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.

The pseudorabies virus UL28 protein enters the nucleus after coexpression with the herpes simplex virus UL15 protein

“…Efforts to better characterize and understand the properties of the six proteins required for encapsidation have recently been made. PRV UL28 had been visualized within the nuclei of infected cells (19), and it was presumed that UL28 contained a nuclear localization signal (NLS) similar to that reported for the simian virus 40 (SV40) T antigen (9). In this work, experiments were performed to demonstrate that UL28 does not possess a classical NLS, that UL28 requires another viral protein in order to enter the nucleus, and that the HSV UL15 protein can fulfill that requirement.…”

“…Subcellular distribution of PRV UL28. PRV UL28 has been shown to accumulate in the nuclei of infected cells (19). A series of experiments was performed to identify sequences required for localization of UL28 to the nucleus.…”

“…This demonstrated that UL28 did not possess a distinct functional NLS and suggested that an interaction with one or more other virus-induced proteins was responsible for the nuclear localization during infection. Previous evidence suggested that UL28 associated with another protein during infection (19). To demonstrate that UL28 was not actively retained in the cytoplasm, a decacodon NLS derived from the SV40 T antigen (9) was inserted within UL28 (Fig.…”

“…Polyclonal rabbit antisera were raised against purified fusion proteins expressed in Escherichia coli. PRV UL28 was expressed as a Cro-UL28 fusion to produce D71 antiserum (19). HSV UL15 was expressed as a maltosebinding protein-UL15 (exon 2) fusion to generate AS9 antiserum (27), and HSV UL25 was expressed as a glutathione S-transferase-UL25 fusion to generate ID1 antiserum.…”

“…Vero cells were plated at 10 to 20% confluence on coverslips and transfected with plasmid DNAs. Plasmid DNAs in serum-free media (Opti-MEM I; Life Technologies) were mixed with a cationic lipid (pFx-3; Invitrogen) in serum-free medium and added to the cell monolayer for 24 h. The medium was replaced with Dulbecco's modified Eagle's medium plus fetal calf serum and antibiotics and incubated at 37°C for an additional 24 h. Cells were fixed with formaldehyde and permeabilized with Nonidet P-40 as previously described (19). The primary antisera (1:250 dilution) in phosphatebuffered saline (PBS) plus 5% nonfat dry milk were incubated with the cells for more than 90 min.…”