Tyrosine 116 of the herpes simplex virus type 1 IEalpha22 protein is an ocular virulence determinant and potential phosphorylation site.
OBJECTIVE
To determine whether tyrosine 116 of the HSV-1 alpha22 protein is involved in virulence and is a potential phosphorylation site.


METHODS
Site-directed mutagenesis was used to revert the Y116C mutation in the alpha22 gene of the strain OD4 to wild type (C116Y), and the effect of virulence was tested by using a marker transfer-infection protocol in mice. Immunoblot analysis, tryptic phosphopeptide mapping, and phosphotyrosine pulldown-immunoblot protocols were used to assess the OD4 alpha22 isoforms.


RESULTS
Reversion of the Y116C mutation resulted in a significant increase in the severity of ocular disease compared with the OD4 virus alone. Reversion of the Y116C and a previously identified mutation (S34A) together did not alter the severity of virulence compared with either mutation alone. Immunoblot analysis revealed a loss or reduction in alpha22 isoforms in the OD4 virus compared with wild type (CJ394 virus). The OD4 virus had numerous alterations in the alpha22 tryptic phosphopeptide pattern, including loss of specific peptides and shifts in the position of several peptides. Phosphotyrosine pulldowns revealed a loss of one or more isoforms and shifts in the apparent size of others.


CONCLUSIONS
The data indicate that Y116 is a determinant of peripheral virulence in mice and that mutations at S34 and Y116 affect virulence independently. The data also show that the S34 and Y116 mutations substantially alter phosphorylation of the alpha22 protein, that Y116 is a potential phosphorylation site, and that the alpha22 protein contains at least two phosphotyrosines. These results are the first to show that mutation of a specific tyrosine in the alpha22 protein is associated with virulence.
Department of Ophthalmology and Visual Sciences, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA. cbrandt@facstaff.wisc.edu
Curtis R
CR
Brandt
Aaron W
AW
Kolb
D000818:Animals; D002522:Chlorocebus aethiops; D003315:Cornea / Q000821:virology*; D000939:Epitopes; D005260:Female; D018259:Herpesvirus 1, Human / Q000472:pathogenicity*; D017874:Immediate-Early Proteins / Q000235:genetics / Q000502:physiology*; D016849:Keratitis, Herpetic / Q000821:virology*; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010449:Peptide Mapping; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000502:physiology*; D020033:Protein Isoforms; D014443:Tyrosine / Q000378:metabolism*; D014709:Vero Cells; D054334:Viral Regulatory and Accessory Proteins; D014774:Virulence
D000939:Epitopes; C057294:ICP22 protein, human herpesvirus 1; D017874:Immediate-Early Proteins; D020033:Protein Isoforms; D054334:Viral Regulatory and Accessory Proteins; D019000:Phosphotyrosine; D014443:Tyrosine