Inhibition of Herpesvirus Ribonucleotide Reductase by the Synthetic Nonapeptide Yagavvndl

Virology

Telford, Elizabeth Anne Reid

University:University of Glasgow

关键词: Virology;   

Others  :  http://theses.gla.ac.uk/77967/1/10999285.pdf

来源: University of Glasgow

【 摘 要 】

Ribonucleotide reductase catalyses the reduction of all four ribonucleotides and is a key enzyme in the de novo synthesis of DNA precursors. A novel ribonucleotide reductase activity is induced in cells infected with herpes simplex virus. The virus-induced ribonucleotide reductase is encoded by the virus and has biochemical properties which are distinct from that of the host cell enzyme and therefore is a potential target for antiviral chemotherapy. The HSV-l-encoded ribonucleotide reductase is composed of two non-identical subunits, termed RRl and RR2, which are dimers of Vmwl36 and Vmw38 polypeptides respectively. Previously it has been shown that a synthetic nonapeptide (YAGAVVNDL) corresponding to the carboxy-terminal nine amino acids of the HSV-l-encoded RR2 subunit inhibits the HSV-l-induced enzyme. The peptide prevents the association of RR2 with RRl by competing with RR2 for a site of interaction on RRl. The inhibitory activity of the nonapeptide was found to decrease upon prolonged incubation with enzyme extract and this observation prompted studies to determine the fate of the nonapeptide in cellular extracts. The synthetic nonapeptide was iodinated with non-radioactive iodine, purified by high pressure liquid chromatography on a reverse phase column and the iodinated peptide was then tritiated by reduction with tritium gas (Amersham International PLC). In a linear gradient increasing from 5% to 95% acetonitrile + 0.1% TFA between 0 and 10 minutes the tritiated peptide eluted from the column at 7 minutes. However, when the tritiated peptide was mixed with HSV-l-infected cell extract, a new species was identified which eluted from the column at 5 minutes. This new species was thought to be a product of cleavage of the nonapeptide by proteases present in the cellular extract. The modified product was purified by HPLC on a reverse phase preparative column. A second modification product was observed using this column and was also purified. The purified modification products were characterised by analysis of amino acid content and relative molecular mass. The results suggested that the synthetic nonapeptide is cleaved into the octamer AGAVVNDL and free tyrosine. This was verified by the synthesis of the octapeptide and the demonstration that it and authentic tyrosine coeluted with the modification products. The octapeptide has a markedly reduced inhibitory potency and therefore a means of protecting the tyrosine-alanine bond from such degradation would be necessary in any compound based on the nonapeptide for it to be useful as an antiviral agent. A range of protease inhibitors were tested to determine whether any could protect the nonapeptide from degradation. Of those tested, only bacitracin offered substantial protection from breakdown of the nonapeptide. Serological evidence for conservation of the carboxy-terminal region of HSV-1 in HSV-2, VZV, PRV and EHV-1 made it likely that the nonapeptide might inhibit a broad range of herpesvirus ribonucleotide reductase activities. This proposal was tested on the novel ribonucleotide reductase activity induced in cells infected with one of two different strains of abortogenic EHV. Rabbit kidney cells were infected with EHV-1 strain Vol939 or strain Kentucky A and virus-induced ribonucleotide reductase activity was partially purified by ammonium sulphate precipitation. The EHV-l-induced ribonucleotide reductase activity was assayed in the presence of various concentrations of the nonapeptide. The results showed the peptide to be inhibitory. Parallel experiments with the HSV-l-induced ribonucleotide reductase established that the concentration of peptide required to inhibit 50% of reductase activity was the same for both enzymes. This data and the finding that the PRV-induced enzyme is inhibited by the nonapeptide support the proposition that an antiviral drug based on the nonapeptide might have activity against a broad range of herpesviruses. The large subunit of ribonucleotide reductase encoded by HSV (RRl) possesses an amino-terminal domain not found in the large subunit of other herpesvirus reductases or reductases from other organisms. It has previously been shown by other workers that truncated forms of the HSV RRl exist which can form a functional enzyme. Oligopeptide-induced antisera were raised against the unique amino domain and the carboxy-terminal domain of HSV-l-induced RRl and these antisera were used by Dr H. Lankinen in experiments to demonstrate that at least part of the unique amino-terminal domain is not necessary for enzyme activity. The antisera which were raised against the HSV-l-induced RRl were used in immunoblotting experiments with EHV-l-infected cell extracts. Antisera directed against the carboxy-domain of RRl reacted specifically with a viral protein Mr 90K. Antisera raised against peptides corresponding to sequences in the unique amino-terminal domain of the HSV-1 RRl failed to react with any viral protein. These results suggest that the EHV-l-induced RRl does not contain the amino-terminal region which is so far unique to HSV-1 and HSV-2.