U(L)31 and U(L)34 proteins of herpes simplex virus type 1 form a complex that accumulates at the nuclear rim and is required for envelopment of nucleocapsids

The herpes simplex virus type 1 (HSV-1) U(L)34 protein is likely a type II membrane protein that localizes within the nuclear membrane and is required for efficient envelopment of progeny virions at the nuclear envelope, wher eas the U(L)31 gene product of HSV-1 is a nuclear matrix-associated phospho protein previously shown to interact with U(L)34 protein in HSV-1-infected cell lysates. For these studies, polyclonal antisera directed against purif ied fusion proteins containing U(L)31 protein fused to glutathione-S-transf erase (U(L)31-GST) and U(L)34 protein fused to GST (U(L)34-GST) were demons trated to specifically recognize the U(L)31 and proteins of approximately 3 4,000 and 30,000 Da, respectively. The U(L)31 and U(L)34 gene products colo calized in a smooth pattern throughout the nuclear rim of infected cells by 10 It postinfection. U(L)34 protein also accumulated in pleiomorphic cytop lasmic structures at early times and associated with an altered nuclear env elope late in infection. Localization of U(L)31 protein at the nuclear rim required the presence of U(L)34 protein, inasmuch as cells infected with a U(L)34 null mutant virus contained U(L)31 protein primarily in central intr anuclear domains separate from the nuclear rim, and to a lesser extent in t he cytoplasm. Conversely, localization of U(L)34 protein exclusively at the nuclear rim required the presence of the U(L)31 gene product, inasmuch as U(L)34 protein was detectable at the nuclear rim, in replication compartmen ts, and in the cytoplasm of cells infected with a U(L)31 null virus. When t ransiently expressed in the absence of other viral factors, U(L)31 protein localized diffusely in the nucleoplasm, whereas U(L)34 protein localized pr imarily in the cytoplasm and at the nuclear rim. In contrast, coexpression of the U(L)31 and U(L)34 proteins was sufficient to target both proteins ex clusively to the nuclear rim. The proteins were also shown to directly inte ract in vitro in the absence of other viral proteins. In cells infected wit h a virus lacking the U(S)3-encoded protein kinase, previously shown to pho sphorylate the U(L)34 gene product, U(L)31 and U(L)34 proteins colocalized in small punctate areas that accumulated on the nuclear rim. Thus, U(S)3 ki nase is required for even distribution of U(L)31 and U(L)34 proteins throug hout the nuclear rim. Taken together with the similar phenotypes of the U(L )31 and U(L)34 deletion mutants, these data strongly suggest that the U(L)3 1 and U(L)34 proteins form a complex that accumulates at the nuclear membra ne and plays an important role in nucleocapsid envelopment at the inner nuc lear membrane.