Identification and characterisation of herpes simplex virus genes required for encapsidation of DNA (1989)

The aim of the study presented in this thesis was to characterise two herpes simplex virus type 1 (HSV-1) DNA positive temperature sensitive (ts) mutants, tsl233 and ts1201, and the genes in which their mutations lie. Electron microscopic examination of thin section preparations of ts1233-infected cells revealed that at the non-permissive temperature (NPT) the nuclei contained large numbers of partially-cored capsids. In contrast to wt virus-infected cells, no dense capsids or empty capsids were detected in the nuclei of ts1233-infected cells at the NPT. This result suggests that the mutant has a block in the assembly of full nucleocapsids. The effect of the ts1233 mutation could not be reversed when mutant virus-infected cells were shifted from the NPT to the permissive temperature (PT) in the presence of a protein synthesis inhibitor. Southern blot analysis of total and encapsidated DNA confirmed that ts1233 failed to encapsidate DNA at the NPT, and showed that the DNA synthesized by ts1233 at the NPT was in an endless state. This information suggested that most of the mutant DNA was in the form of high moleculer weight (mw) concatemers at the NPT. Previous work had located the ts mutation of ts1233 within EcoRI o. Complementation experiments between ts1233 and another HSV-1 mutant tsN20, which also had a lesion in HSV EcoRI o, showed that ts1233, belonged to a different cistron from fsN20. The polypeptide profile of ts1233-infected cells was similar to that of wt virus-infected cells. In contrast to the mutant ts1201, ts1233 processed the structural protein UL26 gene product normally and therefore, the gene in which ts1233 maps is not required for the processing of UL26 gene product. Marker rescue experiments localised the lesion in ts1233 within a 150bp fragment which contains the 5' ends of two genes, UL32 and UL33 oriented in opposite directions. UL32 encodes a 64,000 mw polypeptide and UL33 encodes a 14,000 mw polypeptide. The nucleotide sequence of a 392 base pair (bp) fragment from ts1233 and two ts+ revertants for growth, isolated during this study, was determined. Sequence analysis revealed that ts1233 had a single bp change at residue 69210 of HSV-1 DNA nucleotide sequence within gene UL33. The alteration resulted in the substitution of an isoleucine by an asparagine codon. The nucleotide sequence of the revertants in this region was identical to that of wt virus DNA. The nature of the mutation in ts1233 is consistent with the use of UV-light as a mutagen. Two oligopeptides, one representing a portion of the amino-terminus and the other representing a portion of the carboxy-terminus of UL33 amino acid sequence were synthesised and coupled either to bovine serum albumin (BSA) or to B-galactosidase and injected into rabbits. Antibodies against the peptides were detected by radioimmunoassays. No virus specific bands were detected when the antisera were reacted with virus infected cell extracts on western blots, however, immunoprecipitation experiments with virus-infected cell extracts and the antisera gave a very weak specific reaction with a polypeptide of the apparent mw predicted for UL33 gene product. Attempts to express the UL33 gene product in bacterial expression vectors were unsuccessful. The UL33 gene product was also placed under immediate-early (IE) gene regulation. The IE promoter and upstream regulatory sequence of Vmw175 were inserted in front of the UL33 gene and the UL33 gene containing IE promoter recombined into TK gene of tsK virus, which has a defect in Vmw175. Although novel bands were detected in cells infected with tsK recombinant virus at the NPT, further work is required to determine whether any of these bands are the UL33 gene product. Ts1201, like ts1233 fails to encapsidate DNA at the NPT. Sequence analysis of the 673bp fragment in which the ts1201 lesion mapped revealed that the mutation lies 89bp upstream from the amino terminus of UL26. A single bp change was found at a position corresponding to residue 50897 of HSV-1 11 syn+ nucleotide sequence. This resulted in the substitution of tyrosine with phenyl alanine codon. Both the ts+ revertants analys ed retained the ts1201 mutation and had second site reversions elsewhere within UL26 gene. Three oligopeptides, one representing 9 amino acids at the amino terminus of UL26, one representing 12 amino acids from the second potential AUG, and one representing 14 amino acids of the carboxy terminus were synthesised, coupled to B-galactosidase and injected into rabbits. The antisera all contained oligopeptide antibodies that recognised the peptides which they were raised against. However, in western blot experiments only antibodies against the carboxy terminus of UL26 gene product reacted with a specific virus band in virus infected cell extracts (Abstract shortened by ProQuest.).