The wild-type U L 31, U L 34, and U S 3 proteins localized on nuclear membranes and perinuclear virions; the U S 3 protein was also on cytoplasmic membranes and extranuclear virions. The U L 31 and U L 34 proteins were not detected in extracellular virions. U S 3 deletion caused (i) virion accumulation in nuclear membrane invaginations, (ii) delayed virus production onset, and (iii) reduced peak virus titers. These data support the herpes simplex virus type 1 deenvelopment-reenvelopment model of virion egress and suggest that the U S 3 protein plays an important, but nonessential, role in the egress pathway.Herpes simplex virus type 1 (HSV-1) virions contain a linear double-stranded DNA genome of approximately 152 kb that is packaged into an icosahedral capsid shell. An amorphous tegument layer surrounds the capsid and is, in turn, surrounded by an envelope composed of a host-derived lipid bilayer studded with viral integral membrane proteins. After the viral genome is replicated and packaged into capsids within the nucleus, assembled nucleocapsids acquire a primary lipid envelope by budding through the inner nuclear membrane (INM) into the space located between the inner and outer leaflets of the nuclear envelope (25,33). Whereas the derivation of the primary envelope from the INM is widely accepted, the route of transit of the nascent virions from the perinuclear space to the extracellular space is more controversial. An overview of the key players in herpesvirus egress and a comparison of the salient features of the two proposed envelopment models have been recently published (8,25).A single-step model of herpesvirus envelopment was proposed for the prototypical alphaherpesvirus HSV-1 (6,18,35,44). This model proposes that enveloped virions move through the endoplasmic reticulum (ER) and the Golgi apparatus in transport vesicles with concomitant modification of primary virion glycoproteins. The single-step envelopment model is supported by the observations that (i) enveloped particles within vesicles can be readily detected by electron microscopy and in fracture label studies (35, 44) and (ii) virion egress and virion-associated glycoprotein processing are both inhibited in cells treated with the ionophore monensin (18). On the other hand, neither of these observations can exclude the alternative deenvelopment-reenvelopment model. Such a model is supported by mounting ultrastructural and biochemical evidence (3,10,13,14,30,37,41,46,50) and has been proposed for HSV-1, other alphaherpesviruses such as varicella-zoster virus (VZV) and pseudorabies virus (PrV), and betaherpesviruses such as human cytomegalovirus. In this model, primary envelopment occurs by budding through the INM but the primary envelope surrounding the perinuclear virion is lost, presumably by fusion with the outer lamellae of the nuclear envelope. In a second step, reenvelopment occurs by wrapping of the nucleocapsid and its associated tegument with a lipid bilayer originating from a membranous cytoplasmic organelle bearing viral glycoprotein...
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 ...
Cell 67:117-130, 1991). We show here that production of assembled vRNPs occurs normally in H7-treated cells, and we have used H7 as a biochemical tool to trap vRNPs in the nucleus. When H7 was removed from the cells, vRNP export was specifically induced in a CHO cell line stably expressing recombinant M1. Similarly, fusion of cells expressing recombinant M1 from a Semliki Forest virus vector allowed nuclear export of vRNPs. However, export was not rescued when H7 was present in the cells, implying an additional role for phosphorylation in this process. The viral NS2 protein was undetectable in these systems. We conclude that influenza virus M1 is required to induce vRNP nuclear export but that cellular phosphorylation is an additional factor.Influenza virus has a segmented genome consisting of eight single-stranded, negative-sense RNAs packaged into helical viral ribonucleoprotein (vRNP) complexes (for a review, see reference 19). The nucleoprotein (NP) is the most abundant structural component of the vRNPs. In the virus particle, the vRNPs are connected to each other and with the viral envelope by the matrix protein, M1. The viral NS2 protein is associated with M1-containing vRNPs (37). The vRNPs display bidirectional traffic through the nuclear envelope (33). During virus entry, rapid import of incoming vRNPs occurs from the cytosol to the nucleoplasm through the nuclear pore complexes (22). Replication and transcription of viral RNAs then commence inside the nucleus (see reference 19 for a review). Viral mRNAs are exported to the cytosol and used for translation of nonstructural and structural viral proteins. Many of these proteins are then imported to the nucleus to support continued viral replication and assembly of progeny vRNPs. Replication of viral RNA appears to occur in proximity to the nuclear matrix (3, 20)-the insoluble "skeleton" of the nucleus.Export of vRNPs to the cytosol occurs late in infection. There is evidence that export requires the synthesis of M1 (21), one of several late viral proteins. Why M1 is essential for export of vRNPs from the nucleus to the cytosol is not clear. It may escort the vRNPs from the nucleus and through the nuclear pores, or it may be needed to release the bound vRNPs from the nuclear matrix (38). Another possibility is that, by associating with vRNPs in the cytosol, M1 may prevent their reimport into the nucleus (32).In this study, we have examined the requirement of M1 for nuclear export of vRNPs. We made use of a protein kinase inhibitor, H7 (11), which blocks the synthesis of M1 and other late virus proteins such as hemagglutinin (15,18). In H7-treated cells, synthesis of the early viral proteins, including NP, occurs and viral transcription is not significantly affected (18). The inhibitory effect of H7 is thought to occur at the level of viral mRNA export from the nucleus; while mRNAs for the early viral proteins are apparently exported normally, the mRNAs for the late viral proteins are selectively retained (30). Another effect of H7 is that vRNPs appear to b...
cHerpes simplex virus 2 (HSV-2) is an important human pathogen that is the major cause of genital herpes infections and a significant contributor to the epidemic spread of human immunodeficiency virus infections. The UL21 gene is conserved throughout the Alphaherpesvirinae subfamily and encodes a tegument protein that is dispensable for HSV-1 and pseudorabies virus replication in cultured cells; however, its precise functions have not been determined. To investigate the role of UL21 in the HSV-2 replicative cycle, we constructed a UL21 deletion virus (HSV-2 ⌬UL21) using an HSV-2 bacterial artificial chromosome, pYEbac373. HSV-2 ⌬UL21 was unable to direct the production of infectious virus in noncomplementing cells, whereas the repaired HSV-2 ⌬UL21 strain grew to wild-type (WT) titers, indicating that UL21 is essential for virus propagation. Cells infected with HSV-2 ⌬UL21 demonstrated a 2-h delay in the kinetics of immediate early viral gene expression. However, this delay in gene expression was not responsible for the inability of cells infected with HSV-2 ⌬UL21 to produce virus insofar as late viral gene products accumulated to WT levels by 24 h postinfection (hpi). Electron and fluorescence microscopy studies indicated that DNA-containing capsids formed in the nuclei of ⌬UL21-infected cells, while significantly reduced numbers of capsids were located in the cytoplasm late in infection. Taken together, these data indicate that HSV-2 UL21 has an early function that facilitates viral gene expression as well as a late essential function that promotes the egress of capsids from the nucleus.
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