title: The herpes simplex virus type 2 UL3 open reading frame encodes a nuclear localizing phosphoprotein.
abstract: The herpes simplex virus type 2 (HSV-2, strain 333) UL3 open reading frame (ORF) codes for a protein with a predicted molecular weight of 29,681. Comparisons of the UL3, strain 333 ORF show essentially complete amino acid identity with HSV-2 (strain HG52) and a 75% amino acid identity with HSV type 1 (strain 17). To characterize the expression of this gene, a hydrophilic region of the HSV-2 UL3 gene was cloned into a bacterial expression vector. The resulting fusion protein was used to generate antibodies in rabbits. In vitro translation of HSV-2-derived mRNA followed by immunoprecipitation with the rabbit antisera reveals a major 28,000-Da protein as judged by SDS-polyacrylamide gel electrophoresis. This is consistent with the predicted molecular weight of an unmodified UL3 protein. Pulse-labeling of infected cells, with [35S]methionine, followed by immunoprecipitation and electrophoretic analysis reveals three distinct bands of 28,000, 30,500, and 33,000 Da. Labeling infected cells with [32P]orthophosphate shows that the 30,500- and the 33,000-Da species are post-translationally phosphorylated. The 30,500-Da species can be converted to the 28,000-Da species with alkaline phosphatase treatment. Interestingly, the 33,000-Da species is resistant to this treatment. Immunohistochemical analysis of infected cells reveals that the UL3 protein has a perinuclear location early in infection and at later times becomes associated with the nucleus as discrete particles. A mutant of HSV, which has a major deletion of the UL3 coding region does not show any immunohistochemical staining with the UL3 antisera. The wild-type virus-infected cell staining pattern remains the same subsequent to DNAse and RNAse treatment, indicating that the UL3 protein product is not directly associated with nucleic acid.
authors.affiliation: Department of Molecular Biology, University of Medicine and Dentistry of New Jersey, Stratford 08084.
authors.forename: D M
authors.initials: DM
authors.lastname: Worrad
authors.forename: S
authors.initials: S
authors.lastname: Caradonna
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002417:Cattle; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004279:DNA, Viral; D004591:Electrophoresis, Polyacrylamide Gel; D005814:Genes, Viral; D006367:HeLa Cells; D006801:Humans; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D004279:DNA, Viral; D010750:Phosphoproteins; C082251:UL3 protein, Human herpesvirus 2; D014764:Viral Proteins