Abstract
Abstract: :
Purpose: One factor influencing the severity of HSV keratitis is genes in the virus. Traditionally, these virulence genes are studied individually and very large changes in virulence are sought. In reality, the constellation of genes in a strain work together to determine severity with each gene contributing its own part. We have developed a system with HSV keratitis allowing us to identify and study the contributions of multiple viral genes in pathogenesis. The goal is to map the determinants and study their role in infection. Methods: Mixed infection with HSV strains OD4 and CJ394 generates recombinants with increased virulence, suggesting at least two genes are involved. Subcloning of the CJ394 genome, marker transfer of the clones to OD4, and ocular infections with potential recombinants were used to map the determinants to 2 Kb or less. Automated sequencing was used to detect mutations and site-directed mutagenesis is being used to confirm virulence mutations. Results: Six different regions of the CJ394 genome transferred virulence. From left to right on the genome map, there were US9, UL33, UL36/37, UL39/40, UL41/42, and US1. Eleven amino acid changes have been identified to date, all of which are novel. In the US1 gene the role of the S34A and Y116C mutations was tested by reverting the mutations and testing for virulence by marker transfer/infection. The results showed the reversions restored the ability of OD4 to cause keratitis. Using anti-phosphotyrosine antibodies in pull-down assays and immunoblotting, we have identified Y116 as a phosphorylation site in US1. Conclusion: This novel system for analysis of virulence allowed us to identify i) several novel virulence determinants, ii) the presence of multiple novel changes in the proteins, iii) the UL33 gene as a virulence determinant, iv) Y116 and S34 as being critical for virulence, and v) that Y116 is a phosphorylation site important for virulence. Finally, the fact that some genes transfer ocular virulence only (US9, UL33, US1), while others transfer ocular and neurovirulence (UL36/37, UL39/40, UL41/42) confirm our earlier hypothesis that the two disease phenotypes involve separate virulence genes. This system offers a powerful way to identify and study the role of multiple virulence genes in a large DNA virus.
Keywords: 425 herpes simplex virus • 449 keratitis • 469 microbial pathogenesis: experimental studies