Putative terminase subunits of herpes simplex virus 1 form a complex in the cytoplasm and interact with portal protein in the nucleus

Herpes simplex virus (HSV) terminase is an essential component of the molecular motor that translocates DNA through the portal vertex in the capsid during DNA packaging. The HSV terminase is believed to consist of the U(L)15, U(L)28, and U(L)33 gene products (pU(L)15, pU(L)28, and pU(L)33, respectively), whereas the HSV type I portal vertex is encoded by U(L)6. Immunoprecipitation reactions revealed that pU(L)15, pU(L)28 and pU(L)33 interact in cytoplasmic and nuclear lysates. Deletion of a canonical nuclear localization signal (NLS) from pU(L)15 generated a dominant-negative protein that, when expressed in an engineered cell line, decreased the replication of wild-type virus up to 80-fold. When engineered into the genome of recombinant HSV, this mutation did not interfere with the coimmunoprecipitation of pU(L)15, pU(L)28, and pU(L)33 from cytoplasmic lysates of infected cells but prevented viral replication, most nuclear import of both pU(L)15 and pU(L)28, and coimmunoprecipitation of pU(L)15, pU(L)28, and pU(L)33 from nuclear lysates. When the pU(L)15/pU(L)28 interaction was reduced in infected cells by the truncation of the C terminus of pU(L)28, pU(L)28 remained in the cytoplasm. Whether putative terminase components localized in the nucleus or cytoplasm, pU(L)6 localized in infected cell nuclei, as viewed by indirect immunofluorescence. The finding that the portal and terminase do eventually interact was supported by the observation that pU(L)6 coimmunoprecipitated strongly with pU(L)15 and weakly with pU(L)28 from extracts of infected cells in 1.0 M NaCl. These data are consistent with the hypothesis that the pU(L)15/pU(L)28/pU(L)33 complex forms in the cytoplasm and that an NLS in pU(L)15 is used to import the complex into the nucleus where at least pU(L)15 and pU(L)28 interact with the portal to mediate DNA packaging.