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

Abstract

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, whereas the U L 31 gene product of HSV-1 is a nuclear matrix-associated phosphoprotein previously shown to interact with U L 34 protein in HSV-1-infected cell lysates. For these studies, polyclonal antisera directed against purified fusion proteins containing U Herpes simplex virus type 1 (HSV-1) nucleocapsids, like those of all herpesviruses are assembled in the nucleus and acquire a lipid bilayer envelope by budding through the inner nuclear membrane into the perinuclear space (10). Several viral proteins have been implicated in this initial budding event, including the myristylated U L 11 protein, glycoprotein K, which is necessary for envelopment in nondividing cells, and U L 34 protein (2, 21, 37). Of these, only U L 34 protein has been implicated solely in the initial envelopment step, whereas glycoprotein K and U L 11 also play roles in egress through the cytoplasm towards the extracellular space (2, 21, 37).The U L 34 sequence predicts that the protein is oriented as a type II integral membrane protein with an N-terminal cytoplasmic domain of 247 amino acids and a C-terminal transmembrane domain of 22 amino acids (32,35,37). The type II membrane topology of HSV-2 U L 34 protein has recently been addressed (39). This topology predicts that if the transmembrane domain were anchored in the outer nuclear membrane, the bulk of the protein would lie in the cytoplasm, whereas localization in the inner nuclear membrane would place the bulk of the protein within the nucleoplasm.The exact role of U L 34 protein in the envelopment process remains unclear. One possibility is that U L 34 protein interacts directly with capsids and/or tegument components and the nuclear membrane, thereby mediating wrapping of the capsid in the membrane. Alternatively, U L 34 protein may be responsible for recruiting other viral or cellular factors to the site of envelopment. Both hypotheses predict that U L 34 protein should associate with the nuclear envelope. To date, research on the localization of HSV-1 U L 34 protein and its homologues in other herpesviruses has not yielded consistent results. In baculovirus-transduced cells. HSV-1 U L 34 protein is found at the nuclear envelope and in the cytoplasm (46), whereas in HSV-1-infected cells, U L 34 protein is reportedly detectable at the cell surface (35). HSV-2 U L 34 protein has been reported to localize at the endoplasmic reticulum in transfected and in-* Corresponding author. Mailing address:

Herpes Simplex Virus 1 UL47 Interacts with Viral Nuclear Egress Factors UL31, UL34, and Us3 and Regulates Viral Nuclear Egress

“…In cells infected with an HSV-1 mutant encoding a Us3 kinase-dead mutant or carrying a mutation in the Us3 phosphorylation site in UL47, UL47 accumulated aberrantly in punctate structures at the nuclear membrane (21). During the course of the study, we noticed that the punctate structures containing UL47 induced in the absence of Us3 kinase activity in HSV-1-infected cells were reminiscent of the discrete foci containing the UL31/UL34 complex observed at the nuclear membrane in cells infected with HSV-1 mutants carrying a mutation abrogating either the expression or catalytic activity of Us3 (4,26). These observations raised the possibility that UL47 interacted with the UL31/UL34 complex at the nuclear membrane and modulated the function(s) of the complex in HSV-1-infected cells.…”

“…As described above, Us3 has been reported to phosphorylate UL31, UL34, and UL47 (16,17,21) and to regulate their proper localization at the nuclear membrane in HSV-1-infected cells (4,21). In the absence of the Us3 catalytic activity, the UL31/UL34 complex and UL47 were shown to localize aberrantly in similar punctate structures at the nuclear membrane in HSV-1-infected cells (4,21,26,35).…”

“…It has been well established that a heterodimeric complex of HSV-1 proteins UL31 and UL34, which are conserved in all known herpesviruses, plays a crucial role in HSV-1 primary envelopment (1)(2)(3)(4)(5). In the absence of the HSV-1 UL31/UL34 complex, nucleocapsids accumulate in the nucleoplasm, and progeny virus intermediates and virions are barely detectable in the perinuclear space or cytoplasm or at the cell surface (5,6).…”

“…The HSV-1 UL31/UL34 complex and its homologs in other herpesviruses have been suggested to coordinate multiple events in the primary envelopment of nucleocapsids, including the following: (i) disruption of the nuclear lamina, which has been suggested to facilitate herpesvirus nucleocapsid access to the INM, by recruiting cellular protein kinases, such as protein kinase C isoforms, and by direct binding to components of the nuclear lamina (i.e., lamins A and C) and modifying their conformation (1, 2, 7-11); (ii) recruitment of nucleocapsids into primary envelopes by interaction of the UL31/UL34 complex and the capsid vertex-specific component (CVSC), which consists of the conserved capsid proteins UL17 and UL25 (12,13); and (iii) budding of nucleocapsids into the INM (14,15). In addition to UL31 and UL34, an HSV-1 serine/threonine protein kinase Us3 has been suggested to be involved in HSV-1 primary envelopment since Us3 phosphorylates and regulates proper localization of UL34 and UL31 at the nuclear membrane (4,16,17) and phosphorylates and modifies lamins A and C (7,8,10). UL47 (or VP13/VP14) is a major structural protein in HSV-1 virion tegument (18).…”

Mapping of Key Functions of the Herpes Simplex Virus 1 U S 3 Protein Kinase: the U S 3 Protein Can Form Functional Heteromultimeric Structures Derived from Overlapping Truncated Polypeptides

“…In the absence of the U S 3 protein kinase, capsids are retained in nuclei. Envelopment appears to be limited and occurs at the inner nuclear membrane invaginated into the nucleus (23,24,27). The presence of similar structures in cells infected with a mutant expressing only the U S 3.5 protein kinase suggests that U S 3.5 is less efficient in enabling the restructuring of the nuclear envelope to enable the release of capsids from nuclei (16).…”