- Volume 81, Issue 8, 2000

Infection of CD4+ T lymphocytes with human immunodeficiency virus (HIV) in vitro is accompanied by extensive cytopathicity. The mechanism of cell death is unclear, but may be related to expression of the viral envelope glycoprotein. Here, it is demonstrated that T cell destruction in primary T cells occurs upon contact of infected with uninfected lymphocytes. Cell death was due to the interaction of the envelope glycoprotein with CD4 and subsequent fusion of the cells. Agents that interfered with cell-to-cell fusion such as a monoclonal antibody to CD4 and the peptide T20 prevented T cell death and depletion. In contrast, single-cell lysis due to expression and intracellular processing of the envelope glycoprotein was insignificant. These results suggest that cell-to-cell fusion and concomitant rapid cell death promote the depletion of T cells in HIV-infected individuals.

From analysis of the primary sequence of the hepatitis C virus (HCV) core protein, we have identified three separable regions based on hydrophobicity and clustering of basic amino acids within the protein. Comparison with capsid proteins of related pesti- and flaviviruses suggested that HCV core has a unique central domain (domain 2). Previous findings have revealed that core protein can associate with lipid droplets which are intracellular storage sites for triacylglycerols and cholesterol esters. Confocal analysis of variant forms lacking regions of core indicated that most residues within the unique region are necessary for association of the protein with lipid droplets. A segment within domain 2 (from residues 125 to 144) also was required for stability of the protein and a polypeptide lacking these sequences was degraded apparently by the proteasome. In cells depleted of lipid droplets, core protein remained located in the cytoplasm. Moreover, cleavage of the protein at the maturation site and stability were not affected by inability to bind to lipid droplets.

Since it was first described in Australia in 1994, Hendra virus (HeV) has caused two outbreaks of fatal disease in horses and humans, and an isolated fatal horse case. Our preliminary studies revealed a high prevalence of neutralizing antibodies to HeV in bats of the genus Pteropus, but it was unclear whether this was due to infection with HeV or a related virus. We developed the hypothesis that HeV excretion from bats might be related to the birthing process and we targeted the reproductive tract for virus isolation. Three virus isolates were obtained from the uterine fluid and a pool of foetal lung and liver from one grey-headed flying-fox (Pteropus poliocephalus), and from the foetal lung of one black flying-fox (P. alecto). Antigenically, these isolates appeared to be closely related to HeV, returning positive results on immunofluorescent antibody staining and constant-serum varying-virus neutralization tests. Using an HeV-specific oligonucleotide primer pair, genomic sequences of the isolates were amplified. Sequencing of 200 nucleotides in the matrix gene identified that these three isolates were identical to HeV. Isolations were confirmed after RNA extracted from original material was positive for HeV RNA when screened on an HeV Taqman assay. The isolation of HeV from pteropid bats corroborates our earlier serological and epidemiological evidence that they are a natural reservoir host of the virus.

The nucleotide sequences of RNA segment 7 (nonstructural protein gene; NS) were compared among 34 influenza C virus strains isolated between 1947 and 1992. The results showed that all the NS genes analysed had the potential to encode NS1 and NS2 proteins of 246 and 182 amino acids, respectively. The deduced amino acid sequence of the previously unidentified NS2 was fairly well conserved, although it was more divergent than the NS1 protein sequence. Moreover, immunoprecipitation experiments with rabbit immune serum against a glutathione S-transferase fusion protein containing the C-terminal region of the 182 amino acid NS2 protein revealed synthesis of a protein with an apparent molecular mass of ∼22 kDa in infected cells. A phylogenetic analysis showed that the 34 NS genes were split into two distinct groups, A and B. Comparison of the phylogenetic positions of the individual isolates in the NS gene tree with those in the haemagglutinin–esterase (HE) gene tree suggested that most of the influenza C viruses currently circulating in Japan, irrespective of their HE gene lineage, had acquired group B NS genes through reassortment events that presumably occurred either in the 1970s or in the early 1980s.

Synthetic T7-driven cDNA minigenomes containing the bacterial chloramphenicol acetyltransferase gene as a reporter were derived from the genome of two salmonid novirhabdoviruses, infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHSV). We showed that an exogenous IHNV RNA minigenome transfected into fish cells could be rescued following IHNV infection as it was replicated, encapsidated and transcribed. When cells were infected with a recombinant vaccinia virus expressing T7 RNA polymerase (vTF7-3), transfected with the plasmid carrying the IHNV minigenome (genomic- and antigenomic-sense) and superinfected with IHNV, rescue of the minigenome was more efficient. Heterologous VHSV/IHNV rescue experiments failed. Finally, when the IHNV N, P and L proteins were expressed from cDNAs in cells, the minigenome was also successfully rescued, indicating that the nucleocapsid proteins were biologically functional. These data represent the first example of rescue experiments for non-mammalian rhabdoviruses replicating at a low temperature.

Borna disease virus (BDV) is a non-segmented, negative-strand RNA virus that replicates and transcribes its genome in the nucleus of infected cells. It uses the cellular splicing machinery to generate a set of alternatively spliced mRNAs from the 2·8 and 7·1 kb primary transcripts, each harbouring two introns. To determine whether splicing of these transcripts is regulated by viral factors, the extent of splicing was studied in infected cells and COS-7 cells transiently transfected with plasmids encoding the 2·8 kb RNA of BDV. Unspliced RNA was found to be the most abundant RNA species in infected cells, whereas viral transcripts lacking both introns were only found in minute amounts. In sharp contrast, plasmid-derived 2·8 kb RNA was predominantly intron 1-spliced and double-spliced. Co-expression of the BDV proteins P, N and X did not influence splicing of plasmid-expressed 2·8 kb RNA. Furthermore, the splicing pattern did not change when the 2·8 kb RNA was expressed in BDV-infected cells. Based on these results we speculate that splicing of authentic BDV transcripts is tightly linked to transcription by the viral polymerase.

Expression of the rotavirus non-structural glycoprotein NSP4 in E. coli leads to a decrease in optical density of the culture and release of [3H]uridine into the medium, effects attributable to the ability of NSP4 to perturb the bacterial membrane. To identify a domain of NSP4 responsible, different regions of the polypeptide were expressed in E. coli. Membrane destabilization is associated with a region of the protein located within residues 48–91, which includes a potential cationic amphipathic helix. A second region of NSP4 that contains a coiled-coil oligomerization domain and a sequence reported to function as a viral enterotoxin enhances the membrane-destabilizing activity of residues 48–91, but has no direct effect on the membrane stability. These studies suggest that the membrane-destabilizing and enterotoxic properties of NSP4 may be mediated by different regions of the polypeptide and suggest a possible basis for the cytotoxicity of NSP4 in mammalian cells.

The bluetongue virus ssRNA-binding protein, NS2, is a phosphoprotein that forms viral inclusion bodies in infected cells. Recombinant NS2 was expressed in the baculovirus expression system and purified to homogeneity from insect cells. Purified NS2 bound nucleosides. Further investigation revealed that the protein bound ATP and GTP and could hydrolyse both nucleosides to their corresponding NMPs, with a higher efficiency for the hydrolysis of ATP. The increased efficiency of hydrolysis of ATP correlated with a higher binding affinity of NS2 for ATP than GTP. Ca2+, Mg2+ and Mn2+ were able to function as the required divalent cation in the reactions. The phosphohydrolase activity was not sensitive to ouabain, an inhibitor of cellular ATPases, suggesting that this activity was not the result of a cellular contaminant.

Genital human papillomaviruses (HPV) are considered to be one of the main risk factors for the development of cervical cancer. The P97 promoter at the E6-proximal end of the long control region (LCR) regulates the transcription of viral genes, especially the oncogenes E6 and E7. The LCR contains binding sites of several viral and cellular transcription factors, which either activate or repress the P97 promoter. Intratype variants of HPV-16 belong to six geographically clustered phylogenetic groups distributed all over the world. These variants exhibit differences in E6 protein activities and in tumour progression in vivo. Seven HPV-16 variants were investigated by sequencing the entire LCR (nt 7060–124) and by comparing the transcriptional activities of their P97 promoters. Previously unknown nucleotide variations were identified in all LCRs investigated. In luciferase assays, 3·3- and 2·8-fold increases in P97 promoter activity were detected in the Asian American c and North American 1 variants when compared with the European reference clone. The African variants 1a and 2a exhibited P97 promoter activities comparable to the European reference clone. After recombining different LCR fragments, the region responsible for enhanced transcription in the Asian American c and North American 1 variants could be attributed to the E6-proximal end of the LCR (nt 7619–124).

We previously described two genital carcinomas (IC2, IC4) containing human papillomavirus type 16 (HPV-16)- or HPV-18-related sequences integrated in chromosomal bands containing the c-myc (8q24) or N-myc (2p24) gene, respectively. The c-myc gene was rearranged and amplified in IC2 cells without evidence of overexpression. The N-myc gene was amplified and highly transcribed in IC4 cells. Here, the sequence of an 8039 bp IC4 DNA fragment containing the integrated viral sequences and the cellular junctions is reported. A 3948 bp segment of the genome of HPV-45 encompassing the upstream regulatory region and the E6 and E7 ORFs was integrated into the untranslated part of N-myc exon 3, upstream of the N-myc polyadenylation signal. Both N-myc and HPV-45 sequences were amplified 10- to 20-fold. The 3′ ends of the major N-myc transcript were mapped upstream of the 5′ junction. A minor N-myc/HPV-45 fusion transcript was also identified, as well as two abundant transcripts from the HPV-45 E6–E7 region. Large amounts of N-myc protein were detected in IC4 cells. A major alteration of c-myc sequences in IC2 cells involved the insertion of a non-coding sequence into the second intron and their co-amplification with the third exon, without any evidence for the integration of HPV-16 sequences within or close to the gene. Different patterns of myc gene alterations may thus be associated with integration of HPV DNA in genital tumours, including the activation of the protooncogene via a mechanism of insertional mutagenesis and/or gene amplification.

The E1 protein of bovine papillomavirus type 1 (BPV-1) is the origin recognition protein and is essential for the initiation of viral DNA replication. We reported previously that there is a conserved motif between residues 25 and 60 of all papillomavirus E1 proteins that resembles a casein kinase II (CKII) phosphorylation site. The corresponding serine in BPV-1, serine-48, is an efficient substrate for CKII in vitro. To examine the functional role of this potential phosphorylation site, three amino acid substitutions were constructed at serine-48. Conversion of serine-48 to a glycine (S48G) resulted in a BPV-1 genome that was unable to replicate and had reduced transformation capacity. The S48G E1 protein also failed to support replication of a BPV-1 origin-containing plasmid when expressed from a heterologous vector rather than the viral genome, indicating a direct replication defect. In contrast, conversion of serine-48 to acidic residues (S48D or S48E), which mimic the charge and structure of phosphoserine, maintained the wild-type replication phenotype. These mutational results are consistent with a replication requirement for a negative charge at serine-48, presumably supplied by in vivo phosphorylation. The mechanistic basis for the negative charge requirement was examined by testing several activities of the S48G mutant E1 protein in vivo using yeast one- and two-hybrid systems. No gross defect was observed for stability, origin binding or interaction with E2 or for E1–E1 interaction, although subtle defects in these activities would not likely be detected. Overall, the results suggest that important phosphoregulatory control of E1 replication function is mediated through the N-terminal region of this protein.

Channel catfish virus (CCV), a member of the herpesvirus family, causes a severe haemorrhagic disease in juvenile channel catfish. In this report, we confirm that CCV gene expression is temporally regulated into immediate-early (IE), early and late phases, similar to that of other herpesviruses. The transcriptional regulation of the 14 genes within the direct repeat region of the CCV genome was determined by Northern hybridization analysis of RNA isolated from infected cells in the presence or absence of metabolic inhibitors. Two CCV genes within the direct repeat, ORFs 1 and 3, expressed IE transcripts. Early RNAs were encoded by ORFs 2–9 and 11–14. ORFs 4, 7 and 10–13 expressed late transcripts after the onset of viral DNA replication. A time-course study conducted without metabolic inhibitors confirmed that CCV direct repeat transcription is temporally regulated. The characterization of CCV transcription during cytolytic infection in vitro will provide a foundation for the analysis of CCV gene expression in tissues of acutely and latently infected catfish.

The glycoproteins of herpes simplex virus type 1 (HSV-1) are important targets for the immune system in the control of HSV-1 infections. The humoral and T cell responses to the glycoprotein (g)Ht(His):gL complex of HSV-1 were studied in seven HSV-1-seropositive and three HSV-1-seronegative healthy adults. In addition, responses to HSV-1 gDt were determined. As antigens, purified soluble recombinant forms of the gHt(His):gL complex produced by insect cells and of gDt produced by yeast cells were used. In contrast to seronegative donors, sera of all seropositive donors contained gHt(His): gL-specific IgG. Using peripheral blood (PB) T cells, gHt(His):gL-specific proliferative T cell responses were detected in all seropositive donors. Culture supernatants of PB T cells stimulated with recombinant gHt(His):gL contained high levels of interferon-γ and no detectable interleukin-4, indicating their Th1 phenotype. These results show that naturally acquired HSV-1 infection induces gH:gL-specific humoral and T cell responses.

The mechanisms by which herpes simplex viruses (HSV) mediate fusion between their envelope and the plasma membrane during entry into cells, and between the plasma membranes of adjacent infected and uninfected cells to form multinucleated giant cells, are poorly understood. Four viral glycoproteins (gB, gD, gH and gL) are required for virus–cell fusion, whereas these plus several others are required for cell–cell fusion (syncytium formation). A better understanding would be aided by the availability of a model system, whereby fusion could be induced with a minimal set of proteins, in the absence of infection. A suitable system has now been developed for HSV-2, using transfected COS7, 293 or HEp-2 cells. Insofar as the minimal set of HSV-2 proteins required to cause cell–cell fusion in this system is gB, gD, gH and gL, it would appear to resemble virus–cell fusion rather than syncytium formation. However, the ability of a mutation in gB to enhance the fusion of both transfected cells and infected cells, while having no effect on virus–cell fusion, points to the opposite conclusion. The differential effects of a panel of anti-HSV antibodies, and of the fusion-inhibitor cyclosporin A, confirm that the fusion of transfected cells shares some properties with virus–cell fusion and others with syncytium formation. It may therefore prove useful for determining how these processes differ, and for testing the hypothesis that some viral proteins prevent membrane fusion until the appropriate point in the virus life-cycle, with other proteins then overcoming this block.

The incidence of Kaposi’s sarcoma in South Africa is increasing in parallel with the human immunodeficiency virus type 1 epidemic. An 804 bp region in the ORF75 gene of 40 human herpesvirus-8 (HHV-8) isolates from South Africa was sequenced and the phylogenetic relationships were compared to published sequences. Nineteen strains clustered with subgroup B and 11 with subgroup A; however, the bootstrap values supporting these subgroups were not significant. Three strains grouped significantly with the C subgroup, while eight sequences did not cluster with any of the previously classified subgroups and were termed novel (N). The N subgroup differed from the A, B and C subgroups by DNA distances of 4·8, 4·2 and 4·5%, respectively, although within the N subgroup there was only 0·4% variation. The inclusion of this subgroup increased the number of previously described subgroup-specific polymorphisms from 17 to 47 over an 804 bp region. There was sufficient inter-subgroup genetic diversity for a single-strand conformational polymorphism assay to be used to identify them rapidly. Thus, based on analysis of the ORF75 gene, a unique HHV-8 subgroup, termed N, is present in South Africa, which accounts for 20% of circulating strains. Further studies are required to determine the degree of genetic divergence, distribution and pathogenic potential of this novel subgroup.

Herpesvirus late genes require viral DNA replication for maximal expression. Although late gene expression appears to require DNA replication in cis in alphaherpesviruses, studies in Epstein–Barr virus (EBV) suggest that this cis-requirement might not pertain to the gammaherpesviruses. Based on these findings, a system was created to investigate the elements required for the regulation of Kaposi’s sarcoma-associated herpesvirus (KSHV; human herpesvirus-8) late gene expression. The transcript of a classic late gene encoding the viral assembly protein was characterized and reporter genes driven by the assembly protein promoter region were constructed. Unlike the EBV case, expression of a reporter gene under the control of the assembly protein promoter did not display authentic regulation when removed from the context of the viral genome. Although reporter expression rose in cells displaying lytic replication, this expression was not diminished by specific inhibitors of viral DNA synthesis. Minimal core promoters were similarly unable to reproduce late gene regulation. These results suggest that proper KSHV late gene expression is likely to be dependent upon virus lytic replication in cis and indicate that the regulation of KSHV late genes more closely resembles that observed in herpes simplex virus than that described for EBV.

Kaposi’s sarcoma-associated herpesvirus (KSHV, human herpesvirus-8) is aetiologically associated with Kaposi’s sarcoma and several other lymphoproliferative disorders. The latent nuclear antigen (LNA) encoded by KSHV ORF73 has important functions in virus latent infection and shows molecular polymorphism. Sequence variations were identified in the internal repeat domain (IRD) of ORF73. DNA sequencing of ORF73 from one KSHV-infected cell line, PK-1, revealed that there were 558 bp (30·2%) deletions and 66 (3·6%) point mutations located mainly in repeat region 2, the glutamine-rich region of ORF73 IRD, compared with ORF73 of BC-1 KSHV. Similar sequence variations of ORF73 were also identified in two other isolates. None of the sequence variations caused any translational frame-shift in these four KSHV isolates examined, suggesting that LNA has a conservative function in virus latent infection. The frequent sequence variations in repeat region 2 of ORF73 IRD were also identified by PCR–RFLP genotyping in 26 KSHV isolates, suggesting that this region is a ‘hot-spot’ for genetic variations. Each Kaposi’s sarcoma lesion sample contained one virus genotype with a unique RFLP pattern, indicating that in vivo KSHV infection was established with single predominate genotypes, which was further supported by the presence of invariable genotypes in multifocal lesions from individual KS patients. Four KSHV subtypes were classified based on the RFLP patterns that represent the patterns of DNA sequence variations in the ORF73 IRD. PCR–RFLP genotyping is capable of identifying LNA genetic variations and differentiating individual KSHV isolates, and thus may be useful for KSHV molecular epidemiology studies.