Identification and functional characterization of the equine herpesvirus type 1 IR6 gene.

摘要

Genetic analysis of the 12,766 bp inverted repeat (IR) segment of equine herpesvirus type 1 (EHV-1) has revealed the presence of six ORFs. These include four ORFs encoding known (IR1) or potential (IR2, IR3, IR4) regulatory gene products, one potential structural protein (IR5), and IR6, which is the last gene completely encoded within the IR and is the focus of the research discussed here.;IR6 was shown by Northern blot analysis to encode a 1.2 kb early mRNA that is 3;To identify and characterize the protein product of the IR6 gene, we have generated a specific rabbit antiserum to a TrpE/IR6 fusion protein. The antiserum immunoprecipitated a 33 kDa protein produced by in vitro translation of IR6 specific mRNA and an infected cell protein (ICP), of a similar size, that could be detected as early as 1-2 hrs. p.i. The ICP was also shown to undergo phosphorylation and to be associated with virions and nucleocapsids. Immunofluorescence analysis revealed that the IR6 protein appears to associate with cytoskeletal elements during EHV-1 infection. The IR6 protein does not appear to be N-linked glycosylated, despite the presence of two consensus N-linked glycosylation within the primary amino acid sequence, as determined by results from tunicamycin-blocked glycosylation of ICPs, labeling of ICPs with tritiated glucosamine, in vitro translation of IR6 mRNA in the presence of canine pancreatic microsomes, and pulse-chase experiments.;The unique IR6 gene product does not appear to function in a regulatory role, at least at the level of transcription, during EHV-1 infection based upon results from transient transfection analyses using two representative early and late EHV-1 promoter CAT constructs. The results of the studies presented in the following pages involve the identification of a unique EHV-1 gene and the identification and functional characterization of its protein product.