

Poliovirus isolates were screened for recombinants by combined analysis of two distant polymorphic segments of the poliovirus genome (one in the capsid and the other in the polymerase-coding region). Using a restriction fragment length polymorphism (RFLP) assay, a high number of recombinant genomes was found among vaccine-derived strains excreted by poliovirus vaccine vaccinees or vaccine-associated paralytic poliomyelitis cases. Some of these subjects carried a wild-type poliovirus (non-vaccine-specific) nucleotide sequence in the 3’ part of the genome. Using a similar approach, a collection of wild-type poliovirus strains isolated in South India between 1985 and 1993 was screened for recombinants. Genotypes were defined by the parallel application of RFLP assays and genomic sequencing of the capsid protein VP1 and the 3D polymerase polypeptide. Analyses revealed several instances where the position of an isolate on the phylogenic tree for the capsid protein-coding segment did not agree with its position on the tree for the polymerase-coding region. In this way, several wild-type/wild-type and wild-type/vaccine recombinants could be identified, indicating that recombination is encountered commonly in the natural evolution of poliovirus strains.

The foot-and-mouth disease virus (FMDV) leader (L) proteinase is an important virulence determinant in FMDV infections. It possesses two distinct catalytic activities: (i) C-terminal processing at the L/VP4 junction; and (ii) induction of the cleavage of translation initiation factor eIF4G, an event that inhibits cap-dependent translation in infected cells. The only other member of the Aphthovirus genus, equine rhinitis A virus (ERAV), also encodes an L protein, but this shares only 32% amino acid identity with its FMDV counterpart. Another more distantly related picornavirus, equine rhinitis B virus (ERBV), which is not classified as an aphthovirus, also encodes an L protein. Using in vitro transcription and translation analysis, we have shown that both ERAV and ERBV L proteins have C-terminal processing activity. Furthermore, expression of ERAV L, but not ERBV L, in BHK-21 cells resulted in the efficient inhibition of cap-dependent translation in these cells. We have shown that the ERAV and FMDV L proteinases induce cleavage of eIF4GI at very similar or identical positions. Interestingly, ERAV 3C also induces eIF4GI cleavage and again produces distinct products that co-migrate with those induced by FMDV 3C. The ERBV L proteinase does not induce eIF4GI cleavage, consistent with its inability to shut down cap-dependent translation. We have also shown that another unique feature of FMDV L, the stimulation of enterovirus internal ribosome entry site (IRES) activity, is also shared by the ERAV L proteinase but not by ERBV L. The functional conservation of the divergent ERAV and FMDV proteinases indicates the likelihood of a similar and important role for these enzymes in the pathogenesis of infections caused by these distantly related aphthoviruses.

Taura syndrome virus (TSV) is an important virus infecting penaeid shrimp in the western hemisphere. Genetic variation and immunohistochemical differences of 20 TSV isolates collected from the USA, Taiwan, Mexico and Nicaragua were compared. Capsid protein genes CP1 (546 bp) and CP2 (584 bp) were amplified by RT–PCR and the cDNAs were sequenced. Pairwise comparison of nucleotide sequences showed a 0–2·4% difference in CP1 and a 0–3·5% difference in CP2. Phylogenetic analyses clustered the TSV isolates into two groups: one contained USA, Taiwan and some Mexican isolates, the other contained Mexican isolates only. Immunohistochemical analysis using a TSV-specific monoclonal antibody produced positive results for the USA and Taiwan isolates but negative results for the Mexican and Nicaraguan isolates. Molecular and immunohistochemical data suggest the existence of at least two TSV strains, one of which might have evolved following contact with a new penaeid host, Penaeus stylirostris.

The complete nucleotide sequence of the genomic RNA of an aphid-infecting virus, Aphid lethal paralysis virus (ALPV), has been determined. The genome is 9812 nt in length and contains two long open reading frames (ORFs), which are separated by an intergenic region of 163 nt. The first ORF (5’ ORF) is preceded by an untranslated leader sequence of 506 nt, while an untranslated region of 571 nt follows the second ORF (3’ ORF). The deduced amino acid sequences of the 5’ ORF and 3’ ORF products respectively showed similarity to the non-structural and structural proteins of members of the newly recognized genus Cripavirus (family Dicistroviridae). On the basis of the observed sequence similarities and identical genome organization, it is proposed that ALPV belongs to this genus. Phylogenetic analysis showed that ALPV is most closely related to Rhopalosiphum padi virus, and groups in a cluster with Drosophila C virus and Cricket paralysis virus, while the other members of this genus are more distantly related. Infectivity experiments showed that ALPV can not only infect aphid species but is also able to infect the whitefly Trialeurodes vaporariorum, extending its host range to another family of the order Hemiptera.

The South African isolate of Black queen-cell virus (BQCV), a honey bee virus, was previously found to have an 8550 nucleotide genome excluding the poly(A) tail. Its genome contained two ORFs, a 5’-proximal ORF encoding a putative replicase protein and a 3’-proximal ORF encoding a capsid polyprotein. Long reverse transcription (RT)–PCR was used to produce infectious transcripts for BQCV and to manipulate its genome. Primers were designed for the amplification of the complete genome, the in vitro transcription of infectious RNA and PCR-directed mutagenesis. An 18-mer antisense primer was designed for RT to produce full-length single-stranded cDNA (ss cDNA). Unpurified ss cDNA from the RT reaction mixture was used directly as a template to amplify the full genome by long high-fidelity PCR. The SP6 promoter sequence was introduced into the sense primer to transcribe RNA directly from the amplicon. RNA was transcribed in vitro with and without the presence of a cap analogue and injected directly into bee pupae, which were then incubated for 8 days. In vitro transcripts were infectious but the presence of a cap analogue did not increase the amount of virus recovered. A single base mutation abolishing an EcoRI restriction site was introduced by fusion-PCR, to distinguish viral particles recovered from infectious transcripts from wild-type virus (wtBQCV). Mutant virus (mutBQCV) and wtBQCV were indistinguishable by electron microscopy and Western blot analysis. The EcoRI restriction site was present in wtBQCV and not in mutBQCV.

The simian immunodeficiency virus (SIV) Nef protein contains a consensus Src-homology 3 (SH3) binding motif. However, no SH3-domain proteins showing strong binding to SIV Nef have yet been found, and its potential capacity for high-affinity SH3 binding has therefore remained unproven. Here we have used phage-display-assisted protein engineering to develop artificial SH3 domains that bind tightly to SIV strain mac (SIVmac) Nef. Substitution of six amino acids in the RT loop region of Hck-SH3 with the sequence E/DGWWG resulted in SH3 domains that bound in vitro to SIVmac Nef much better than the natural Hck- or Fyn-SH3 domains. These novel SH3 domains also efficiently associated with SIVmac Nef when co-expressed in 293T cells and displayed a strikingly differential specificity when compared with SH3 domains similarly targeted for binding to human immunodeficiency virus type 1 (HIV-1) Nef. Thus, SIVmac Nef is competent for high-affinity SH3 binding, but its natural SH3 protein partners are likely to be different from those of HIV-1 Nef.

Maedi–visna virus (MVV) causes encephalitis, pneumonia and arthritis in sheep. In vitro, MVV infection and replication lead to strong cytopathic effects characterized by syncytia formation and subsequent cellular lysis. It was demonstrated previously that MVV infection in vitro induces cell death of sheep choroid plexus cells (SCPC) by a mechanism that can be associated with apoptotic cell death. Here, the relative implication of several caspases during acute infection with MVV is investigated by employing diverse in vitro and in situ strategies. It was demonstrated using specific pairs of caspase substrates and inhibitors that, during in vitro infection of SCPC by MVV, the two major pathways of caspase activation (i.e. intrinsic and extrinsic pathways) were stimulated: significant caspase-9 and -8 activities, as well as caspase-3 activity, were detected. To study the role of caspases during MVV infection in vitro, specific, cell-permeable, caspase inhibitors were used. First, these results showed that both z-DEVD-FMK (a potent inhibitor of caspase-3-like activities) and z-VAD-FMK (a broad spectrum caspase inhibitor) inhibit caspase-9, -8 and -3 activities. Second, both irreversible caspase inhibitors, z-DEVD-FMK and z-VAD-FMK, delayed MVV-induced cellular lysis as well as virus growth. Third, during SCPC in vitro infection by MVV, cells were positively stained with FITC-VAD-FMK, a probe that specifically stains cells containing active caspases. In conclusion, these data suggest that MVV infection in vitro induces SCPC cell death by a mechanism that is strongly dependent on active caspases.

Herpes simplex virus type 1 (HSV-1) DNA has been shown to exist as a linear, double-stranded molecule in the virion and as a non-linear (endless), episomal, nucleosomal form in latently infected trigeminal ganglia. The kinetics of the formation and appearance of endless viral genomes and the stability of linear genomes in neuronal cells are not well understood. Nerve growth factor (NGF)-differentiated PC12 cells can sustain long-term, quiescent infections with HSV-1. In this report, the structure and stability of HSV-1 viral DNA in NGF-differentiated PC12 cells was studied as a function of time following infection using both wild-type and replication-defective virus. Unexpectedly, unencapsidated linear genomes were stable in the nucleus of NGF-differentiated PC12 cells for up to 2–3 weeks following infection, beyond the period at which there is no detectable viral gene expression. However, following infection with wild-type HSV, the majority of quiescent viral genomes were in an endless form after 3–4 weeks. These data suggest that the stability and fate of HSV-1 DNA in non-mitotic neuronal-like cells is different from that in productively infected cells and thus there is a significant cellular role in this process. The relevance to the virus life-cycle in neurones in vivo is discussed.

Early during infection, the herpes simplex regulatory protein ICP0 promotes the proteasome-dependent degradation of a number of cellular proteins and the loss of a number of SUMO-1-modified protein isoforms, including PML. Recently, ICP0 has been shown to induce the accumulation of conjugated ubiquitin and function as a ubiquitin E3 ligase. However, certain aspects of the biochemistry, cell biology and the links between SUMO-1 conjugation/deconjugation and protein degradation remain unclear. For example, it is not currently known whether SUMO-1 deconjugation is a prerequisite for ubiquitination or degradation and, if so, by what mechanism this may occur. To help address these questions, a SUMO-specific protease (SENP1) was cloned and its expression and localization in relation to ICP0 examined. A cell line was established which constitutively expresses SUMO-1 to facilitate studies of localization and biochemistry. SENP1 localized to the nucleus mainly in discrete subdomains, a subset of which co-localized with the PML bodies. Both ICP0 and SENP1 protease promoted the loss of SUMO-1 from the nucleus, observed both for the endogenous species and the cell line expressing the epitope-tagged SUMO-1. The tagged SUMO-1 was recruited into high molecular mass conjugates in the cell line, and expression of SENP1 promoted loss of these species, including the modified species of PML. Finally, in co-transfection experiments ICP0 promoted the recruitment of SENP1 to nuclear domains, a result which was also observed early during infection. The significance of these findings is discussed in relation to the function of ICP0.

Transfection of bovine cells with bovine herpesvirus-1 genomic DNA yields low levels of infectious virus. Cotransfection with the bICP0 gene enhances productive infection and virus yield because bICP0 can activate viral gene expression. Since the latency-related (LR) gene overlaps and is antisense to bICP0, the effects of LR gene products on productive infection were tested. The intact LR gene inhibited productive infection in a dose-dependent fashion but LR protein expression was not required. Further studies indicated that LR gene sequences near the 3’ terminus of the LR RNA are necessary for inhibiting productive infection. When cotransfected with the bICP0 gene, the LR gene inhibited bICP0 RNA and protein expression in transiently transfected cells. Taken together, these results suggest that abundant LR RNA expression in sensory neurons is one factor that has the potential to inhibit productive infection and consequently promote the establishment and maintenance of latency.

The potential of CpG-enhanced plasmid DNA vectors encoding a truncated secreted form of bovine herpesvirus-1 (BHV-1) glycoprotein D (tgD) to induce enhanced immune responses in cattle was investigated. We created tgD expression plasmids containing 0, 40 or 88 copies of the hexamer 5’ GTCGTT 3’, a known pan-activating CpG motif in several species. The total tgD-specific IgG titre of calves immunized with these plasmids did not correlate with the CpG content of the plasmid backbone. However, the pBISIA88-tgD-vaccinated group showed a significantly lower IgG1:IgG2 ratio than calves immunized with pBISIA40-tgD or pMASIA-tgD, which has no CpG motifs inserted. Antigen-specific lymphocyte proliferation and IFN-γ secretion by peripheral blood mononuclear cells correlated positively with the CpG content of the vectors. In contrast, calves that received a killed BHV-1 vaccine had an IgG1-predominant isotype and low lymphocyte proliferation and IFN-γ levels. Following challenge, the pBISIA88-tgD-immunized group developed the greatest anamnestic response, the highest BHV-1 neutralization titres in serum and a significantly lower level of virus shedding than the saline control group. However, there were no significant differences in clinical symptoms of infection between the DNA-immunized groups and the saline control group. These data indicate that CpG-enhanced plasmids induce augmented immune responses and could be used to vaccinate against pathogens requiring a strong cellular response for protection.