Analysis of the HSV-1 UL2 and UL1 Genes by Insertional Mutagenesis

Mullaney, James (1990) Analysis of the HSV-1 UL2 and UL1 Genes by Insertional Mutagenesis. PhD thesis, University of Glasgow.

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Insertional mutagenesis has been used to investigate the functional role of the HSV-1 UL1 and UL2 gene products in the lytic cycle of the virus. The genes were mutagenised by disruption of the protein coding sequences, which was achieved by inserting a segment of foreign DNA into a plasmid-cloned copy of the target gene. The insert contained a marker gene, the Escherichia coli LacZ gene under the direction of the SV40 early promoter, and was tranferred into the wt genome by homologous recombination of the flanking sequences. Recombinant progeny were isolated by the addition of X-gal, a chromogenic substrate for B-galactosidase (the product of the LacZ gene) to an agar overlay. Plaques of recombinant virus, which appear blue using this screening technique, were subjected to plaque purification. This technique was used in attempts to construct three different recombinants. In all three cases, the cotransfection gave rise to blue plaques, and in two cases the recombinants were purified. The first of these (designated in1601) carried the insert in the coding region of the gene UL2 as verified by restriction endonuclease analysis. Earlier mapping studies had indicated a possible function for the product of this gene, namely uracil-DNA giycosylase, a DNA repair enzyme which appears to be ubiquitous. Enzyme assays performed on extracts of tissue culture cells infected with inl601 were consistent with this assignation, as they demonstrated that in1601-infected extracts had only mock-infected levels of uracil-DNA glycosylase activity, but wt levels of three other virus-induced enzymes. Final confirmation came with comparison between the amino acid sequence of the UL2 gene product and the amino acid sequences translated from other known uracil-DNA glycosylase genes (E. coli, Saccharomyces cerevisiae and human). Immunoblotting analysis, using antisera raised against a synthetic oligopeptide representing the carboxy terminus of the protein, showed a reaction with a protein of approximate Mr 39,000, which is similar to enzymes from other sources. The mutant was readily isolated and possesses growth characteristics very similar to wt, indicating that the enzyme does not appear to be required for lytic growth, at least in the tissue culture system employed for growth and maintenance of the recombinant. This is in contrast with the observations that a high degree of amino acid conservation exists between the enzymes from different sources, and that the gene is found in all of the herpesviruses sequenced to date. The latter would tend to imply an important role in vivo for this enzyme. It is accepted that this in vivo function is the removal of uracil from DNA, brought about by deamination of cytosine. This is a potentially mutagenic event, and if left uncorrected, would result in a G.C to A.T transition mutation. The second recombinant which was purified was designated in1602 and was constructed by cotransfection of the mutated plasmid-borne copy of the UL2 gene with a virus containing an insertion in the gene encoding the virus induced dUTPase. This enzyme is thought to be responsible for minimising the misincorporation of dUTP into DNA during replication, by lowering the pool of dUTP. Although this also results in the presence of uracil in the DNA, it will not result in mutagenesis if left uncorrected, as subsequent replication will not alter the sequence of the DNA. Misincorporation of dUTP into DNA and in situ deamination of cytosine are the two major mutagenic events which result in the presence of uracil in DNA. Therefore the recombinant in1602 is deficient in both of these genes, as confirmed by restriction analysis, and in both of these activities, as confirmed by enzyme assays. The third recombinant, whose construction was attempted unsuccessfully, represented insertion of the LacZ sequences into the coding sequence of the gene UL1. Although the initial co-transfection also yielded blue plaques, these failed to grow through subsequent rounds of plaque purification. The co-transfection was repeated twice and repeated attempts were made to titrate the blue plaques obtained from the co-transfection, all without success. Control experiments done in tandem with these titrations indicate that the procedure was functioning normally. The two most likely explanations for this phenomenon are: (a) The recombinant was unstable due to other, unpredicted alterations to the genome. This has been observed in the construction of other recombinant viruses, but usually when the construction has involved duplication of virus DNA sequences, (b) There is a stringent requirement for the UL1 gene product in the replicative cycle of the virus. At present there is no direct evidence which would support either of these explanations at the expense of the other. Searches of amino acid sequence databases failed to identify any sequences which could be homologues of this protein, so there is no further functional information about this protein.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Virology
Date of Award: 1990
Depositing User: Enlighten Team
Unique ID: glathesis:1990-78103
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 30 Jan 2020 15:40
Last Modified: 30 Jan 2020 15:40

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