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Curtis R. Brandt, Aaron W. Kolb, Hermann Bultmann; Sequence Variation and Phosphorylation Sites in the Herpes Simplex virus Type-1 Us1(α 22) Ocular Virulence Determinant. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5863.
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The HSV-1 Us1 gene has been identified as a virulence determinant for keratitis. Studies on the role it plays in disease are hampered by the lack of data on sequence variability between strains and the location of phosphorylation sites on the different isoforms in infected cells. The goal of this study was to determine the degree of sequence variability and physically identify sites of phosphorylation in the HSV-1 α22(Us1) ocular virulence determinant.
The Us1 gene from 6 low passage ocular HSV-1 isolates as well as KOS and F were sequenced directly from the genome and clones using specific PCR primers. Bioinformatics were used for phylogenetic and MEME analysis, and to identify potentially important structural features. Globplot2 was used to predict ordered areas of the protein. NanoLC-MS/MS was used to identify phosphorylated residues in immunoprecipitated FLAG-tagged proteins from strains CJ394 and OD4.
Strains 17, F, CJ394, and CJ311 were identical. With the other strains, the majority of the variability was in the N-terminal half of the protein which had 32 differences compared to only 11 in the C-terminal half. Six residues W187, F202, R219, G229, W240, and L256 were absolutely conserved in all strains. The maximum sequence divergence was 2%. MEME analysis identified a 49 residue core sequence located in the center of the protein. Serines 5, 22, and 167 were phosphorylated in both the CJ394 and OD4 proteins. Globplot was only able to fold the central third of the protein which overlapped the 49 residue core.
The data suggest that the α22 protein has a core scaffold to which amino- and carboxy-terminal domains, that may have phosphorylation dependent structures, are attached. This information will be useful in designing structure-function relationship studies of the protein and the role it plays in ocular HSV infections.
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