Overexpression in bacterial and identification in infected cells of the pseudorabies virus protein homologous to herpes simplex virus type 1 ICP18.5

Abstract

The ICP18.5 gene (UL28) of herpes simplex virus type 1 is a member of a well-conserved gene family among herpesviruses and is thought to play a role in localization of viral glycoproteins. We have cloned, sequenced, and expressed the entire pseudorabies virus (PRV) ICP18.5 open reading frame in Escherichia coli as a Cro-ICP18.5 fusion protein. Rabbit antiserum against Cro-ICP18.5 immunoprecipitated a 79-kDa protein from PRV-infected cells as well as a 79-kDa protein from in vitro translation of a T7 RNA polymerase transcript of the ICP18.5 gene. ICP18.5 could be detected in infected cells by 2 h postinfection. Analysis by indirect immunofluorescence demonstrated that ICP18.5 became associated with the nucleus. Subcellular fractionation confirmed that ICP18.5 synthesized during a pulse-chase experiment appeared in the nuclear fraction with time and was stable for at least 2.5 h after synthesis. Pulse-chase analysis revealed that ICP18.5 was synthesized as a monomer during a 2-min pulse labeling but formed faster sedimenting complexes which were sensitive to sodium dodecyl sulfate (SDS) treatment. The majority of ICP18.5 appeared in complexes with an antigenically unrelated 70-kDa protein. Immunoblot analysis of total infected-cell extracts using polyvalent anti-ICP18.5 serum demonstrated that a 74-kDa cellular protein in addition to the 79-kDa ICP18.5 was detected. This cellular protein was present at similar levels in uninfected cells and in PRV-infected cells at least 12 h into the infectious cycle.