- Volume 71, Issue 4, 1990

We have developed a cell-free system derived from measles virus-infected cells that supported the transcription and replication of measles virus RNA in vitro. The data suggest that tubulin may be required for these reactions, since an anti-β-tubulin monoclonal antibody inhibited viral RNA synthesis and the addition of purified tubulin stimulated measles virus RNA synthesis in vitro. Tubulin may be a subunit of the viral RNA polymerase, since two different anti-tubulin antibodies, one specific for the β- and another specific for the α-subunit of tubulin, coimmunoprecipitated the measles virus L protein as well as tubulin from extracts of measles virus-infected cells. Other experiments further implicated actin in the budding process during virus maturation, as there appeared to be a specific association of actin in vitro only with nucleocapsids that have terminated RNA synthesis, which is presumably a prerequisite to budding.

BioBreeding Worcester diabetes-prone (BBdp) rats develop insulin-dependent autoimmune-driven diabetes mellitus spontaneously and intravenous administration of 1 × 107 p.f.u. of lymphocytic choriomeningitis virus (LCMV) to young adult mice prevents disease. The virus is lymphotropic, binding to and replicating in such cells. BBdp rats fail to generate virus-specific major histocompatibility complex-restricted cytotoxic T lymphocyte (CTL) responses when challenged with this dose or other doses of LCMV, Pichinde virus or vaccinia virus. Yet such rats clear virus effectively and show no evidence of persistent infection. Associated with this clearance of virus and establishment of immunity is the production of neutralizing antibodies.

In contrast, diabetes-resistant (BBdr) rats generate virus-specific CTL responses. Furthermore LCMV binds to fewer lymphoid cells of BBdr rats (in comparison to those of BBdp rats) and replicates in fewer lymphocytes (by one order of magnitude) from these rats. Thus, unlike mice in which CTLs play a dominant role in the control of LCMV infection, BBdp rats do not overcome this infection via CTLs. In addition, both the BBdp rats and their BBdr counterpart may provide useful models for determining whether or how individual lymphocyte subsets function in the induction of CTL responses, for the analysis of virus-induced immunosuppression and for the use of viruses or their products as therapeutic modalities.

The dsRNA profiles of all nine African horsesickness virus (AHSV) serotypes were compared by agarose gel electrophoresis and PAGE. The agarose profiles were identical, but a unique profile was obtained for each of the nine serotypes by PAGE. Nine of the 10 dsRNA genome segments of AHSV-3 were cloned and the clones were used in dot-spot and Northern blot hybridization experiments to determine intra- and inter-serogroup nucleic acid similarities. Segments 1, 3, 4, 5, 7 and 8 were highly conserved in the AHSV serogroup and no genetic relationship with any of the other orbiviruses was observed. Of these segments 3, 5 and 8 showed the largest degree of cross-hybridization to the cognate genes of all the serotypes. These clones did not cross-hybridize to other orbiviruses such as epizootic haemorrhagic disease virus, bluetongue virus or equine encephalosis virus and are therefore recommended for use as group-specific probes for the identification of the AHSV serogroup. Genome segments 6 and 10 showed an intermediate degree of conservation, whereas segment 2 is serotype-specific and therefore probably codes for the outer capsid protein VP2.

Infection of human embryonic kidney cells with adenovirus type 5 (Ad5) induces aberrations (gaps and breaks) in the cell chromosomes. We have conducted a study utilizing a large number of Ad5 mutants to identify the viral functions that are responsible for the occurrence of cytogenetic damage. The results of our investigation have indicated that expression of the gene products of the Ad5 early region 1A (E1 A) is necessary for the induction of chromosomal aberrations and that other early viral gene products do not appear to contribute to this phenotype. We have also shown that expression of both the major E1 A gene products, the 243 amino acid and the 289 amino acid proteins, is required for induction of damage at wild-type levels, although the 289 amino acid protein appears to retain detectable activity on its own. Lastly, we have observed that deletions in the amino-terminal region of the E1 A proteins and in the transactivating domain of the 289 amino acid protein prevent the occurrence of cytogenetic damage, whereas mutations elsewhere in the proteins do not affect this process.

The SK-v cells, established from a premalignant vulvar lesion, contain human papillomavirus type 16 (HPV- 16) sequences integrated at a single cellular site and derive from a cell clone present in vivo. Transcription of the HPV-16 genome in SK-v cells was analysed by cDNA heteroduplex mapping and sequencing, and by RNase mapping. Viral sequences were shown to be transcribed into virus-cell fusion messengers. The two major transcripts have a coding capacity for a truncated E6 protein, an E7 protein and an E1-E4 fusion protein, but differ in their 3′ virus-cell junction. Minor transcripts have a coding capacity for a full-length E6 protein and another truncated version of E6. The transcription pattern in the E6-E7 region was found to be the same both in SK-v cells and in CaSki cells, a line derived from an invasive cervical carcinoma. Immuno- precipitation experiments showed that the E6 protein (18K) and, predominantly, the E7 protein (20K) are expressed in SK-v cells as in CaSki cells. The E7 protein was found in a two- to threefold lower amount in SK-v cells, but showing the same half-life (about 1 h).

Chicken anaemia agent (CAA) was purified using differential centrifugation and successive cycles of equilibrium density gradient centrifugation using sucrose and CsCl. The purification method was dependent on the use of an antigen-detecting ELISA based on a CAA-specific monoclonal antibody. Virus particles banded at a density of 1·33 to 1·34 g/ml in CsCl and measured 23·5 ± 0· 8 nm in diameter. Purified preparations contained one major polypeptide (M r 50000) and a single-stranded, circular DNA (2.3 kb). CAA shares some of the biochemical characteristics possessed by porcine circovirus and the virus associated with psittacine beak and feather disease.

The rat pheochromocytoma cell line, PC 12, undergoes neuron-like morphological, biochemical and electrophysiological differentiation, in the presence of low concentrations of nerve growth factor (NGF). NGF- treated PC 12 cells have been shown previously to support 139A scrapie agent replication. In the present report we extended these findings and analysed the cellular conditions necessary for agent replication. Following the infection of differentiated PC 12 cells, scrapie replicated to relatively high titres as determined by an incubation period assay. The removal of NGF, which causes the gradual dedifferentiation of PC 12 cells, resulted in the inability of scrapie to replicate. The scrapie infectivity detected in PC 12 cultures is cell- associated and not released into the medium. Cells in infected cultures did not show any change in morphology when compared to cells in mock-infected cultures. Titration studies of scrapie infectivity in PC 12 cells have indicated that up to 4 LD50 units per cell can be obtained although a yield of 1 LD50 per cell was more common. Using an approximate m.o.i. of 1, only differentiated PC 12 cells supported 139A scrapie agent replication when compared to two other differentiated, neuronal cell types, indicating that PC 12 cells are more susceptible to agent replication. These studies support further the suitability of using differentiated PC 12 cells as an in vitro model to study scrapie agent replication.

PrP is a glycoprotein found in normal brain. In brain affected by scrapie it forms scrapie-associated fibrils (SAF). PrP from SAF shows considerable heterogeneity of size and charge on two-dimensional gels. It separates into six major regions, the three more acidic regions arising as a result of partial proteolytic degradation. The two more basic higher M r forms (M r 34000 and 29000) of PrP can be reduced in apparent M r to a lower M r form (M r 25000) with Peptide-N- glycosidase F. In addition, a series of lectins has been found to bind to PrP. Some bind preferentially to the higher M r forms whereas others bind more strongly to the lower M r form. Some of the heterogeneity of PrP is therefore due to differential N-glycosylation. We suggest that one or two N-linked carbohydrate chains are bound to the protein causing some of the differences in M r The major cause of heterogeneity of PrP is therefore proteolytic cleavage combined with differential glycosylation at the two potential N-glycosylation sites. The glycolipid moiety attached to PrP may be responsible for some lectin binding to all three bands. Using lectins as a probe to study potential differences in N-glycosylation we have looked at their binding to PrP isolated from SAF, from different strains of scrapie and from different regions of the same brain. No major differences in the N-glycan moieties were found.

We have sequenced a 5·4 kb region of the genome from the avian herpesvirus infectious laryngotracheitis virus (ILTV) and identified genes homologous to the thymidine kinase (TK) gene, the capsid p40 gene of herpes simplex virus type 1 (HSV-1), a gene encoding a protein antigenic in Epstein-Barr virus infections plus one other highly conserved gene encoding a protein implicated in cell fusion in HSV-1. Computer analysis of the TK gene and the upstream overlapping gene sequence is used to produce trees showing potential evolutionary relationships between the herpesvirus subfamilies.

Vmw175, the product of herpes simplex virus type 1 immediate early (IE) gene 3, is essential for viral replication. It is required for the activation of transcription from both early and late gene promoters and also for the repression of IE gene expression. Vmwl75 is able to bind specifically to certain DNA sequences, some of which (including that at the cap site of IE gene 3) contain the consensus sequence ATCGTC. The presence of this sequence at the cap site has been correlated with the ability of Vmw175 to autoregulate its own promoter. This report describes the characterization of five viruses with temperature-sensitive (ts) lesions in Vmwl75. Four of these mutants express Vmw175 which is ts in its ability to bind to DNA in vitro and to autoregulate IE-3 gene expression in the infected cell. Although Vmw175 produced by the remaining mutant, ts1225, fails to autoregulate IE-3 expression at the non-permissive temperature (NPT) its DNA-binding properties are indistinguishable from those of the wild-type protein. This suggests that the ability of Vmw175 to bind to the IE-3 cap site (as measured in vitro) is insufficient for autoregulation (in vivo). All five newly characterized ts mutants are partially permissive for early gene transcription at the NPT, although Vmw175 expressed by four of them is unable to bind to the IE-3 cap site sequence at elevated temperatures. This suggests that binding to one class of recognition sequences by Vmw175, as measured in vitro, is not absolutely required for the activation of early gene promoters during virus infection. The lesions in these five ts mutants lie in the carboxy- terminal third of the polypeptide; three of the mutations (those in tsl219, ts1221 and ts1225) were identified by DNA sequence analysis and were found to affect amino acid residues that are conserved in the homologous proteins from varicella-zoster virus and pseudorabies virus.

Mouse L cell lines constitutively expressing glycoproteins B or D of herpes simplex virus type 1 (LTKgB and LTKgD respectively) were used to study the longevity of the immune response to these viral glycoproteins in mice. Two immunizations with the cell lines were necessary to induce a persisting antibody response (present for over 200 days). Only LTKgD induced a neutralizing antibody response in mice and this also remained at high titres over 200 days after two inoculations. The presence in mice of precursor cytotoxic T lymphocytes specific for gB expressed in the L cells was also shown up to 270 days after immunization. Mice immunized with the cell lines showed an increased rate of virus clearance from the ear pinna, inoculation with LTKgD resulting in more clearance than LTKgB at 7 days post-immunization. This type of protection was reduced with time after inoculation, until by day 161 there was no significant difference in virus titres between immunized and control groups. However, LTKgD immunization protected against the establishment of latent infections in the ganglia of mice even up to 186 days postinoculation.

The gene encoding the major envelope glycoprotein complex, gp55–116 (gB), of human cytomegalovirus (HCMV) was expressed at high levels in insect cells utilizing a recombinant baculovirus. The mature intracellular form of the insect-derived gp55–116 was a protein of M r 150K which contained approximately 50K of N-linked oligosaccharides. The oligosaccharide linkages were almost exclusively endoglycosidase H- sensitive. The 150K protein was processed, presumably by proteolytic cleavage, to yield at least one of the previously defined cleavage products of the gp55–116. This processing step was significantly less efficient in insect cells than the analogous step in mammalian cells. Finally, the insect-derived gp55–116 was highly immunogenic in experimental animals and readily recognized by antibodies contained within HCMV- immune human serum, suggesting that this recombinant protein warrants further study as a potential HCMV subunit vaccine candidate.

A panel of 11 monoclonal antibodies (MAbs) raised against pseudorabies virus (PRV) was used to map epitopes on the virus glycoprotein I (gl). We employed three approaches to map epitopes on gl. By a competition binding assay, six groups of MAbs were defined as reacting with distinct antigenic domains on gl. To identify regions along the gl polypeptide chain encompassing the domains recognized by these MAbs, DNA fragments derived from the gI-coding region were cloned into pEX expression plasmids. The antigenic reactivity of the fusion proteins expressed in Escherichia coli was analysed by immunoblotting. Five antigenic domains were mapped within the first 238 amino acids of gl: domains A, B and D were mapped between amino acids 52 and 123 and domains C and E between amino acids 78 and 238. One MAb, representing domain F, did not react with the expressed fusion proteins. To assess the precise location and amino acid sequences of the epitopes, overlapping nonapeptides covering the amino acid sequence 52 to 238 were synthesized. The antibody-binding activity of these peptides was tested by an ELISA (Pepscan-method). Three antigenic domains were mapped: domain A was localized to amino acids 64 to 73 and 75 to 84, domain B to amino acids 52 to 67 and domain D to amino acids 68 to 82. Four MAbs representing antigenic domains C, E and F did not react in the Pepscan. Finally, sera from pigs infected experimentally with PRV reacted with the fusion protein of plasmid ps1 (amino acids 52 to 238).

We have previously shown that the herpes simplex virus type 1 (HSV-l)-specified glycoprotein C (gC-1) produced in epitheloid cells contains epitopes of peptide nature, which are dependent on galactose of oligosaccharides for their expression. In the present communication we report that these epitopes are expressed in a mouse neuroblastoma cell line (Cl300) with low levels of galactosyl transferases. However, in place of galactose the glycoprotein from Cl300 cells was found to contain oligosaccharides with additional fucose units. Fucosidase treatment, but not galactosi-dase treatment, abolished the antigenic activity of the carbohydrate-dependent epitopes. Altogether the results indicated that the carbohydrate-dependent epitopes of gC-1 from Cl300 cells were stabilized by peripheral sugars of N-linked oligosaccharides rather than O-linked ones and that fucose could substitute for terminal galactose in promoting the activity of the carbohydrate-dependent epitopes. This is the first demonstration of the involvement of fucose in the establishment of a carbohydrate-dependent epitope of peptide nature. The results also demonstrated that reversible carbohydrate-peptide interactions were responsible for the activity of the carbohydrate-dependent epitopes.

The cis-acting DNA sequences and trans-acting proteins that control the expression of the major immediate early (IE) gene of varieella-zoster virus (VZV) were investigated. The location of the IE mRNA 5′ terminus was determined by primer extension and S1 nuclease analyses and the functional activities of DNA sequences upstream of this site were analysed by a transfection assay. The VZV IE promoter exhibited low activity in BHK and HeLa cells, but was transactivated by the herpes simplex virus type 1 (HSV-1) virion protein Vmw65. DNA sequences between positions −131 and +57 were responsible for promoter activity, whereas sequences between −410 and −131 mediated the response to Vmw65. Two short elements in the −410 to −131 region formed protein-DNA complexes with HeLa cell nuclear proteins and formed a ternary complex when Vmw65 was added. One of the elements, ATGTAAATGAAAT, possessed a strong similarity to the HSV-1 TAATGARAT. The VZV homologue of Vmw65, encoded by open reading frame (ORF) 10, failed to trans-activate expression from HSV-1 or VZV IE promoters and did not form a ternary complex with functional TAATGARAT elements and HeLa cell proteins. Therefore, stimulation of VZV IE transcription by Vmw65 can occur by a mechanism similar to that employed by HSV-1, but VZV ORF 10 does not function as a trans-activator of IE gene expression.

Replication of varicella-zoster virus (VZV) was studied in a range of human cells (Chang conjunctival, HEp2 and Vero) and compared with that in HEL cells. Evidence for virus-specific protein and nucleic acid synthesis was obtained from each of these cell lines. In some cell lines, virus proteins were clearly visible by polyacrylamide gel electrophoresis both with and without immunoprecipitation. Multiple passage of infected cells enhanced both virus-specific protein synthesis and shutoff of host protein synthesis. No qualitative differences in the patterns of VZV-specific protein synthesis were detected. Approximately 2000 copies of the VZV genome were detected per infected HEL cell.

In peripheral sensory ganglia latently infected with herpes simplex virus type 1 (HSV-1) transcription is restricted. A set of viral latency-associated transcripts, the LATs, have been characterized by Northern blotting and in situ hybridization. These transcripts have previously been mapped to a 3 kb region of the viral genome within the repeat long region. However, transcription from adjacent regions of the genome can be detected by in situ hybridization, which cannot be detected by Northern blotting. These RNAs are termed minor LATs or m-LAT. In this study we show that in ganglia latently infected with the HSV-1 variant 1704, which is deleted in one complete copy of the LAT gene and in the promoter and 5′ portion of the other copy, m-LATs are not detected by in situ hybridization. Furthermore, the levels of DNA in nervous system tissue latently infected with the parental and the 1704 variant virus are similar. Thus we propose that the sequence elements necessary for initiating transcription or stabilizing m-LATs are within the region deleted in variant 1704 that codes for the promoter and the 5′ end of the LATs.

A 5 bp insertion was introduced into the BstEII site at nucleotide 2815 in DNA of hepatitis B virus (HBV) and a mutant HBV genome was produced, which coded for envelope and core proteins, but not for DNA polymerase, due to a frameshift. Cultured hepatoma cells (HepG2) were simultaneously transfected with a plasmid harbouring a tandem dimer of the mutant HBV DNA and another plasmid harbouring a tandem dimer of DNA of woodchuck hepatitis virus or duck hepatitis B virus. The replication of mutant HBV DNA, incapable of encoding DNA polymerase, was accomplished by cotransfecting woodchuck hepatitis virus DNA, but not by duck hepatitis B virus DNA. These results indicated a trans-complementation of the C and P genes in mammalian hepadnaviruses beyond a species barrier.