UL20 Protein Functions Precede and Are Required for the UL11 Functions of Herpes Simplex Virus Type 1 Cytoplasmic Virion Envelopment

Abstract

Egress of herpes simplex virus type 1 (HSV-1) from the nucleus of the infected cell to extracellular spaces involves a number of distinct steps, including primary envelopment by budding into the perinuclear space, de-envelopment into the cytoplasm, cytoplasmic reenvelopment, and translocation of enveloped virions to extracellular spaces. UL20/gK-null viruses are blocked in cytoplasmic virion envelopment and egress, as indicated by an accumulation of unenveloped or partially enveloped capsids in the cytoplasm. Similarly, UL11-null mutants accumulate unenveloped capsids in the cytoplasm. To assess whether UL11 and UL20/gK function independently or synergistically in cytoplasmic envelopment, recombinant viruses having either the UL20 or UL11 gene deleted were generated. In addition, a recombinant virus containing a deletion of both UL20 and UL11 genes was constructed using the HSV-1(F) genome cloned into a bacterial artificial chromosome. Ultrastructural examination of virus-infected cells showed that both UL20-and UL11-null viruses accumulated unenveloped capsids in the cytoplasm. However, the morphology and distribution of the accumulated capsids appeared to be distinct, with the UL11-null virions forming aggregates of capsids having diffuse tegument-derived material and the UL20-null virus producing individual capsids in close juxtaposition to cytoplasmic membranes. The UL20/UL11 double-null virions appeared morphologically similar to the UL20-null viruses. Experiments on the kinetics of viral replication revealed that the UL20/UL11 double-null virus replicated in a manner similar to the UL20-null virus. Additional experiments revealed that transiently expressed UL11 localized to the trans-Golgi network (TGN) independently of either gK or UL20. Furthermore, virus infection with the UL11/UL20 double-null virus did not alter the TGN localization of transiently expressed UL11 or UL20 proteins, indicating that these proteins did not interact. Taken together, these results show that the intracellular transport and TGN localization of UL11 is independent of UL20/gK functions, and that UL20/gK are required and function prior to UL11 protein in virion cytoplasmic envelopment.Herpes simplex virus type 1 (HSV-1) morphogenesis occurs in multiple stages within infected cells. Initially, the virion capsid assembles within the nucleus and the virion acquires an initial envelope by budding into the perinuclear spaces (39). Subsequently, these enveloped virions fuse with the outer nuclear lamellae, leading to the accumulation of unenveloped capsids in the cytoplasm. Within the cytoplasm, a number of additional tegument proteins attach to the capsid and the fully tegumented capsids bud into cytoplasmic vesicles, which mostly likely originate from the trans-Golgi network (TGN). Enveloped virions are ultimately secreted to extracellular spaces through the utilization of cellular vesicular trafficking systems (7,19,20,33,40,43). The process by which the tegumented cytoplasmic capsids bud into TGN-derived vesicles is not well unders...

Effects of Simultaneous Deletion of pUL11 and Glycoprotein M on Virion Maturation of Herpes Simplex Virus Type 1

“…Our ultrastructural analyses of the HSV-1 mutants yielded no evidence for an impairment of nuclear egress, as had been described for a previous UL11 deletion mutant of HSV-1 (1). Thus, they are in line with more recent investigations of another HSV-1 UL11 deletion mutant (14), as well as of UL11-negative PrV (23), which revealed inhibition of virion formation in the cytoplasm but not of nuclear egress. These differences may be explained by the fact that the HSV-1 UL11 deletion mutant used in the first study was still able to express the N-terminal part including a dileucine motif, which has been reported to contribute to binding of pUL11 to pUL16 (32).…”

“…Also, pUL11 was reported to associate with pUL16 in HSV-1 and pseudorabies virus (PrV) (31), and it has been suggested that pUL11 could be involved in recruiting viral proteins to the site of virus assembly at the TGN. In line with this hypothesis, deletion of pUL11 homologs resulted in impairment of secondary envelopment in HSV-1, PrV, and human cytomegalovirus (HCMV) (2,14,23,56,57). However, in PrV and HSV-1 the absence of pUL11 reduced titers only approximately 10-fold, whereas the homol-ogous ppUL28 (pUL99) in HCMV is essential for viral replication (55,56), which may be indicative of functional differences between these proteins.…”

Herpes Simplex Virus Capsid-Organelle Association in the Absence of the Large Tegument Protein UL36p

“…Alphaherpesvirus capsid-membrane association and envelopment are driven by numerous cooperative, and in some cases redundant, interactions between multiple tegument and envelope proteins (8)(9)(10)(11)(12)(13)73) and the cellular ESCRT machinery (14)(15)(16)(17)(18)(19). While it is clear that UL36p is essential for envelopment, the simplest interpretation of our data is that the association of some HSV capsids with the surface of cytoplasmic organelles does not absolutely require UL36p.…”

“…The apparatus that could mediate UL36p-independent capsid-membrane docking is unknown, but one attractive candidate is the Golgi network-localized membraneanchored UL11p-UL16p-UL21p complex (74)(75)(76). This heterotrimer facilitates cytoplasmic envelopment in both HSV and PRV (8,73,77) perhaps via recruitment of envelope proteins and outer tegument components, including gE and VP22 (54,78). Importantly, UL16p assembles onto HSV capsids in the absence of UL36p (79), raising the possibility that UL11p-UL16p-UL21p could connect capsids to the envelopment membrane and outer tegument independently of UL36p/ UL37p function.…”

The Amino Terminus of Herpes Simplex Virus 1 Glycoprotein K Is Required for Virion Entry via the Paired Immunoglobulin-Like Type-2 Receptor Alpha

“…Viral plaques were visualized by immunohistochemistry as we have previously described (29)(30)(31)(32). Analysis of one-step growth kinetics was performed as described earlier (20,22,33).…”