Localization of the Us protein kinase of equine herpesvirus type 1 is affected by the cytoplasmic structures formed by the noval IR6 protein.
Previous work revealed that the Us (unique short) segment of equine herpesvirus type-1 (EHV-1), like that of other alphaherpesviruses, encodes a serine/threonine protein kinase (PK). Experiments were carried out to identify the PK encoded by the EHV-1 EUS2 gene (ORF 69) and to ascertain its time course of synthesis and cellular localization. Western blot and immunoprecipitation analyses of EHV-1-infected cell extracts using a PK-specific polyclonal antibody generated against a bacterially expressed TrpE/PK fusion protein identified the Us PK as a 42- to 45-kDa phosphoprotein. The PK protein is first synthesized at 3 hr postinfection, is produced throughout the infection cycle, and is incorporated into EHV-1 virions. Interestingly, immunoprecipitation analyses revealed that the PK protein within the cytoplasm is associated with the 33-kDa IR6 novel protein of EHV-1, is expressed abundantly as an early protein, and is present in the large rod-like structures formed by the IR6 protein (ORF67 protein) within the cytoplasm of infected cells. Confocal microscopic examination of cells stained with fluorescein-labeled antibody clearly showed that the PK protein colocalized with the cytoplasmic IR6 rod-like structures and remained associated with these unique structures during infection. In contrast, in cells infected with the EHV-1 RacM strain in which the IR6 protein harbors four amino acid substitutions that prevent formation of the rod-like structures (Osterrieder et al., 1996, Virology 217, 442-451), the PK protein localized predominantly to the nucleus. The possible significance of the association of the IR6 and PK proteins in EHV-1 replication is discussed.
Colle
C F
CF
Department of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport 71130, USA.
O'Callaghan
D J
DJ
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D002460:Cell Line; D002467:Cell Nucleus / Q000821:virology; D003593:Cytoplasm / Q000378:metabolism; D004861:Herpesvirus 1, Equid / Q000201:enzymology*; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D010750:Phosphoproteins / Q000378:metabolism* / Q000648:ultrastructure; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism* / Q000648:ultrastructure; D011817:Rabbits; D014764:Viral Proteins / Q000378:metabolism* / Q000648:ultrastructure
D000914:Antibodies, Viral; C099910:EUS2 protein, Equine herpesvirus 1; D010750:Phosphoproteins; D014764:Viral Proteins; C078423:IR6 protein, Equine herpesvirus 1; D017346:Protein Serine-Threonine Kinases