Lock, Matthew James; (2000) Quantitation of human herpesvirus 8 load and functional analysis of the viral thymidine kinase. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Human herpesvirus 8 (HHV-8) is the latest member of the Herpesviridae to be described, following its discovery in Kaposi's sarcoma tissue from AIDS patients, in 1994, by Chang et al. using representational difference analysis (RDA). Qualitative and quantitative-competitive PCR (QCPCR) methods were developed for the detection and quantification of HHV-8 DNA in patient samples. In a study looking at various post-mortem tissues of AIDS patients 32/153 (21%) tissues were found to be positive for HHV-8, whereas 0/47 control tissues were positive. HHV-8 viral loads were shown to vary from <10^1 to 10^5.6 genome copies/μg DNA. There was a significant difference in the viral loads between patients with KS and patients without KS, however there was no significant difference in HHV-8 load in tissue samples when correlated with HIV proviral DNA presence or load. In order to gain further insight into the molecular mechanisms that govern the antiviral susceptibility of HHV-8, functional studies of the HHV-8 thymidine kinase (TK) homologue were performed. The recombinant HHV-8 TK was shown to be functional for the phosphorylation of deoxythymidine. Sequence homology of the HHV-8 TK with the HSV TK identified three amino acid residues which may be of importance to the TK function, that were targeted for mutagenesis. These mutants were expressed and shown to have limited or no thymidine kinase activity. The affinity of the HHV-8 TK for known antiviral drugs was examined. Inhibition studies demonstrated that the anti-herpesvirus drugs GCV and ACV were unable to inhibit phosphorylation of dT by HHV-8 TK, whereas the anti-HIV drugs AZT and d4T, and the nucleoside analogue BrdU were competitive inhibitors of dT phosphorylation. In addition, AZT and d4T were shown to be phosphorylated by the HHV-8 TK, however GCV was not. In conclusion, established anti-herpetic agents are extremely poor substrates for the HHV-8 TK, although agents used for the treatment of HIV infection both inhibit the HHV-8 TK and are phosphorylated by its action.

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