Characterization of the Functional Domains of the Herpes Simplex Virus (HSV-2) Strain HG52 RL1 Gene

Bdour, Salwa (1995) Characterization of the Functional Domains of the Herpes Simplex Virus (HSV-2) Strain HG52 RL1 Gene. PhD thesis, University of Glasgow.

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The long repeat region of HSV-1 contains the diploid RL1 gene which encodes the protein ICP34.5, a 43K polypeptide of 263 amino acids in HSV-1 strain F and a 37K polypeptide of 248 amino acids in HSV-1 strain 17+ infected cells. The role of RL1 in virulence of HSV-1 has been studied by Chou et al (1990), MacLean, A et al (1991) and McKie et al (1994). The discovery of the RL1 gene in HSV-1 was followed by sequencing data of the corresponding region of HSV-2 strain HG52 which indicated that HG52 possesses a counterpart of the HSV-1 RL1 gene (McGeoch et al., 1991). The organization of HG52 RL1 differs from that of HSV-1 although both initiation codons are in equivalent positions. The coding sequence of HG52 RL1 lacks the (PAT)io repeat element and is disrupted by a reiterated sequence proposed to be an intron. Removal of this intron results in a second exon containing a 63-amino acids HSV-l/HSV-2 conserved region, being in frame. To characterise the functional domains of the HSV-2 RL1 gene, three mutant viruses have been constructed and characterized: (1) 2620, with a stop codon within the conserved region, 46 amino acids upstream of the 3' end of RL1 (2) 2621, with an in frame stop codon 9bp downstream of the initiating ATG and (3) 2622, in which the proposed intron has been apparently deleted. The mutant viruses 2620, 2621 and 2622 were characterized in vivo following intracerebral inoculation of Balb/c mice and in vitro using BHK21/C13 and 3T6 mouse embryo fibroblast cells. The mutant 2621 was avirulent with a LD50 >107 p.f.u./mouse compared to 3.16x106 p.f.u./mouse for 2604 and 102 p.f.u./mouse for HG52. The 2620 mutant was also avirulent with a LD50 of 2.37x106 p.f.u./mouse. The 2622 mutant was intermediate with a LD50 of 105 p.f.u./mouse. The growth of the mutants 2620 and 2621 was not impaired in BHK21/C13 but was impaired in 3T6 cells. The growth of 2622 was comparable to that of the wild-type HG52 in BHK21/C13 cells and intermediate between HG52 and the avirulent deletion mutant 2604 in 3T6 cells. Rescuants of the mutants (R2620, R2621, R2622) were generated by in vitro and in vivo marker rescue. Those generated by in vitro marker rescue demonstrated an intermediate virulence phenotype. Rescuants, generated by in vivo marker rescue, except for R2620 were restored to the full virulence phenotype of the wild-type HG52 pi. 17. A secondary mutation in the genome of 2620 and its rescuants generated by in vitro marker rescue could explain failure of the R2620 rescuants to return to wild-type virulence. The secondary mutation could be due to passage of the virus during purification and/or virulence heterogeneity within the HG52 pi. 17 stock. Individual plaques of HG52 pi. 17 demonstrated variable virulence phenotypes. The rescuants generated in vitro and individual plaques of HG52 pi. 17 were characterised by growth in 3T6 cells which differentiates between ICP34.5 positive and negative virus. The 3T6 growth and plaque morphology strongly discount a possible mutation in RL1 which should be confirmed when an antiserum against ICP34.5 of HG52 becomes available. It seems that the growth phenotype of HG52 variants in vitro does not always mimic the in vivo phenotype thus emphasising the necessity of the animal model to determine virulence. The demonstrated heterogeneity of the HG52 pl. 17 stock emphasises the necessity of in vivo marker rescue experiments to confirm the phenotype of constructed mutants.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Moira Brown
Keywords: Virology, Genetics
Date of Award: 1995
Depositing User: Enlighten Team
Unique ID: glathesis:1995-74646
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 13 Nov 2019 15:58
Last Modified: 13 Nov 2019 15:58

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