Phosphorylation of the U _L 31 Protein of Herpes Simplex Virus 1 by the U _S 3-Encoded Kinase Regulates Localization of the Nuclear Envelopment Complex and Egress of Nucleocapsids

Abstract

The U L 31 and U L 34 proteins of herpes simplex virus 1 (HSV-1) form a complex that accumulates at the inner nuclear membrane (INM) of infected cells (26,27). This complex is essential for the budding of nucleocapsids through the INM into the perinuclear space (26,28). pU L 34 is a type 2 integral membrane protein with a 247-amino-acid nucleoplasmic domain that binds pU L 31 and holds the latter in close approximation to the INM (16,19,26,31,36,37). Both proteins become incorporated into nascent virions, indicating that they directly or indirectly interact with nucleocapsids during the budding event (27). Interestingly, the coexpression of the pseudorabies virus homologs of HSV pU L 31 and pU L 34 are sufficient to induce budding from the INM in the absence of other viral proteins (13).The most prominent model of nuclear egress proposes that the step following primary envelopment involves the fusion of the perinuclear virion envelope with the outer nuclear membrane (ONM), allowing subsequent steps in which the deenveloped capsid engages budding sites in the Golgi or trans-Golgi network (20, 32). The U S 3 protein is a promiscuous kinase that phosphorylates pU L 31, pU L 34, and several other viral and cellular components (1,2,5,11,15,(21)(22)(23)25). In the absence of pU S 3 kinase activity, (i) virions accumulate within distensions of the perinuclear space that herniate into the nucleoplasm (14, 27, 29), (ii) the pU L 31/pU L 34 complex is mislocalized at the nuclear rim from a smooth pattern to discrete foci that accumulate adjacent to nuclear membrane herniations (12,14,27,29), and (iii) the onset of infectious virus production is delayed (21,29).Aberrant accumulations of perinuclear virions similar to those observed in cells infected with U S 3 kinase-dead viruses have been observed in cells infected with viruses lacking the capacity to produce glycoproteins H and B (gH and gB, respectively) (8). Because these proteins are required for fusion with the plasma membrane or endocytic vesicles during HSV entry (3,4,9,10,18,30,33), it has been proposed that the accumulation of perinuclear virions in the absence of gH and gB reflects a failure in the apparatus that normally mediates the fusion between the nascent virion envelope and the ONM (8). By extension of this hypothesis, pU S 3 might act to trigger or otherwise regulate this perinuclear fusion event.The substrate(s) of the pU S 3 kinase responsible for the altered localization of the pU L 31/pU L 34 complex and the aberrant accumulation of perinuclear virions were heretofore unknown. In one study to identify such a substrate, it was determined that precluding the phosphorylation of pU L 34 was not responsible for the nuclear egress defects induced by the absence of pU S 3 or its kinase activity (29). The current study was therefore undertaken to investigate the hypothesis that the pU S 3-mediated phosphorylation of pU L 31 is critical to regulate nuclear egress. The presented evidence indicates that aspects of the U S 3 kinase-dead phenotype, including the ...

Selection of HSV capsids for envelopment involves interaction between capsid surface components pU L 31, pU L 17, and pU L 25

“…We were also interested to determine whether pU S 3, a kinase that phosphorylates pU L 31 and thereby regulates virus egress from the nucleus (30), and pU L 34, the integral membrane protein that normally interacts with pU L The data presented herein suggest a model in which enrichment of pU L 17 and pU L 25 on C capsids helps select these capsids for envelopment by interacting with pU L 31 of the NEC, eventually leading to capsid budding at the nuclear membrane. Data supporting this model include the observations that capsids do not exit the nuclei of cells infected with U L 17 and U L 25 null mutants, pU L 25 is more abundant on DNA-containing capsids as opposed to other capsid types, and pU L 31 is required for optimal envelopment of capsids at the INM (4,5,10,21,22,31,32).…”

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

“…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). To examine whether UL47 colocalized with the UL31/ UL34 complex in the presence or absence of the Us3 catalytic activity in HSV-1-infected cells, Vero cells were infected with YK524(mRFP1-UL47)encodingmRFP1-UL47,YK527(mRFP1-UL47/Us3K220M) encoding mRFP1-UL47, and Us3 with the kinase-dead K220M mutation or YK528 (mRFP1-UL47/ Us3K220M-repair) in which the Us3 K220M mutation in YK527 was repaired ( Fig.…”

Time-resolved Global and Chromatin Proteomics during Herpes Simplex Virus Type 1 (HSV-1) Infection

“…Previous studies suggested that viral US3 kinase may affect the lamin A/C (LMNA) disrupting function of the envelopment apparatus by UL31 phosphorylation (59). The phosphorylated form of UL31 (at S26, S27, and S43) was suggested to be associated with the inner nuclear membrane, thereby preventing UL31/UL34 aggregation and premature budding of virus (107). Interestingly, the trends of US3 and UL31 expression patterns and identified UL31 phosphosites (S24, S26, S27) (supplemental Fig.…”