Xba I Site Loss Mutants and Deletion/Duplication Variants of Herpes Simplex Virus Type 1: Isolation, Characterization and Recombination Studies

MacLean, Alasdair Roderick (1988) Xba I Site Loss Mutants and Deletion/Duplication Variants of Herpes Simplex Virus Type 1: Isolation, Characterization and Recombination Studies. PhD thesis, University of Glasgow.

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The aim of this project was to isolate a herpes simplex virus type 1 Glasgow strain 17 genome lacking all four Xba I restriction enzyme sites and to use the sites as non-selected markers to study intratypic HSV recombination. However, a large part of the work involved the analysis of variant genomes which were identified during the isolation of Xba I site negative viruses. The parent virus used in these studies was the variant X2, from which the 0.07 map unit (m.u.) and 0.29 m.u. Xba I sites had been removed by selection enrichment (Brown et al., 1984). The remaining two Xba I sites were deleted as follows s the Xba I site at 0.45 m.u. , which lies within the gene (UL 33) encoding a predicted polypeptide of 14,000 molecular weight (mol. wt.), was removed by selection enrichment; while the Xba I site at 0.29 m.u., which lies within the gene (UL22) encoding the essential glycoprotein H (Buckmaster et al., 1984), was removed by site-directed mutagenesis. The variant devoid of Xba I sites (1702) shows normal growth characteristics in vitro and its polypeptide profile is indistinguishable from wild-type virus apart from the absence of the thymidine kinase (tk) polypeptide, a feature which is believed to be unrelated to the loss of the Xba I sites. During in vitro latency experiments, Cook and Brown (1987) isolated a variant of X2 containing a novel Xba I site at 0.74 m.u. This virus was used in intratypic recombination experiments with wild-type strain 17 to generate a HSV-1 strain 17 variant (1708) containing a fifth Xba I site, thus increasing the number of non-selectable markers between this virus (5) and 1702 (0). Different temperature-sensitive (ts) lesions were introduced into 1702 and 1708 as selectable markers. A time-course of recombination was carried out at the permissive temperature and the appearance of non-ts recombinants assayed at the non-pemissive temperature. Recombination was first detected at 4 h post infection, following the onset of DNA replication, and rapidly increased to 15% by 24 h post infection. The distribution of the non-selectable markers, ie. the Xba I sites, in ts+ recombinant molecules was analyzed. From the experimental results a number of conclusions were drawn: (i) there was an increase in the complexity or number of crossovers in recombinants arising at later time-points, confirming the previous hypothesis of Ritchie et al. (1977) that both parental and progeny molecules take part in HSV recombination; (ii) in ts+ recombinants, recombination was very high outwith the selected recombination region, suggesting that correct alignment of genomes plays an important role in determining the overall but not the relative rate of recombination; and (iii) due to the large distance between the Xba I sites, little can be determined about recombinational hotspots. The HSV-1 genome consists of two unique sequences - the long unique (UL) and the short unique (US) - flanked by inverted repeat elements known as the internal repeats (IRL/IRS) and the terminal repeats (TRL/TRS). During the isolation of 1702, a variant (1703) was isolated which has a deletion of approximately 5x10e6 mol. wt. in the UL and IRL regions of its genome, such that one copy of the immediate-early (IE) gene 1 and two unique open reading frames coding for predicted polypeptides of 20,000 mol. wt. and 22,000 mol. wt. (UL55 and UL56) are deleted. The variant 1703 synthesizes reduced levels of VmwIE110, the product of IE gene 1, and under immediate-early conditions apparently fails to synthesize VmwIE63, at both the polypeptide and RNA levels, despite there being no apparent deletion in the coding or controlling regions of the IE2 (VmwIE63) gene. 1703 also fails to synthesize the thymidine kinase polypeptide, although this is unrelated to either the deletion or the failure to synthesize VmwIE63. The variant 1703 exhibits normal growth characteristics in vitro. During the analysis of eighty plaque isolates from one recombination experiment, fourteen variants with rearrangements around the long repeats were detected. Of these, eleven have extensive variation (up to 0.4x10e6 mol. wt.) in the size of the long repeats outwith the 'a' sequence. The remaining three variants have large scale deletion or duplication of both unique and repeat sequences.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Virology, Genetics
Date of Award: 1988
Depositing User: Enlighten Team
Unique ID: glathesis:1988-77721
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jan 2020 11:53
Last Modified: 14 Jan 2020 11:53
URI: https://theses.gla.ac.uk/id/eprint/77721

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