Isolation and Structural Analysis of Genomic Variants of Herpes Simplex Virus Type 2

Harland, June Elizabeth (1990) Isolation and Structural Analysis of Genomic Variants of Herpes Simplex Virus Type 2. PhD thesis, University of Glasgow.

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The original aim of the project was to study recombination in HSV-2 strain HG52 using restriction enzyme sites as unselected markers. As no relevant DNA sequence data was available for HSV-2 it was decided to isolate site deletion mutants by enrichment selection of spontaneously occurring variants. The restriction enzyme Xba I was chosen as it makes only four staggered cuts in the HSV-2 genome and a virus, HG52X163X3X53, lacking all four Xba I sites was isolated following three rounds of enrichment selection. However, during the screening procedures involved in the isolation of HG52X163X3X53, a large number of variants with genomic alterations was identified and the work described in this thesis has concentrated on the analysis and characterization of these variants. Of the variants isolated after the initial enrichment selection of HG52 DNA five have been studied in detail. Three (HG52X85/4, HG52X85/5 and HG52X86) have deletions from IRL ranging in size from 2. 5x10e6 daltons to 6x10e6 daltons. Under immediate early conditions VmwIE64 production by HG52X85/5 and HG52X86 in which only the 3' part of IE1 is removed is reduced despite their deletions ending approximately 1kb downstream from the 3' end of UL54 which encodes this polypeptide. HG52X85/4 which also has a deletion ending approximately 1kb downstream of UL54 but in which an entire copy of IE1 is removed makes VmwIE64 in normal amounts. Of the two remaining variants one, HG52X192, has a deletion of approximately 1x10 6 daltons in each copy of RL. The final variant, HG52X19, has a deletion of approximately 9x10e6 daltons which removes the entire internal copy of the long repeat and half of the short repeat. The long segment of the genome is fixed in the prototype orientation whilst the short region inverts inefficiently through the undeleted part of the repeats (the internal copy of the 'a' sequence being deleted). The genome has non-HSV DNA inserted across the deletion. The inserted DNA has been reiterated to give different copy numbers and hence a heterogeneous genome population. It has been demonstrated that the inserted DNA is of bovine origin -presumably the result of recombination with the calf thymus DNA used as a carrier during transfection. To investigate if the enrichment selection procedure was responsible for the production of the unexpectedly high proportion of variants, isolates from untreated HSV stocks were studied. Of the fifty plaques of HG52 analysed, twelve differed significantly from the wild type structure. However, the plaques, five of HG52/5 and seven of HG52/10, represented only two variants with the same genome structures as HG52X86 and HG52X192 respectively. No significant variation was found in the stocks of other HSV strains examined (ie. HSV-2 strains 333 and 186 and HSV-1 strains 17 and KOS or plaque purified HG52 isolates (eg. tsl)). Variation was found in HSV-1 strain McKrae but its relevance was impossible to assess as the history of the stock is unknown. The conclusion has been drawn that variation has arisen in HG52 in the absence of enrichment selection although the structure of HG52X19 must have resulted from the transfection procedure. Three variants, HG52X163X12, HG52X163X14 and HG52X163X21, were isolated following the second round of enrichment selection using as the parent HG52X163 which lacks the 0.7m.c. Xba I site. HG52X163X12 has a deletion removing sequences from 0.94m.c. to 0.99m.c. (ie. part of U S and almost all of TRS) . The isolation of this variant demonstrates that genes US 10, 11, 12 and one copy of IE3 and one copy of oriS are non essential, at least in vitro. The two remaining variants, HG52X163X14 and HG52X163X21, both have deletions removing the same region as from HG52X163X12. However, the deleted sequences are replaced by sequences between 0.83m.c. -0.91m. c. in the inverted orientation thereby effectively deleting US between 0.94-0.96m.c. while extending the short repeats by 6kb. The only differences found between HG52X163X14 and HG52X163X21 were the loss of the 0.91m.c. Xba I site and a small insert containing a Hind III and EcoR I site at the 0.94m.c. Xba I site in the latter. It is possible that the enrichment selection procedure was responsible for the generation of these three variants as the rearrangements appear to be associated with the Xba I sites at 0.91 and 0.94m.c.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Virology
Date of Award: 1990
Depositing User: Enlighten Team
Unique ID: glathesis:1990-78093
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 30 Jan 2020 15:41
Last Modified: 30 Jan 2020 15:41

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