- Volume 73, Issue 10, 1992

Subunit approaches to vaccines against viral diseases have resulted in the development of a number of methods for presentation of defined epitopes to the immune system. We have exploited a highly immunogenic presentation system based on hepatitis B core antigen (HBcAg) particles to produce a number of candidate vaccines against simian immunodeficiency virus (SIV). Recombinant particles have been produced in bacteria which carry multiple copies of defined or predicted neutralizing epitopes of SIV at a number of different sites within the particle. In parallel, a number of synthetic peptide-based SIV vaccines have been produced based on homology to reported neutralizing epitopes in human immunodeficiency virus. Although potent immune responses were elicited against both particulate and peptide forms of the antigen, neutralizing antibodies were not induced as judged by available assays.

To determine whether the two major core proteins (VP3 and VP7) of bluetongue virus can interact in vitro to form morphological structures, linearized VP3 and VP7 cDNA clones were transcribed using SP6 polymerase and the resultant transcripts were co-translated using rabbit reticulocyte lysates. The structures derived were isolated by sedimentation through a sucrose gradient and found to resemble VP3–VP7 core-like particles (CLPs) expressed in vivo. Reacting CLPs synthesized in vivo with outer capsid proteins translated in vitro (VP2 or VP5) indicated that each outer capsid protein has the capacity to bind to a preformed CLP. This was confirmed by in vivo expression of the appropriate genes using baculovirus vectors. The interaction of VP2 or VP5 with the CLP was analysed by electron microscopy and by using immunogoldlabelled monoclonal antibody.

Comparison of sequence data for Broadhaven (BRD) virus, a tick-borne orbivirus, and bluetongue virus (BTV), the type species of the genus, indicated that RNA segments 2 and 7 of BRD virus encode the two structural core proteins, VP2 and VP7, respectively. Segment 2 is 2792 nucleotides in length with a coding capacity for a protein (VP2) of 908 amino acids and a net charge of +8.5 at neutral pH. Segment 7 is 1174 nucleotides in length with a coding capacity for a protein (VP7) of 356 amino acids and a net charge of +11.5 at neutral pH. Comparison of the two sequences with BTV serotype 10 revealed amino acid identity of 35% between the product of segment 2 and BTV VP3, and 21% between the product of segment 7 and BTV VP7. The core proteins therefore show evidence of significant evolutionary divergence compared with that shown between different insect-borne orbiviruses. In particular, the amino terminus of BRD virus VP7 differed markedly from the equivalent region in VP7 of BTV and African horse sickness virus. This region is thought to interact with the outer capsid layer of insect-borne orbiviruses.

The haemagglutinin—esterase gene (HE) of bovine coronavirus (BCV) encodes a major viral membrane glycoprotein that elicits BCV-neutralizing antibodies. The BCV HE gene was cloned into a human adenovirus serotype 5 (Ad5) transfer vector in place of early transcription region 3, and a helper-independent recombinant virus was constructed by rescue of the transcription unit by homologous in vivo recombination between the vector and Ad5 genomic DNA. The BCV HE polypeptide expressed by this recombinant Ad was characterized in vivo and in vitro. A 65K polypeptide was identified using an anti-BCV antibody in both human (293) and bovine (MDBK) cells infected with the recombinant Ad. In the absence of a reducing agent, migration of the 65K polypeptide was shifted to 130K, indicating that the recombinant HE polypeptide existed in a dimeric form. The HE polypeptide was glycosylated, as demonstrated by labelling with [3H]glucosamine, and was immunoreactive with three distinct groups of conformation-specific anti-HE monoclonal antibodies (MAbs). Cells infected with recombinant Ad expressing BCV HE exhibited both haemadsorption activity and acetylesterase activity. In addition, the anti-HE group A MAbs HC10-5 and KD9-40 inhibited both the haemadsorption activity and esterase activity of the recombinant HE polypeptide, suggesting that the antigenic domain responsible for BCV neutralization may overlap (or is closely associated with) the domain(s) responsible for haemagglutination and/or acetylesterase activities. When mice were inoculated intraperitoneally with live recombinant Ad, a significant level of BCV-neutralizing HE-specific antibody was induced. These results indicate that the recombinant Ad replicates and directs the synthesis of the BCV HE polypeptide in vivo.

The in vitro viral lymphoproliferative response of pigs vaccinated against foot-and-mouth disease virus (FMDV) has been characterized. Peripheral blood mononuclear cells from immunized animals up to 1 year post-immunization (p.i.) showed a time-dependent FMDV-specific response, as assayed by virus-specific cellular blastogenesis. The optimum viral concentration decreased with time (around 20 weeks p.i.), and the response was faster and weaker. Lymphoproliferation appeared to be mainly due to CD4+ T cells. The response was heterotypic, being induced by all FMDV serotypes tested (C, A and O) after only two vaccinations with FMDV of serotype C (C-S8). Each individual structural protein assessed (VP1, VP2 and VP3) induced proliferation, with VP3 and VP1 being more effective stimulators. In vitro serum neutralization activity and FMDV-specific IgG production were found to be active even at 1 year p.i.

The complete nucleotide sequence of the sixth largest segment of ssRNA (RNA-6) of the tick-borne orthomyxo-like Dhori/India/1313/61 virus was determined by using cloned cDNA derived from infected cell mRNA and dideoxynucleotide sequencing of viral RNA. RNA-6 contains 962 nucleotides and is predicted to encode a protein of 270 amino acids with an M r of 30498 in its first open reading frame (ORF). This protein is likely to represent the viral membrane (M1) protein, based on its predicted M r of 29000 (estimated by PAGE), its relatively high abundance in infected cells and amino acid composition analysis. In addition, a second ORF was found which overlaps the M1 protein gene sequence by 327 nucleotides. This additional reading frame, in the +3 frame, potentially can encode a protein of 141 amino acids. However, S1 nuclease analysis of RNA-6 mRNA from infected cells indicated that there was only a single abundant RNA species corresponding in size to the full-length genomic RNA (0.96 kb). Further studies are needed to determine whether expression of the second ORF occurs and how that expression might arise.

Measles virus (MV) has a very limited host range, humans being the only natural reservoir of the virus. This restriction may be due to the absence of an MV receptor on the surface of non-primate cells. We have studied the MV-binding ability of several cell lines and attempted to characterize the receptor by studying the binding of 35S-labelled MV and by a rosette formation technique. We confirmed that all the human cell lines examined (HeLa, Raji and Jurkat) bound MV and that the murine cell lines (BW and L) did not. The glycoprotein nature of the receptor activity was demonstrated by the fact that it could be removed from the cell membrane using proteolytic enzymes and by its failure to be re-expressed in the presence of a protein synthesis inhibitor or an N-glycosylation inhibitor. A monoclonal antibody isolated after immunization of mice with Raji cells specifically inhibited MV binding and infection of human cells, and recognized human and simian but not murine cells. Depending on the cell line (HeLa, Raji, Jurkat or Vero), this antibody immunoprecipitated one or two glycoproteins with apparent M rs of 57K and/or 67K from human and simian cells, but not from murine cells.

The location of the epitope recognized by monoclonal antibody (MAb) 63G on the primary structure of the human respiratory syncytial virus G glycoprotein was determined by testing the reactivity of synthetic peptides with the MAb. The role of individual amino acids in this epitope was determined by using a set of 13-mer peptides containing sigle residue deletions. Residues 204 to 209 were found to be essential for antibody binding. These results are in full agreement with previous sequence data for escape mutants selected with MAb 63G. Several peptides, free or bound to keyhole limpet haemocyanin (KLH), were used to raise antisera in rabbits. The antipeptide antibodies reacted with the G protein in Western blots. However, only peptide G1-KLH (residues 187 to 200 bound to KLH) induced antibodies that reacted with the intact G protein and inhibited infectivity. These findings are discussed in terms of the antigenic structure of the G glycoprotein and the molecular engineering of peptide antigens.

We examined the effect of DNA template topology on the transcription of immediate early (E1a), early (E1b) and late (pIX) adenovirus genes in vitro. Transcription in whole cell extracts was measured by quantitative hybridization to end-labelled DNA and protection of hybrids from S1 nuclease digestion. Two- to fourfold more E1a RNA was synthesized from supercoiled, compared to linear, DNA templates. Similarly, transcription of the E1b gene was stimulated three- to sevenfold when the template was supercoiled. In contrast, RNA synthesis from the late pIX gene was found to be independent of DNA topology. These results show that DNA topology affects transcription in a promoter-specific manner.

Amplification of bovine papillomavirus type 1 (BPV-1) DNA in growth-arrested mouse cell cultures appears to mimic the process of induction of vegetative BPV-1 DNA synthesis in cells of the stratum spinosum in productively infected bovine warts. In both cases, cells permissive for viral DNA amplification express large amounts of viral E2 protein which accumulates within the cell nucleus. Whereas in latently infected virus-transformed cells truncated transcriptional repressor forms of E2 predominate, our previous studies have demonstrated that the full-length E2 transcriptional trans-activator protein is preferentially expressed during the period of maximal BPV-1 DNA amplification in growth-arrested cell cultures. To investigate the role of the full-length E2 gene in the induction of viral DNA amplification in this experimental viral replication system we have used a mutant BPV-1 genome (BPVE2-ts1) containing an E2 gene which is temperature-sensitive (ts) for transcriptional trans-activation. This mutant genome has also been shown to be ts for stable viral plasmid DNA replication and for the induction of cell transformation. We show here that viral DNA amplification was not severely impaired when BPVE2-ts1-transformed cells were tested at the restrictive temperature, indicating that the transcriptional trans-activating function of E2 was not essential for viral DNA amplification in division-arrested cells and, moreover, that the trans-activation and replication functions of E2 were separable. Consistent with this hypothesis, amplification of the BPVE2-ts1 genome at the restrictive temperature was still associated with the accumulation of large amounts of nuclear E2 antigen, showing that the mutation did not disrupt nuclear transport or render the E2 protein highly unstable. Furthermore, C127 cells harbouring ts E2 and full-length E1 expression constructs supported transient plasmid replication of a BPV origin vector at the restrictive temperature. These observations imply that E2 functions primarily as a viral replication factor in the vegetative phase of BPV-1 DNA replication, and suggest a fundamental difference in the genetic regulation of stable BPV-1 plasmid DNA replication in mitotic cells and viral DNA amplification in postmitotic cells.

Human papillomaviruses (HPVs) are a heterogeneous group of small dsDNA viruses which cause a variety of proliferative epithelial lesions at specific anatomical sites. Although more than 65 different virus types have been cloned and characterized, no uniform classification system exists. In order to classify HPV DNA types, phylogenetic trees were constructed based on nucleotide sequence alignments using parsimony and distance matrix algorithms. The resulting phylogenetic trees provide a classification of the HPVs into specific groups encompassing the known tissue tropism and oncogenic potential of each HPV type. The implications of a phylogenetic taxonomy on the diagnostic detection of HPVs and the concept of different HPV species are discussed.

We have derived a restriction enzyme map for the fowlpox virus FP9 strain. Sites for BamHI, PvuII, PstI and NcoI have been mapped mainly by Southern blotting. The size of the genome derived from the restriction maps (254 kb) corresponds to the figure of 260 ± 8 kb determined from analysis of genomic DNA by pulsed-field electrophoresis. The map can be compared with a previously published map for a different strain of fowlpox virus using the PstI digest which is common to both studies. Some 65 kb of fowlpox virus sequence, in 11 blocks, as well as individual M13 clones have been aligned with the map. Where those blocks correspond with blocks of homologous genes in vaccinia virus, it is possible to compare the genomic locations for those genes in the two viruses. This comparison reveals that, whereas there are blocks of sequence within which genes exist in the same relative position in the two viruses, the genomic location of those sequence blocks differs widely between the two viruses.

A 610-bp region of the JC virus (JCV) genome sequenced from brains of 11 progressive multifocal leukoencephalopathy (PML) patients contains 20 sites of point mutations that allow reliable classification of JCV isolates into two types. These type-determining sites were located in the region extending from position 2131 in the VP1 gene, through the intergenic region, to position 2740 in the T antigen gene. At these 20 sites the presence of different nucleotides creates two distinct patterns of substitution, with six isolates having the Type 1 pattern and five having the Type 2 pattern. Only four of the 11 isolates had ‘crossovers’ to the opposite type consensus DNA sequence at a small number of sites, indicating a very high type specificity. Additionally, three type-determining sites occur in the non-coding region to the left of the origin of replication. Other mutations occurred at random sites, making each strain unique, although one strain, 105, is identical to the Type 1 consensus. The JCV prototype strain Mad-1 was found to be Type 1 and differs from the consensus sequence at five sites. The other previously sequenced JCV strain, GS/B, is Type 2. At three sites out of five in the T antigen C terminus there is a type-specific amino acid substitution; however, none of the type-determining mutations in the VP1 gene cause an amino acid substitution. Comparison of each type’s consensus DNA sequence to that of BK virus suggests that Type 2 represents the ancestral JCV sequence from which Type 1 diverged during human evolution.

Brefeldin A (BFA) was found to interfere with specific events of human cytomegalovirus (HCMV) maturation in human fibroblasts. Ultrastructural as well as biochemical studies suggested that short-term exposure of infected cultures to BFA during the late infectious cycle primarily prevented Golgi-dependent processes, e.g. envelopment of naked cytoplasmic nucleocapsids in the trans-Golgi network (TGN) and normal processing of glycoprotein B. In contrast, the nuclear phase of viral morphogenesis, e.g. transport budding at the nuclear envelope, was not impaired. These observations were compatible with the interpretation that HCMV morphogenesis may involve sequential budding events at the nuclear envelope and at cisternae of the TGN. BFA treatment during the early infectious cycle efficiently inhibited HCMV-DNA synthesis and thus late viral functions, preventing production of viral progeny. Cytotoxicity was excluded as a cause for these findings.

The human cytomegalovirus (HCMV) UL75 gene product is the homologue of herpes simplex virus type 1 (HSV-1) glycoprotein H (gH), a virion glycoprotein that is essential for infectivity and which is conserved among members of the alpha-, beta- and gamma-herpesviruses. It has previously been shown that HSV-1 gH forms a stable complex with HSV-1 gL, the product of the UL1 gene, and the formation of this complex facilitates the cell surface expression of gH. None of the open reading frames within the HCMV genome encode a product with discernible sequence homology with HSV-1 gL, but an examination of the arrangement of conserved genes in HCMV suggested that the UL115 gene is a ‘positional homologue’ of HSV-1 UL1 which, like UL1, encodes a small secreted glycoprotein. Co-expression of HCMV gH (the UL75 gene product) and the UL115 gene product revealed that these proteins form a disulphide-linked complex and that the formation of this complex results in cell surface expression of gH. This complex is analogous to the gH:gL complex of HSV-1 and the HCMV UL115 gene product is therefore the functional homologue of HSV-1 gL.

Monoclonal antibody (MAb) E-13 to human cytomegalovirus is used widely for diagnostic and fundamental studies, and has been shown to be directed against an immediate early (IE) protein(s). To determine which viral antigen is detected by MAb E-13, four subfragments from the open reading frame encoded by exons 2, 3 or 4 of IE-1 were cloned in the bacterial expression vector pROS. The resulting fusion proteins contained amino acids 77 to 491 encoded by mainly exon 4, amino acids 25 to 78 encoded by exon 3, amino acids 1 to 85 encoded by exons 2 and 3, and amino acids 1 to 24 encoded by exon 2. The reactivity of MAb E-13 with the fusion proteins was assayed by Western blotting. MAb E-13 was shown to react exclusively with proteins encoded by exon 2 and therefore recognizes IE proteins which contain the N-terminal amino acid sequence encoded by exon 2, namely the major 72K IE protein, the 82K to 86K IE-2 protein and the 52K to 55K IE-2 protein. MAb E-13 can be used to detect both IE-1- and IE-2-encoded proteins, which share the polypeptide encoded by exon 2.

The neutralization of human cytomegalovirus (HCMV) after adsorption to the cell surface at 4 °C was studied using two neutralizing monoclonal antibodies (C-23 and C-41) recognizing glycoprotein 130/55. HCMV adsorbed to cells was neutralized by C-23 (complement-independent), but not by C-41 (complement-dependent). Furthermore, the virus remained sensitive to C-23 for 120 min after shifting up from 4 °C to 37 °C, suggesting that C-23 might block an early stage of virus penetration into cells, and also that transition from virus attachment to virus penetration might be quite slow. The cell-to-cell infection of HCMV was also blocked only by C-23, and not by C-41. On the basis of the results presented here, we suggest that C-41 blocks the attachment of virus to the cell surface whereas C-23 prevents the penetration of virus into the cell.