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Title: Characterization of the HSV-1 strain 17+ neurovirulence gene RL1 and its expression in a bacterial system
Author: McKie, Elizabeth Ann
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1993
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Abstract:
In HSV-1 strain F, one gene product had previously been localised to the region of the genome between RL2 and the 'a' sequence, and shown to be a determinant of HSV-1 strain F neurovirulence. This protein was designated ICP34.5. Sequence analysis of the HSV-1 strain 17+ genome in this region revealed that in HSV-1 strain 17+, the putative ORF ascribed to ICP34.5 was thoroughly disrupted by a 2bp insert, which rendered 60% of the coding-region out of frame. This did not correlate however, with data obtained by several laboratories which clearly demonstrated that in HSV-1 strain 17+ this locus encoded an important neurovirulence determinant. The initial stage of this project involved reinvestigation of the HSV-1 strain 17+ sequence at the terminus of the long repeat, where the expected frame-shift occurred. By comparison of two newly sequenced clones with the original clone used to sequence this region of the genome, it was demonstrated that the original sequence obtained for this locus of HSV-1 strain 17+ was inaccurate and came from an atypical plasmid clone. Correction of the strain 17+ sequence opens the reading frame proposed by Chou and Roizman to encode a protein of 248 amino acids. This ORF has been designated RLl. Having confirmed that HSV-1 strain 17+ encodes an ICP34.5 homologue, we wished to confirm the importance of this gene in neurovirulence following intracerebral inoculation of HSV-1 strain 17+. Verification that lack of synthesis of ICP34.5 alone, was responsible for a non-neurovirulent phenotype was achieved through the construction and characterization of a HSV-1 strain 17+ RLl variant, 1771, with a stop-codon in only the ICP34.5 reading frame, 9 base pairs downstream from the initiating ATG. 1771, did not produce ICP34.5 as demonstrated by Western blotting using an ICP34.5 polyclonal antiserum which was generated during the course of this study. It was not impaired in growth in tissue culture, and following intracerebral inoculation of BALB/c mice it was totally non-neurovirulent, with a LD50 of >106 p.f.u./mouse, compared to 7 p.f.u./mouse for the wild-type virus HSV-1 strain 17+. This lack of neurovirulence was shown to correlate with an inability to replicate in mouse brain, and clearly demonstrated the crucial role played by ICP34.5 in HSV-1 strain 17+' following intracerebral inoculation of mice. Previously, peptide antisera had been raised against 7 different regions of the predicted HSV-1 strain 17+ RLl ORF, but none of these, until recently, had successfully identified ICP34.5 in HSV-1 strain 17+ infected cells. To demonstrate that the HSV-1 strain 17+ RLl ORF was capable of expressing a protein, it was placed under the control of a strong T7 promoter and over-expressed in E.coli BL21(DE3) cells. A two-step process was developed for partial purification of the E.coli-expressed ICP34.5 which involved initial ammonium sulphate precipitation followed by anion-exchange chromatography. The partially purified protein was used as an immunogen for the production of a rabbit polyclonal anti-ICP34.5 serum. This antiserum successfully recognised ICP34.5 in HSV-1 strain 17+ and HSV-1 F infected cell extracts and revealed that previous results which had indicated that ICP34.5 was underproduced in HSV-1 strain 17+ were inaccurate, and the protein was produced in similar quantities by both strains. Using this polyclonal antiserum, and 1771 as a negative control, ICP34.5 was specifically localised to the cytoplasm of HSV-1 strain 17+ infected cell extracts. An antiserum, raised against a peptide corresponding to 10 copies of a Proline-Alanine-Threonine amino acid repeat predicted by the HSV-1 strain F RLl ORF, had been previously shown to recognise a polypeptide of Mr 39K in HSV-1 strain F infected cell extracts, but only recently, following extensive optimization of our assay conditions has it specifically recognised ICP34.5 in HSV-1 strain 17+ infected cells. A strain F mutant, F11, with a deletion in RL1 was constructed to confirm the specifity of this antiserum, to use as a negative control in ICP34.5 localization studies, and to show that loss of ICP34.5 expression specifically correlated with a loss of neurovirulence. Fll was shown to be totally non-neurovirulent following intracerebral inoculation of mice with a LD50 of >107 p.f.u./mouse, compared to < 102 p.f.u./mouse for the parental wild- type HSV-1 strain F. Unlike the strain 17+ mutant 1716 (which has an identical deletion in its genome) F11 was not significantly impaired , compared to the wild-type virus, in reactivating from latency. Using Western blot analysis we were able to demonstrate that lack of synthesis of ICP34.5 correlated with a loss of neurovirulence. Using F11 as a negative control, ICP34.5 was localised to the cytoplasm of HSV-1 strain F infected cells by immunofluorescence and cell fractionation studies. During the analysis of single plaques from a transfection, a variant designated 1772, was isolated which had a large deletion in Us. It was observed that this variant had a small plaque morphology on BHK21/C13 cells which was indicative of a defect in cell-to-cell spread.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.788560  DOI: Not available
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