- Volume 70, Issue 5, 1989

The analysis of replicative form (RF) DNA of Aleutian disease virus (ADV) by alkaline gel electrophoresis revealed that all RF DNA species segregate into DNA single strands which represent integral multiples of a genome equivalent. This demonstrates that as with other autonomous parvoviruses, the virion and complementary DNA strands are frequently linked by hairpin structures and that also, nicks are present at subterminal sites. Approximately 50 % of the 5′-terminal hairpins contain a subterminal nick whereas no nick is detectable in the 3′-terminal hairpin. This finding together with the presence of nicks in the 3′ palindrome sequence of the dimer RF DNA (D RF DNA) bridge fragment is the first experimental proof for the so far hypothetical substrate specificity of a nickase. A novel DNA structure was identified in the monomer (M) RF DNA population. This molecule, designated ‘monomer covalently closed linear RF DNA’ (Mccl RF DNA), consists of a continuous, self-complementary, circular polynucleotide chain of twice the genome length. It was directly visualized by electron microscopy that denatured ADV M RF DNA is a single-stranded circular molecule of twice the genome length with covalently closed terminal hairpins on either end. Alkaline gradient centrifugations, enzymic assays and electrophoretic techniques confirmed the proposed structure. Moreover, evidence was obtained that the D RF DNA species contains an analogous Dccl RF DNA. It is suggested that the newly described Mccl RF DNA form is an important intermediate common to the DNA replication of all autonomously replicating parvoviruses.

The growth of the Sabin strain of type 3 poliovirus is reduced at high temperatures compared to that of its virulent precursor strain Leon. Recombinant viruses have been generated from infectious cDNA clones and demonstrate that the temperature-sensitive (ts) phenotype is mainly attributable to a difference in residue 91 of the virion protein VP3. Examination of non-ts mutants derived in vitro or in vivo reveals the existence of second site mutations some of which are clearly able to suppress the ts phenotype. The location of residue 91 of VP3, and of a number of candidate suppressor mutations, in the atomic structure of the virion suggests that the ts phenotype may result in destabilization of the particle and that the suppressors may function by stabilizing specific interfaces. It is not yet clear whether the ts phenotype is expressed at the level of the particle or in the form of defects in assembly or uncoating of the virion, or all three.

Twenty-one monoclonal antibodies reactive with Junin virus structural proteins were produced and characterized. Using radioimmunoprecipitation and Western blot assays, 13 were found to react with the nucleoprotein, seven with the surface glyco-protein and one failed to react, but showed a fluorescent antibody staining pattern consistent with other glycoprotein-specific antibodies. In radioimmunoprecipitation assays, glycoprotein-specific monoclonal antibodies reacted not only with the 35K structural glycoprotein, but also with what is presumed to be the glycoprotein precursor. Four of seven glycoprotein-specific antibodies neutralized Junin virus to high titres. Cross-reactivity with other arenaviruses was found to be restricted to nucleoprotein-specific monoclonal antibodies and occurred only with New World arenaviruses. Cross-reactivity also shows the Junin virus to be most closely related to Machupo and Tacaribe viruses.

Four hybridoma cell lines producing monoclonal antibodies (MAbs) to bovine papillomavirus type 1 (BPV-1) L2 open reading frame (ORF) gene products have been established from mice immunized with a BPV-1 L2-β-galactosidase fusion protein. Hybridomas were selected and cloned (from over 700 hybridomas) on the basis of specific reactivity of supernatant fluids with BPV-1 L2 epitopes on disrupted BPV-1 particles and L2-β-galactosidase fusion proteins by ELISA and Western blotting, and with acetone-fixed frozen sections of BPV-1-induced fibropapillomas by immunofluorescence. These MAbs were not reactive with intact BPV-1 particles or BPV-1 L1-β-galactosidase fusion proteins by ELISA or with β-galactosidase by ELISA and Western blotting. The four MAbs detected viral structural proteins of M r 76K, 68K and possibly 55K in purified BPV-1 preparations by Western blotting. Two of the four MAbs were cross-reactive with BPV-2-induced fibropapillomas. These findings suggest that (i) the BPV-1 L2 ORF encodes the minor capsid protein(s), (ii) the gene products of the BPV-1 L2 ORF have M r values of 76K, 68K and possibly 55K, (iii) minor capsid epitopes are internal to the BPV-1 particle, and (iv) MAbs reactive with genetically engineered truncated BPV-1 L2 ORF gene products can distinguish between BPV-1 and BPV-2 productive infections.

Alcelaphine herpesvirus type 1 (AHV-1) is a causative agent of the fatal lymphoproliferative disease malignant catarrhal fever in deer and cattle. The genomes of the attenuated WC11 isolate and the virulent C500 isolate have been studied. The genome of WC 11 comprises a region of unique DNA of approximately 130 kbp, which has a G + C content of 50%, and approximately 30 kbp of additional tandem direct repeat sequences with a G + C content of 72 %. WC11 possesses a major repeat sequence of 950 bp interspersed with a small number of related sequences of different length; these sequences are probably terminal in location. DNA from the C500 isolate has a similar restriction profile to that of WC11 in the unique region, but only one repeat sequence of 1050 bp is present. We propose, on the basis of biological and structural properties, that AHV-1 be included within the γ 2 group of herpes viruses of which herpesvirus ateles is the prototype.

We have sequenced a gene in human cytomegalovirus and a homologous gene in human herpesvirus 6 which could specify a product related to protein kinases. This gene appears to be generic in the herpesvirus family as homologues were found in three other human herpesviruses. The five sequences were aligned and found to be quite divergent. Some of the differences occur at amino acid positions which are functionally important and highly conserved in known protein kinases. Hence these genes may represent a significant departure from known protein kinases in terms of structure and/or function.

Equine herpesvirus types 1 and 4 (EHV-1 and EHV-4) labelled with [14C]glucosamine were purified from infected cell culture medium and profiles of their structural proteins were obtained that enabled identification of the major glycoproteins. Nine glycosylated polypeptides were identified for each virus. Preparations of the purified viruses each contained a glycoprotein which was linked by disulphide bonds, as determined by diagonal gel electrophoresis under reducing/non-reducing conditions. High M r forms of this glycoprotein were detected for EHV-1 when the sample was not heated. The EHV-1 protein consisted of three polypeptides of M r 108K, 76K and 58K and the EHV-4 protein consisted of three polypeptides of M r 112K, 74K and 61K. Western blotting and immunoprecipitation with monoclonal antibodies confirmed that the EHV-1 gB homologue migrates with an apparent M r of 108K (140K under non-reducing conditions) but is cleaved to give glycoproteins of 76K and 58K which are held together by disulphide bonds. The EHV-4 gB homologue consists of a 112K glycoprotein which is cleaved to give glycoproteins of 74K and 61K which are also linked by disulphide bonds.

Following intraperitoneal or intranasal inoculation of the Syrian hamster with equine herpesvirus type 1 (EHV-1), strain Kentucky D, virus replicated in the liver and lungs reaching a peak at 4 days post-infection (p.i.). By day 6 p.i. virus titres in these organs had reduced and the spleen contained virus-specific cytotoxic cells. This cytotoxicity was mediated by T cells since treatment of effector cells with a monoclonal antibody to hamster T lymphocytes inhibited the effect. An antiviral humoral immune response was present by day 4 when antibodies capable of lysing EHV-1-infected target cells in the presence of complement were detected. The transfer of serum from recovered hamsters into naive recipients 24 h before challenge prevented virus infection, whereas serum transferred 24 h after challenge reduced the titre of virus recovered from target organs. Inoculation of hamsters with monoclonal antibodies directed to glycoproteins 13, 14 and 17/18 of a subtype 1 virus (Army 183) before virus challenge protected hamsters. This hamster model will prove useful for studying the immune response to EHV-1 and evaluating the immunogenicity of individual virus components.

Transcription from the early and late classes of the herpes simplex virus type 1 (HSV-1) promoters requires prior immediate early (IE) gene expression. Although the product of IE gene 1, Vmw110, is not absolutely essential for virus growth in tissue culture, transfection experiments have demonstrated that Vmw110 can activate gene expression both by itself and in a synergistic manner with the product of IE gene 3, Vmw175. This paper describes the construction of 10 mutant HSV-1 viruses with deletion and insertion mutations in Vmw110. The mutant viruses were then studied in single-step growth curve experiments, by assaying for plaques in a variety of cell types and by analysis of viral polypeptide synthesis during productive infection at high and low multiplicities. The results show that mutations in Vmw110 reduce the efficiency of plaque formation by HSV-1; the extent of this reduction depends on cell type and the position of the mutation in the polypeptide. In particular, a potential zinc finger domain is crucial for Vmw110 function. The patterns and amounts of viral polypeptide synthesis during high multiplicity infections with mutant and wild-type viruses were similar in all cell types. At low multiplicity, mutations in Vmw110 reduced viral gene expression in the least permissive cell type. The data suggest that the role of Vmw110 during virus infection in tissue culture is at a very early stage of low multiplicity infections; its inactivity leads to the failure to express viral genes so that the virus does not enter the lytic cycle.

Epstein-Barr virus (EBV) DNA sequences were detected in four established monoblast or early monocytic cell lines (CM-S, ROV-S, CV-S and AD-S) obtained from bone marrow of children suffering from maturation defects of haematopoiesis. EBV is present in these cells in a latent state. The viral DNA in these cell lines was analysed by Southern blot hybridization, using a set of cloned EBV DNA fragments from the EBV strain B95-8 as probes. A common spectrum of highly related but distinguishable EBV DNA restriction enzyme sequences was found, suggesting some genomic diversity. Propagation of the cells in long-term culture revealed a gradual decrease of EBV copies per cell in all lines with some minor changes in the restriction pattern of the EBV DNA. These findings demonstrate that human precursor monocyte cells may be susceptible to infection by EBV.

The Epstein-Barr virus nuclear antigen 2 (EBNA 2) shows serotype variation and two serologically distinct groups of viruses have been identified. These correspond to the two hybridization groups of viruses (A and B) that are distinguished by a highly substituted nucleic acid sequence in the middle of the open reading frame of the EBNA 2 gene. An epitope survey of the EBNA 2-coding region was carried out using a new prokaryotic expression vector tailored to express DNA fragments from the M13 sequencing libraries of the B95-8 (type A) and Jijoye (type B) prototype virus strains. Short overlapping stretches of EBNA 2 sequence were expressed as fusion proteins and used in Western blotting with human sera that contained serotype-specific antibodies. The type A-specific epitope was located between residues 378 and 435 of the B95-8 EBNA 2 polypeptide and the type B-specific epitope mapped between residues 390 and 454, at the carboxy terminus of the Jijoye polypeptide chain. All of the type-specific anti-EBNA 2 sera tested reacted with fusion proteins containing one or other of these epitopes. Despite the direct correlation between the hybridization and serological phenotypes, the type-specific epitopes appear to lie in the relatively conserved carboxy-terminal region of EBNA 2. There was no indication that the residues of the non-homologous region contributed to the formation of antibody-combining sites.

The complete coding sequence of the Epstein–Barr virus strain B95-8 latent membrane protein (LMP) was cloned using a Raji cell cDNA library and genomic B95-8 DNA. The clone was characterized by sequencing and then used to make a recombinant vaccinia virus. This virus (VLMP) was shown to express a relatively high level of LMP in an authentic fashion. Antisera raised in rabbits against VLMP were shown to react with B95-8 LMP as well as cross-reacting with a 50K cellular protein.

The coding region of the glycoprotein complex gII of pseudorabies virus (PRV) is located in the unique long part of the genome on SalI subfragments 1A and 1G of BamHI fragment 1 (map units 0·105 to 0·130). Fragment 1G which includes the 3′ end of the gII gene displays a size heterogeneity among different PRV strains and also within plaque isolates of a given strain. To reveal the cause of this heterogeneity and whether it might affect the gII-coding region we sequenced different 1G fragments of the PRV strains Ka, Phylaxia and Dessau, and determined the 3′ end of the gII mRNA by SI analysis. These data show that the size heterogeneity is caused by the presence of a variable number of tandemly repeated DNA sequence downstream but adjacent to the coding region of the glycoprotein gII gene. The 3′ end of the gII mRNA was mapped about 24 bp upstream of the first repeat unit. A 15 bp sequence 5′ GGGACGGAGGGGAGA 3′ is repeated from three to over 50 times in different 1G fragments. It is the only repeat unit present in strain Ka, whereas the Phylaxia and Dessau strains show additional modifications.

We have isolated reactive clones from a λgt11 expression library of human cytomegalovirus (HCMV) DNA using HCMV-positive human sera. Among the recombinant clones obtained, one carried a fragment encoding a portion of p52, the major non-structural DNA-binding protein of 52K (p52) and another carried a part of the gene coding for p150, the major structural phosphoprotein. These two fusion proteins were examined by immunoblot analysis to test their ability to bind specific antibodies in human sera. The results showed that high titres of antibody to the DNA-binding protein are present in sera of patients undergoing acute HCMV infection, whereas high titres of antibodies to the structural phosphoprotein are widespread in the healthy HCMV-seropositive population. The use of these fusion proteins as antigens for differential screening of serum as a way of detecting recent HCMV infection is discussed.

The ability of mice to survive infection with murine cytomegalovirus (MCMV) is known to be influenced by genes of the major histocompatibility complex (MHC). One hypothesis to account for this association is that MHC-linked resistance to MCMV is an ‘immune response’ gene effect, caused by differences in the strength of the MHC-restricted T cell response of mouse strains with different MHC haplotypes. Therefore, removal of T cell responses in mouse strains differing only at the MHC should render them equally susceptible to the virus infection. To test this hypothesis, the immunosuppressive drug cyclosporin (CsA) was used to reduce T cell responses in inbred congenic mouse strains carrying either a resistant or susceptible MHC haplotype. CsA reduced the delayed-type hypersensitivity (DTH) response to MCMV in both resistant and susceptible mouse strains to background levels, equivalent to control uninfected mice. CsA treatment had little effect on the susceptibility of C57BL/10 and B10. BR mice to the virus and the differences in susceptibility between these strains remained. In contrast, CsA increased the susceptibility of the genetically susceptible BALB/c mice (H-2d) by 100-fold and increased the susceptibility of resistant BALB.K mice (H-2k) by 15-fold. Thus the H-2-determined difference in susceptibility between these strains was increased after CsA treatment. The results obtained with congenic strains show that MHC-linked resistance patterns to MCMV are not eliminated by CsA and suggest therefore that T cells are not responsible for this phenomenon. Interestingly, the mean time to death was delayed for CsA-treated BALB/c mice compared with untreated mice given equivalent virus doses. In addition, although CsA prevented DTH responses in both genetically susceptible A/J (H-2a) and resistant CBA (H-2k) mice, CsA treatment markedly increased the susceptibility of A/J mice (32-fold) but had little effect on the susceptibility of CBA mice to the virus.

Primary human epithelial cells were transfected with a subgenomic fragment of DNA cloned from a cervical carcinoma, containing the putative transforming genes E6 and E7 from the human papillomavirus type 16 prototype. Several immortalized cell lines were generated, all retaining tumour DNA and expressing the viral genes, suggesting that either one or both of these genes is sufficient for the immortalization of primary human genital keratinocytes.

Using plasmids containing sequences complementary to the genes that code for oligo-2′,5′-adenylate synthetase, actin and H4 histone, we have shown that although interferon does not affect the accumulation of RNA transcripts of the actin and H4 histone genes, it activates the accumulation of RNA transcripts of the oligo-2′,5′-adenylate synthetase gene. However, interferon inhibits the accumulation of RNA transcripts of simian virus 40 (SV40) genes in SV40-infected cells.

5′ deletion mutants of the Autographa californica nuclear polyhedrosis virus very late p 10 gene promoter have been prepared and subjected to a transient expression assay in infected Spodoptera frugiperda cells. The control plasmid contained the chloramphenicol acetyltransferase (CAT) reporter gene under the control of the p 10 promoter, which was included in a 230 bp sequence upstream from the p 10 translation initiation codon. The control plasmid also contained a segment of the hr5 enhancer downstream from the CAT gene. Promoter activity was unaffected by 5′ deletion to position –77, which lies about 11 bp upstream from the p10 cap site. However, deletion of 12 more bp completely eliminated p10 promoter activity. Thus, the 5′ border of the p10 promoter lies downstream from position –77, and the region between positions –77 and –65 contains an element that is important to promoter activity. This is the region that is conserved near the cap sites of late baculovirus genes. Our studies also show that transient expression of CAT under the control of the p10 promoter and hr5 enhancer is higher when transfection occurs prior to infection by virus.

The nucleotide sequence of the small (S) RNA segment of the Bunyamwera virus genome has been determined. The S RNA is 961 bases in length and, in common with other bunyaviruses, encodes two proteins, N and NSs, in overlapping reading frames. A six-way alignment of the amino acid sequences of the N and NSs proteins of viruses representing three serogroups within the Bunyavirus genus indicates regions which are strongly conserved, and provides targets for future analysis of protein function.