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Title: Antisense RNA transcription regulates IE2 expression in the HSV-1 deletion variant 1703
Author: Sinclair, Mary Catherine
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1992
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The aim of this project was to further characterize the HSV-1 strain 17+ deletion variant 1703. Initial characterization after isolation (MacLean & Brown, 1987a) demonstrated that 1703 had a deletion of approximately 7500 base pairs (bp); (4. 9x10 6 mol. wt. ) in the UL/IRL region of the genome. By restriction enzyme analysis, the deletion was shown to affect genes UL55, UL56 and one copy each of IE1 and LAT. Polypeptide analysis demonstrated that, although the deletion terminated an estimated 500 base pairs downstream of the 3' end of IE2, the IE2 gene product Vmw63 was apparently not produced during immediate-early times of infection. Further characterization of 1703 was achieved by: 1. The dideoxynucleotide sequence analysis of 1703 DNA fragments in which IE2 and the end points of the deletion were located, 2. The analysis of 1703 IE2 gene products, 3. The in vivo characterization of 1703 and 4. The construction of a 1703 wild-type recombinant. Following the suggestion by Dr John McLauchlan that IE2 mRNA synthesis in 1703 infected cells may be controlled by the production of transcripts initiating from the promoter of the IRL copy of IE1 which was antisense to IE2 RNA, the project was extended to determine if this could be substantiated. Dideoxynucleotide sequence analysis of IE2 demonstrated that the promoter, promoter associated, terminator, terminator associated signals and most of the open reading frame were homologous to the published wild-type sequence (McGeoch et al. , 1988a). Sequencing of the deletion end points has shown that it spans the region between np (nucleotide position) 123623 and np15839, removing UL56 and 343 base pairs of the 3' end of UL55 thus leaving 555 base pairs between the 3' end of IE2 and the deletion end point. The methods of polypeptide analysis, Western blot analysis and S1 nuclease mapping were used to detect IE2 gene products at both protein and RNA levels. These techniques demonstrated that IE2 mRNA and Vmw63 synthesis were reduced, but not totally absent, at immediate-early times of infection. Western blot analysis of 17+ immediate-early polypeptide extracts titrated in mock infected extracts compared to 1703 immediate-early polypeptide extracts demonstrated that, at most, Vmw63 production in 1703 infected cells was 1/8 that produced by 17+. At early and late times of infection, Vmw63 synthesis by 1703 was equivalent to 17+. The in vivo effect of the loss of UL55, UL56, one copy of IE1 and LAT and the reduction in synthesis of Vmw63 during immediate-early times of infection was examined. Inoculation of 3 week old mice with 1703 via the intracranial route demonstrated that 1703 was as virulent as the wild-type virus. The latency characteristics of 1703 were also shown to be equivalent to those of 17+ indicating that the products of the genes mentioned above are not required for intracranial virulence or for the establishment, maintenance or reactivation of latent genomes. Construction of a wild-type recombinant of 1703 was achieved by recombination of the 1703 DNA fragment in which the end points of the deletion were located into 17+ DNA. The characterization of the resultant recombinant's IE2 gene products indicated equivalence to those produced by 1703 and hence that the deletion was responsible for the underproduction of Vmw63 during immediate-early times of infection. To examine the possibility of antisense transcripts controlling the production of IE2 mRNA, a polyadenylation signal was cloned between the 3' end of IE2 and the 5' end of the IRL copy of IE1 in the correct orientation to terminate the synthesis of a potential antisense transcript before IE2 coding sequences. A HSV-2 strain HG52 polyadenylation signal was chosen for this and, since the surrounding sequences were heterologous to 1703 DNA, two 1703 fragments were cloned around the polyadenylation signal and the construct recombined into 1703 DNA. The resultant recombinant was called 1703PA and analysis of 1703PA IE2 gene products demonstrated that IE2 mRNA and Vmw63 synthesis had returned to wild-type levels. The detection of the novel transcript generated as a result of the insertion of the polyadenylation signal substantiated the conclusion that antisense transcripts initiating from the IRl copy of IE1 had the potential to control the production of IE2 mRNA in 1703 infected cells.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available