Investigation of ICP34.5 and Its Role in HSV Pathogenicity

Holman, Holly A (2000) Investigation of ICP34.5 and Its Role in HSV Pathogenicity. PhD thesis, University of Glasgow.

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The aims of this project were to determine the role of two antisense genes, RL1 and ORF P in herpes simplex virus (HSV) virulence and to identify the HSV-2 strain HG52 ICP34.5 expression. The first overall aim was to define individual phenotypes associated with ICP34.5 and ORF P in HSV-1 strain 17+. HSV-1 contains an ORF in the long repeat sequences named RL1 (also called ?134.5) which expresses the polypeptide ICP34.5, One RL1 deletion variant, 1716, is avirulent upon intracranial inoculation in the central nervous system (CNS) and the peripheral nervous system (PNS). Replication of 1716 in vitro was shown to be cell type and cell state dependent. The avirulent phenotype of 1716 based on deletion of this region of the HSV-1 genome was originally attributed solely to the RL1 gene. However, transcripts in the opposite direction and antisense to RL1 in HSV-1 were identified in 1994. From these transcripts two open reading frames (ORF O and ORF P) mapped antisense to RL1. This led to the hypothesis that ORF P may be at least partly responsible for the avirulent phenotype of ICP34.5 negative mutants. 1716 was used as the backbone to generate three recombinant viruses containing either RL1 or ORF P in the nonessential UL43 gene. Two lengths of RL1 were cloned due to uncertainty about the position of the putative RL1 promoter sequence and role of the 5' untranslated leader in expression. One RL1 fragment contained only the ORF while the other fragment contained the ORF and 134 bp additional upstream sequence. The RL1 or ORF P genes were individually sub-cloned downstream of the HSV-1 gD promoter. This promoter was used because it has similar kinetics to that of the natural ICP34.5 promoter. These RL1 or ORF P cassettes also contained the lacZ gene under the SV40 promoter in the opposite orientation for detection of recombinant virus. The recombinant virus, 1622, contained only the RL1 open reading frame and demonstrated an 8-fold overexpression of ICP34.5 by Western blotting and immunoprecipitation. 1622 regained wild type replication kinetics in vitro in cell lines non- permissive for ICP34.5 negative viruses. In certain cell types, an in vitro function of ICP34.5 is to maintain protein synthesis by using a protein kinase (PKR) pathway. 1716 showed host protein synthesis shut-off in human neuroblastoma and HeLa cells confirming the previously reported data for the HSV-1 strain F ICP34.5 deletion mutant, R3616. 1622 infected cells demonstrated protein synthesis similar to 17+ by SDS-PAGE. 1622 demonstrated a small reduction in neurovirulence compared with 17+ by intracranial inoculation but was 40-fold more virulent than its parent following infection via the snout and trigeminal ganglia as assayed by in vivo replication and immunohistochemsitry. The recombinant virus, 1623, by Western blotting expressed very low levels of ICP34.5 compared to 17+. Based on 2-fold dilutions of 1623 infected BHK cell extracts, ICP34.5 is expressed eight times less than in 17+ and thus 64-fold less than in 1622. Interestingly, this low level of expression still enabled 1623 to maintain wild type levels of protein synthesis in neuroblastoma and HeLa cells. However, the replication kinetics in vitro from 1623 was slower than 1716. This suggests that there may be a second mutation elsewhere in the genome. The recombinant virus, 1624, contained ORF P under the HSV-1 gD promoter. Due to difficulties in cloning the ORF P gene, final purification and characterisation of this mutant was not carried out. However, expression of ORF P from 17+ infected cells was analysed by an antiserum from a GST/ORF P fusion protein. By Western blotting, ORF P was detected in 17+ infected cells during normal lytic infection in vitro. Cells infected with a RL1 epitope tagged virus, 1621, demonstrated expression of a slightly larger form of ORF P. The temperature sensitive virus, tsK, when grown at 39.5

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: A Maclean
Keywords: Virology
Date of Award: 2000
Depositing User: Enlighten Team
Unique ID: glathesis:2000-74894
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 27 Sep 2019 15:28
Last Modified: 27 Sep 2019 15:28

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