- Volume 78, Issue 11, 1997

Fourteen T cell lines of rabbits were infected with herpes simplex virus type 1 (HSV-1) and examined for their susceptibility to lytic infection and ability to support virus replication. T cell lines of CD4 8 phenotype were more vulnerable to lysis and supported higher levels of virus replication than those of other phenotypes. Cell lines of CD4 8 and CD4 8 phenotypes continued to proliferate, while remaining productively infected, for more than a month. These latently infected cell lines could be established by treatment with anti-HSV-1 serum and complement. Viral genes were only partially expressed and the CD8 membrane antigen was down-regulated. Infected cell lines, as well as peripheral blood lymphocytes, were shown to induce meningoencephalitis when inoculated intravenously into syngeneic hosts, suggesting a possible role for infected lymphocytes in HSV-1 transport in vivo.

Although herpes simplex virus type 1 (HSV-1) does not induce apoptosis in infected HEp-2 cells, in the presence of cycloheximide infection induced apoptosis with characteristic morphological changes as well as endonucleosomal DNA cleavage. The induction of apoptosis without de novo protein synthesis suggests that a structural protein of the HSV-1 virion is responsible for the observed apoptosis.

Apoptosis or programmed cell death (PCD) is a type of animal cell death in which cells die by an active cellular process under the control of a genetically encoded cell suicide programme Apoptosis is characterized morphologically by cell shrinkage, plasma-membrane blebbing, chromatin condensation and nuclear fragmentation and biochemically by degradation of chromosomal DNA into oligonucleosome- sized fragments (Kerr & Harmon, 1991) . Although many animal viruses are known to induce an apoptotic response in infected cells (Clem & Miller, 1994; Shen & Shenk, 1995; White & Gooding, 1994), cells infected with wild-type herpes simplex virus type 1 (HSV-1) do not show apoptotic characteristics . However, based on the finding that mutant virus which lacks the γ34.5 gene induces PCD in infected human cells (Chou & Roizman, 1992; Chou et al. , 1994), the lack of apoptosis in the HSV-1-infected cells has been considered to be an outcome of expression of a viral antiapoptosis gene whose product inhibits one of the steps in the virus-induced signalling leading to PCD . Later, HSV-1 was found to carry, as well as the γ34.5 gene, an antiapoptosis gene which can suppress the cellular apoptotic response induced by hyperthermia (Leopardi & Roizman, 1996) or sorbitol treatment (Koyama & Miwa, 1997) .

The crucial role of herpes simplex virus type 1 immediate early protein Vmw175 (ICP4) in the regulation of all classes of viral genes has been established by extensive analysis of temperature- sensitive, insertion and deletion mutants. It has long been known that Vmw175 binds to selected DNA sequences, and recent studies have shown that it interacts with components of the basal transcription machinery. However, the role of DNA binding in its mechanism of action has been controversial. Despite the presence of Vmw175 recognition sites at numerous locations throughout the viral genome, it has proved difficult to establish that these sites are important for the activation of early and late promoters. In this study we have analysed the ability of a large number of insertion and single point mutant derivatives of Vmw175 to bind to DNA and to support virus replication. Vmw175 mutants which were unable to bind to DNA were also incapacitated in terms of their ability to activate gene expression in transfection assays and to complement the growth of a Vmw175 deletion mutant virus. These results strongly support the hypothesis that interaction with DNA is an essential feature of the mechanism of action of Vmw175.

We have identified the herpes simplex virus type 1 (HSV-1) US10 gene product using rabbit polyclonal antisera raised against a recombinant 6xHis-US10 fusion protein expressed in Escherichia coli. The antiserum reacted specifically with 34 and 36 kDa proteins in HSV-1 KOS-infected cells as shown by Western blotting and immunoprecipitation experiments. The 36 kDa protein was immunoprecipi- tated with the US10 antiserum from 32P-labelled lysates of Vero cells infected with HSV-1 KOS, demonstrating that the US10 protein was phos- phorylated. Indirect immunofluorescence studies localized the US10 protein mainly to nuclei as large discrete particles at later times post-infection (p.i.), and nuclear fractionation studies revealed that the protein was tightly associated with the nuclear matrix. Moreover, analysis of isolated intracellular capsids showed that both phosphorylated and unphosphorylated forms of the US10 product were also associated with the capsid/tegument. These results indicate that the US10 gene of HSV-1 encodes a capsid/tegument-associated phospho- protein which copurifies with the nuclear matrix.

To facilitate the evaluation of vaccines against bovine herpesvirus type 1 (BHV-1), a cotton rat model of intranasal (i.n.) BHV-1 infection was established. Cotton rat lung cells were similar to bovine cells in their ability to support BHV-1 replication in vitro. Furthermore, i.n. inoculation of cotton rats with BHV-1 resulted in pulmonary lesions comparable to BHV-1 infection in cattle. Using this model, the potential of i.n. and gastrointestinal (g.i.) immunization was examined with recombinant human adenoviruses expressing glycoprotein D (gD) of BHV-1 to induce protective immunity against BHV-1.The replication-competent virus (gD-dE3) was more efficient than the replication-defective virus (gD-dE1E3) in inducing gD- specific antibody in the serum and in the respiratory tract. Furthermore, i.n. immunization with gD-dE3 stimulated antigen-specific antibody-secreting cells in the lung 12 weeks following immunization. Protection against BHV-1 challenge correlated with gD-specific antibody levels such that i.n. immunization with gD-dE3 conferred complete protection, while g.i. immunization conferred only partial protection of the lungs of most animals against BHV-1 challenge. In comparison, immunization with gD-dE1E3 by either route resulted in only a partial reduction of BHV-1 titre in the respiratory tract. The results obtained demonstrate that mucosal immunization with replication-competent recombinant adenovirus expressing gD of BHV-1 can induce immunity and protection against BHV-1 challenge.

Infectious laryngotracheitis virus (ILTV) is an alpha- herpesvirus that causes severe upper respiratory infections in chickens. Although ten putative ILTV glycoprotein genes have been identified by sequence analysis, no ILTV glycoprotein has been extensively characterized. In order to delineate the synthesis and processing pathway of ILTV glycoprotein B (gB), rabbit polyclonal antibodies were raised against a Cro-gB- β-galactosidase fusion protein. Through immunoprecipitation analysis of ILTV-infected chicken embryo liver cells it was determined that ILTV gB is initially synthesized as a 110 kDa monomeric precursor protein which rapidly assembles into homodimers composed of 100 kDa subunits. The dimer form of ILTV gB is rapidly cleaved to form two disulphide-linked species of 58 kDa. The apparent reduction in mass (from 110 to 100 kDa) of the mature form of gB during processing in the Golgi apparatus appears to be a common feature of avian herpesvirus gB proteins.

The human cytomegalovirus (HCMV) UL98 gene is predicted to encode a homologue of the conserved herpesvirus alkaline nuclease. To determine if the HCMV UL98 gene product is functionally homologous to other herpesvirus alkaline nucleases, the HCMV UL98 protein was purified and its activity characterized in vitro. Extracts of HCMV-infected cells were fractionated using Q Sepharose, phos- phocellulose and native DNA cellulose chromatography. UL98 immunoreactivity copurified with alkaline pH-dependent nuclease activity. The purified protein migrated at its predicted size of approximately 65 kDa in denaturing polyacrylamide gels, and displayed nuclease activity in an activity gel assay. Enzyme activity was characterized by a high pH optimum, an absolute requirement for divalent cation, salt sensitivity, and 5′ to 3′ exonuclease activity. DNA digestion resulted in 5′ monophos- phoryl mono- and oligodeoxyribonucleotides. Kinetic analyses revealed a turnover rate of greater than 200 per min, and similar apparent affinity and rate constants on single- and double-stranded DNA. These results indicate that a functional alkaline nuclease activity is conserved among distant members of the herpesvirus family, and are consistent with a highly conserved role in the virus life cycle.

A cis-acting sequence within the rat cytomegalovirus (RCMV) genome (or/Lyt) that directs initiation of lytic-phase DNA replication is identified in this report. RCMV or/Lyt was localized within a 4 3 kb NcoI fragment that is situated immediately upstream of the gene encoding the major DNA-binding protein. The activity of or/Lyt was investigated in a transient replication assay, in which the ability of plasmid constructs to promote DNA replication was tested. Replication of or/Lyt-containing plasmids was autonomous and resulted in the generation of high-molecular-mass concatemers of head-to-tail- linked plasmid oligomers. or/Lyt-mediated replication was found to depend on viral DNA polymerase activity supplied by RCMV infection. The sequence required for or/Lyt function was found to reside within a 3·3 kb HincII-NcoI fragment. The RCMV or/Lyt sequence is highly complex, containing 23 direct repeats (DRs) and 16 inverted repeats (IRs) of lengths greater than 10 bp. Two of the DRs (DR21 and DR22) are exceptionally large, being 80 and 88 bp in length, respectively. In addition, two sequence elements (of 127 and 120 bp) with dyad symmetry were identified within or/Lyt. Although the sequence similarity of RCMV or/Lyt with its human cytomegalovirus counterpart is limited, there is a striking resemblance in the overall organization of several IRs and DRs within both sequences.

The Epstein-Barr virus latent membrane protein 1 (LMP-1) is essential for virus-induced B cell immortalization and can protect B lymphoma cell lines from apoptosis signals in vitro via induction of cellular Bcl-2 expression. However, we have reported that high-level expression of LMP-1 in epithelial cells (RHEK-1 cells) itself induces apoptosis. This apoptotic event occurs in the absence of detectable Bcl-2 expression in the LMP-1- transfected epithelial cells. In this study, we transfected the bcl-2 gene into the LMP-1-containing cells and examined the effect of Bcl-2 upon LMP-1- mediated apoptosis, and upon the growth phenotype of the transfected cells. The results show that ectopic expression of Bcl-2 specifically blocks apop- totic death induced by LMP-1. This was observed from cell culture viability and from gel electrophoresis and flow cytometric assays of the degree of DNA fragmentation in cultured cells. Furthermore, co-expression of LMP-1 and Bcl-2 in RHEK-1 cells enabled the cells to grow under low-serum conditions and to form colonies in semi-soft agar medium. These results suggest, therefore, that these two proteins play important complementary roles in the process of EBV-associated epithelial cell transformation. It appears significant, therefore, that LMP-1 and Bcl-2 are frequently co-expressed in the malignant cells of an EBV-positive epithelial tumour, nasopharyngeal carcinoma.

BHRF1, a component of the restricted early antigen (EA) complex of the Epstein-Barr virus (EBV) lytic cycle, encodes a 17 kDa putative transmembrane protein with both sequence and functional homology to the Bcl-2 proto-oncogene. To determine whether there was any sequence variation over the BHRF1 open reading frame (ORF), 15 EBV isolates from different geographical regions and from both healthy donors and patients with EBV-associated diseases were sequenced. A small number of base changes which resulted in amino acid substitutions in the BHRF1 protein were found relative to the prototype B95.8 EBV sequence and these were predominantly clustered near the amino terminus of the BHRF1 protein outside conserved domains identified in the Bcl-2 homologues. In transient transfection assays none of the mutations in the BHRF1 ORF from eight different EBV isolates had a

significant effect on BHRF1 protein localization compared to the B95.8 BHRF1 protein. However, transient expression of the adenovirus 12 19K protein or Bcl-2 resulted in localization patterns distinct from that observed with BHRF1 protein. Whilst all eight EBV isolates and E1B-19K gave comparable levels of protection to the DNA-damaging agent c/s-platin, Bcl-2 did not afford significant protection. Thus, despite several amino acid changes in the BHRF1 ORF of some of the EBV isolates studied, the ability of the protein to protect against cis-platin induced apoptosis is conserved. The highly conserved nature of BHRF1 amongst different EBV isolates at both the sequence and functional level supports the proposed important role of BHRF1 in delaying cell death, thereby maximizing the production of progeny virus and facilitating the establishment of virus persistence.

Bovine papillomavirus type 4 (BPV-4) does not possess an E6 ORF. The E6 oncoprotein of human papillomavirus (HPV) binds and degrades the tumour suppressor protein p53, thus contributing to tumour progression. Since BPV-4 lacks E6, it is unknown how the virus evades the tumour suppressor properties of p53 in the induction of tumours of the gastrointestinal tract. Mutations in the p53 gene have been detected both in papillomas and carcinomas, suggesting that p53 dysfunction plays a part in these neoplasias. BPV-4 can transform primary foetal bovine cells (PalFs) in cooperation with an activated ras gene, but the transformed cells are neither immortal nor tumori- genic. Co-transfection with the HPV-16 E6 (16E6) ORF confers immortality but not tumorigenicity. To investigate the role of p53 in BPV-4 cell transformation in vitro, we transfected PalFs and p53- null mouse fibroblasts with BPV-4 DNAin combinations with ras, 16E6 ORF and mutant (V143A) p53 cDNA. Transfection of PalFs with BPV-4 DNA, ras and mutant p53 led to cell immortalization, indicating that 16E6 and mutant p53 are functionally equivalent in conferring immortality. However, cotransfection of PalFs with BPV-4 DNA, ras, and both mutant p53 cDNA and 16E6 ORF resulted in cells which were fully transformed to tumorigenicity. In p53-null mouse fibroblasts, BPV-4 DNA induced transformation by itself, but the transformed cells were incapable of suspension growth. The cotransfection of BPV-4 DNA with 16E6ORF produced many more transformed colonies and the cells were capable of growing in suspension. In this system, therefore, 16E6 confers anchorage-independence to BPV-4-transformed cells in a p53-independent fashion.

Human papillomavirus type 16 (HPV-16) is a DNA tumour virus that has been implicated in the development of cervical cancer. In non-transformed HPV-infected cells, the HPV E2 protein regulates transcription of the viral E6 and E7 oncogenes. Malignant transformation is usually accompanied by disruption of the E2 gene and consequent deregulated expression of E6 and E7. Here we show that re-introduction of the HPV-16 E2 protein into an HPV-16-transformed cervical carcinoma cell line results in a decrease in growth rate and, in the absence of serum growth factors, cell death via apoptosis. E2 expression increases E6/E7 mRNA levels. This brings about an increase in E7 protein levels, which in turn leads to an increase in free E2F, a condition that has previously been shown to induce apoptotic cell death. Despite the increase in E6 mRNA there is no detectable E6 protein in these cells and E2 expression does not reduce the activity of a p53-responsive promoter. Our data suggest that disruption of the E2 gene produces HPV- transformed cells that are less liable to undergo apoptosis and, therefore, more likely to form cervical tumours.

Recombinant vaccinia virus with tumour cell specificity may provide a versatile tool either for direct lysis of cancer cells or for the targeted transfer of genes encoding immunomodulatory molecules. We report the expression of a single chain antibody on the surface of extracellular enveloped vaccinia virus. The wild-type haemagglutinin, an envelope glycoprotein which is not required for viral infection and replication, was replaced by haemagglutinin fusion molecules carrying a single chain antibody directed against the tumour-associated antigen ErbB2. ErbB2 is an epidermal growth factor receptor-related tyrosine kinase overexpressed in a high percentage of human adenocarcinomas. Two fusion proteins carrying the single chain antibody at different NH2-terminal positions were expressed and exposed at the envelope of the corresponding recombinant viruses. The construct containing the antibody at the site of the immunoglobulin-like loop of the haemagglutinin was able to bind solubilized ErbB2. This is the first report of replacement of a vaccinia virus envelope protein by a specific recognition structure and represents a first step towards modifying the host cell tropism of the virus.

Transcription of the HBV 2·1 kb RNAs is regulated by the major surface antigen promoter. Previously, transient transfection analysis identified regulatory sequence elements in this promoter located between 189 and 1 which govern the level of transcription from this promoter and appear to bind only ubiquitous transcription factors including NF1, Sp1 and NF-Y. However, in vivo transcription analysis in transgenic mice has demonstrated that the expression of the HBV 2·1 kb RNAs is largely restricted to hepatocytes. In this study, the presence of a functional HNF3 transcription factor binding site located between 231 and 240 in the major surface antigen promoter suggests that the in vivo liver-restricted expression of the 2·1 kb RNAs may be governed by this liver-enriched transcription factor. The identification of a functional HNF3 binding site upstream of the DNA polymerase open reading frame also supports the contention that transient transfection analysis may fail to detect all of the cis-acting regulatory sequence elements involved in modulating the level of transcription from the viral promoters.

The human helper virus-dependent parvovirus adeno-associated virus (AAV) has been found in human female genital tissues including material from first trimester miscarriage. In the latter case, AAV type 2 (AAV-2) DNA and viral proteins were detected mainly in the trophoblast cell layer of placenta. In this report, we present evidence that AAV DNA is also present in established human trophoblast cell lines (JEG-3, JAr, BeWo) and in the human amnion cell line FL. In cells of these lines, AAV-2 DNA could be detected both by PCR and Southern blot analysis. Restriction enzyme analysis indicated that AAV DNA was integrated into the host cell genome. Although the cell lines supported AAV replication when infected with AAV-2 and adenovirus type 2 (Ad2) as a helper virus, superinfection with Ad2 alone did not induce replication of AAV DNA, i.e. it failed to rescue AAV from its integrated state. This is probably due to rearrangements within the integrated AAV genome. The presence of AAV DNA in cells derived from human embryonic tissue corroborates the suggestion that human embryonic tissue may be one of the targets of AAV infection.

The 3′ untranslated regions (UTRs) of the three genomic RNAs of alfalfa mosaic virus consist of a 3′ homologous sequence of 145 nt and upstream unique sequences 18–34 nt in length. Mutations were made in the 3′ UTR of a cDNA clone of RNA3. Point mutations in five AUGC motifs which interfere with specific binding of coat protein to the 3′ UTR had no effect on template activity of RNA3 for minus-strand RNA synthesis in vitro by purified viral RNA-dependent RNA polymerase (RdRp). Deletion analysis showed that the 3′ homologous sequence of 145 nt was sufficient for a low level of template activity in the in vitro RdRp assay and a similarly low level of RNA3 accumulation in plants. The presence of an additional sequence of nucleotides 145–165 from the 3′ end of RNA3 enhanced template recognition by RdRp in vitro and accumulation of RNA3 in vivo to wild-type levels.

Transgenic potato plants were generated which express a dicistronic mRNA corresponding to the 8 kDa protein and coat protein (CP) genes of potato virus X (PVX). Northern blot analysis of RNA isolated from these plants indicated that both the 8 kDa protein and the CP are translated from this dicistronic mRNA. Expression of both of these proteins in transgenic plants was demonstrated by Western blot analysis, and suggests that translation of CP takes place either by initiation by internal entry of ribosomes or by a termination/reinitiation mechanism. CP was detected in protoplasts electroporated with RNA transcripts of the dicistronic construct in the presence or absence of a stable hairpin structure at the 5′ terminus, suggesting that the former model is more likely. Similar results were obtained when a DNA fragment containing the PVX CP leader sequence was placed between two reporter genes in the transient assay system. These results suggest that potexvirus CP expression may take place from the genomic RNA as well as from the subgenomic RNA.