John H. Subak-Sharpe

Genetic Studies with Herpes Simplex Virus Type 1. The Isolation of Temperature-sensitive Mutants, their Arrangement into Complementation Groups and Recombination Analysis Leading to a Linkage Map

Summary Nine 5-bromodeoxyuridine-induced temperature-sensitive (ts) mutants were isolated from stocks of a syncytial plaque type mutant (syn) itself derived from the non-syncytial wild-type (syn +) of the glasgow strain 17 of herpes simplex virus type 1. The nine ts mutants have been assigned to eight cistrons on the basis of different complementation tests. Non-syncytial revertants isolated from the original ts syn mutants were used in reciprocal three-factor crosses of the type: tsX syn × tsY syn + and tsX syn + × tsY syn. With recombination data from these crosses a linkage map has been constructed. The provisional map which locates nine cistrons is linear and spans about 25 recombination units. The regular occurrence of plaques with mixed syn/syn + morphology in the progeny from crosses is reported.

Biochemical Studies on the Herpes Simplex Virus-specified Deoxypyrimidine Kinase Activity

Summary The response to various stimuli of herpes simplex virus-specified deoxypyrimidine kinase activity differs significantly from that of the thymidine kinase and deoxycytidine kinase of mammalian cells. However, the two phosphorylating activities of the HSV deoxypyrimidine kinase are themselves quite distinct in their biochemical behaviour. It has been impossible to separate the HSV-coded thymidine and deoxycytidine phosphorylating activities by their sedimentation, electrophoretic or size characteristics. The evidence suggests that both deoxypyrimidine nucleosides are phosphorylated at the same active site.

The polypeptide and the DNA restriction enzyme profiles of spontaneous isolates of herpes simplex virus type 1 from explants of human trigeminal, superior cervical and vagus ganglia.

Analysis of the infected cell polypeptides and the DNA restriction profiles of 31 HSV-1 isolates from the trigeminal, superior cervical and vagus ganglia from 17 individuals (12 U.S.A., 2 Japanese, 3 Norwegian) could be classified as 15 different virus strains. With the exception of the three Norwegian isolates which gave identical profiles, virus isolates from the ganglia of different individuals could all be distinguished from one another. In contrast virus isolates from the trigeminal, superior cervical and vagus ganglia of the same individual, or virus isolates from the left and right ganglia of the same individual or multiple isolates from different explants of a single ganglion were indistinguishable. In conclusion, a single virus strain infects each individual initially and virus descended from this event subsequently infects and becomes latent in different cells of the same ganglion as well as in different ganglia.

ISOLATION OF HERPES SIMPLEX VIRUS FROM HUMAN TRIGEMINAL GANGLIA, INCLUDING GANGLIA FROM ONE PATIENT WITH MULTIPLE SCLEROSIS

Herpes-simplex virus (H.S.V.) was isolated from 18 of 39 trigeminal ganglia (T.G.) obtained within 12 h of death. The virus was isolated from ten persons who had died of trauma, from one case of lymphoma, and from one case of multiple sclerosis. In the cadaver with histologically confirmed multiple sclerosis, large bilateral areas of demyelination were present near the points of entry of the nerve root, and the possibility that H.S.V. migration to the root entry zone caused demyelination cannot be excluded.

Resistance of herpes simplex virus to acycloguanosine—genetic and physical analysis

Abstract Resistance of herpes simplex viruses (HSV-1 and HSV-2) to acycloguanosine (ACG) is determined by the two genetic loci of HSV coding for deoxypyrimidine kinase (thymidine kinase) and DNA polymerase activity. Mutants of HSV-1 which induce defective deoxypyrimidine kinase (dPyK − mutants) activity can participate in interallelic complementation, and show variability in their resistance to acycloguanosine (ACG R ). This allows for subdivision of dPyK − mutants based on their resistance to ACG. Mutants dPyK − and dPyK −11 are in one interallelic complementation group and both are 100-fold more resistant to ACG in a plaque reduction assay than is wild type herpes simplex virus type 1 (HSV-1). (The 50% inhibitory concentrations (ID 50 ) are 6.0 and 7.0 μM of ACG compared to 0.06 μM .) Mutant MDK (Kit) which cannot complement any other mutant is 500-fold more resistant to ACG (ID 50 = 50 μM ). Other dPyK − mutants fall in between in their sensitivity to ACG. The map position of the mutations in the DNA polymerase locus which result in resistance to ACG ( acg r ) was located by correlating the ACG sensitivity of HSV-1IHSV-2 intertypic combinants with restriction endonuclease analysis of their DNA. The mutations acg r and paa r (phosphonoacetic acid resistance) are contained in the same 1.3 kbp region of DNA (map units 40.2 to 41.0). The level of sensitivity to ACG is type specific, differing in HSV-1 and HSV-2. This type specificity is determined by DNA sequences within map units 39.6 to 41.0. Thus the DNA sequences for type-specific sensitivity and resistance to ACG are shown to be distinct and clustered in the same region as the HSV DNA polymerase locus.

HSV molecular biology: general aspects of herpes simplex virus molecular biology.

Comparison of the herpes simplex virus type 1 (HSV-1) DNA sequence with that of other α, β and γ-herpesviruses, allied with molecular genetic studies have greatly increased understanding of the HSV genome and the functions encoded by individual virus genes and has facilitated the development of rational antiviral strategies. Here we review the coding content of the HSV-1 genome and identify: genes encoding structural components of the capsid, tegument or envelope; genes whose products are essential for growth in tissue culture; and genes that are conserved between members of the α, β and γ-herpesvirinae. The HSV lifecycle and the main regulation cascade is discussed and genes that present targets for antiviral intervention identified. The protein content of the infectious virion particle is reviewed and compared with that of two additional non-infectious HSV-related particles species (L-particles and pre-DNA replication particles (PREPs)). The potential of HSV-1 L particles and PREP particles as DNA-free HSV-1 vaccine candidates and the desirability of deleting specific gene products from live HSV vaccines is discussed.

Generation of anti-peptide and anti-protein sera effect of peptide presentation on immunogenicity

The technique of Fmoc chemistry has been applied successfully to the synthesis of branched peptides. The immunogenicity of branched peptides has been compared quantitatively with those of protein-conjugated and resin-linked peptides. Six different peptide sequences were used to immunise rabbits and both antipeptide and anti-protein titres were determined for each serum. The data show that the titres of sera from rabbits immunised with branched peptides were higher than those of rabbits immunised with protein-conjugated peptides which in turn were higher than those immunised with resin-linked peptides. The effect was demonstrated with two strains of rabbits.

DNA sequence of the gene encoding a major secreted protein of vaccinia virus, strain Lister

Infection of tissue culture cells with vaccinia virus results in the specific secretion of several polypeptides into the medium. Previous studies identified a protein of approximate Mr 35,000 (35K) which was secreted in large amounts at both early and late times after infection with the Evans strain. We now show that a related protein is secreted by the Lister strain but not by WR, Wyeth nor Tian Tan. The gene encoding the Lister strain 35K protein was mapped within the inverted terminal repeats of the genome. The DNA sequence of this region showed that the ends of this gene are very similar to previously published sequences flanking a gene of WR which encodes a protein of approximate Mr 7,500 (7.5K). Our results suggest that the 7.5K polypeptide of WR may have arisen as a result of a deletion event and is a truncated form of the 35K Lister protein. Site-directed mutagenesis demonstrated that the 35K secreted protein encoded by Lister is not essential for growth in tissue culture.

Genetic Interactions Between Temperature-sensitive Mutants of Types 1 and 2 Herpes Simplex Viruses

Summary It has been shown by infectious centre assay of mixedly infected cells that ts mutants of HSV type 1 and type 2 are able to complement each other in many, but not all combinations. This complementation pattern has some unusual and unexpected features. Progeny tests on virus from infectious centres showed the presence of both parental types and also that of recombinants some of which have a novel phenotype. Successive progeny tests demonstrated that a substantial proportion of genomes retain the potential for segregating with respect to the ts and or the plaque morphology markers. The one recombinant so far tested behaved as intermediate between types 1 and 2 in neutralization tests.

Latency Competence of Thirteen HSV-1 Temperature-sensitive Mutants

Thirteen temperature-sensitive (ts) mutants of HSV-1 were analysed for their capacity to establish latent infections in the brains of mice. Eleven of the mutants could be classified as latency-positive or -negative; two could not be assigned to either group. Leakiness of mutants in the brain and differences in particle/infectivity ratios were found not to play a role in the results. Ts+ revertants of selected latency-negative mutants regained the capacity to establish latent infections, indicating that it was the ts lesion in these agents which was involved in latency. Ultrastructural studies of neuroblastoma cells infected with various mutants and maintained at the restrictive temperature showed that no absolute correlations could be made between capacity to establish latent infection and synthesis of various morphologically identifiable virus products. Finally, from a comparison of latency characteristics with previously established polypeptide phenotypes of mutants it was concluded that one immediate early and one or more later virus functions are necessary for establishment and/or maintenance of the latent state.