Brefeldin A arrests the maturation and egress of herpes simplex virus particles during infection.

Herpes simplex virus (HSV) requires the host cell secretory apparatus for transport and processing of membrane glycoproteins during the course of virus assembly. Brefeldin A (BFA) has been reported to induce retrograde movement of molecules from the Golgi to the endoplasmic reticulum and to cause disassembly of the Golgi complex. We examined the effects of BFA on propagation of HSV type 1. Release of virions into the extracellular medium was blocked by as little as 0.3 microgram of BFA per ml when present from 2 h postinfection. Characterization of infected cells revealed that BFA inhibited infectious viral particle formation without affecting nucleocapsid formation. Electron microscopic analyses of BFA-treated and untreated cells (as in control cells) demonstrated that viral particles were enveloped at the inner nuclear membrane in BFA-treated cells and accumulated aberrantly in this region. Most of the progeny virus particles observed in the cytoplasm of control cells, but not that of BFA-treated cells, were enveloped and contained within membrane vesicles, whereas many unenveloped nucleocapsids were detected in the cytoplasm of BFA-treated cells. This suggests that BFA prevents the transport of enveloped particles from the perinuclear space to the cytoplasmic vesicles. These findings indicate that BFA-induced retrograde movement of molecules from the Golgi complex to the endoplasmic reticulum early in infection arrests the ability of host cells to support maturation and egress of enveloped viral particles. Furthermore, we demonstrate that the effects of BFA on HSV propagation are not fully reversible, indicating that maturation and egress of HSV type 1 particles relies on a series of events which cannot be easily reconstituted after the block to secretion is relieved.     

The pseudorabies virus US3 protein is a component of primary and of mature virions

Herpesviruses acquire a primary envelope by budding of capsids at the inner leaflet of the nuclear membrane. They then traverse into the cytoplasm after fusion of the primary envelope with the outer leaflet of the nuclear membrane. In the alphaherpesvirus pseudorabies virus (PrV), the latter process is impaired when the US3 protein is absent. Acquisition of final tegument and envelope occurs in the cytoplasm. Besides the capsid components, only the UL31 and UL34 gene products of PrV have unequivocally been shown to be part of primary enveloped virions, whereas they lack several tegument proteins present in mature virions (reviewed by T. C. Mettenleiter, J. Virol. 76:1537-1547, 2002). Using immunoelectron microscopy, we show that the US3 protein is present in primary enveloped as well as in mature virions. It is also detectable in intracytoplasmic inclusions produced in the absence of other viral tegument components or envelope-associated glycoproteins. In particular, inclusions formed in the absence of the inner tegument protein UL37 contained the US3 protein. Thus, the US3 protein is a tegument component of both forms of enveloped alphaherpes virions. We hypothesize that US3 protein in primary virions modulates deenvelopment at the outer leaflet of the nuclear membrane and is either lost from primary virions during nuclear egress and subsequently reacquired early during tegumentation or is retained during transit of the nucleocapsid through the nuclear membrane.     

Egress of alphaherpesviruses: comparative ultrastructural study

Egress of four important alphaherpesviruses, equine herpesvirus 1 (EHV-1), herpes simplex virus type 1 (HSV-1), infectious laryngotracheitis virus (ILTV), and pseudorabies virus (PrV), was investigated by electron microscopy of infected cell lines of different origins. In all virus-cell systems analyzed, similar observations were made concerning the different stages of virion morphogenesis. After intranuclear assembly, nucleocapsids bud at the inner leaflet of the nuclear membrane, resulting in enveloped particles in the perinuclear space that contain a sharply bordered rim of tegument and a smooth envelope surface. Egress from the perinuclear cisterna primarily occurs by fusion of the primary envelope with the outer leaflet of the nuclear membrane, which has been visualized for HSV-1 and EHV-1 for the first time. The resulting intracytoplasmic naked nucleocapsids are enveloped at membranes of the trans-Golgi network (TGN), as shown by immunogold labeling with a TGN-specific antiserum. Virions containing their final envelope differ in morphology from particles within the perinuclear cisterna by visible surface projections and a diffuse tegument. Particularly striking was the addition of a large amount of tegument material to ILTV capsids in the cytoplasm. Extracellular virions were morphologically identical to virions within Golgi-derived vesicles, but distinct from virions in the perinuclear space. Studies with gB- and gH-deleted PrV mutants indicated that these two glycoproteins, which are essential for virus entry and direct cell-to-cell spread, are dispensable for egress. Taken together, our studies indicate that the deenvelopment-reenvelopment process of herpesvirus maturation also occurs in EHV-1, HSV-1, and ILTV and that membrane fusion processes occurring during egress are substantially different from those during entry and direct viral cell-to-cell spread.     

Nuclear envelope breakdown can substitute for primary envelopment-mediated nuclear egress of herpesviruses

Herpesvirus nucleocapsids assemble in the nucleus but mature to infectious virions in the cytoplasm. To gain access to this cellular compartment, nucleocapsids are translocated to the cytoplasm by primary envelopment at the inner nuclear membrane and subsequent fusion of the primary envelope with the outer nuclear membrane. The conserved viral pUL34 and pUL31 proteins play a crucial role in this process. In their absence, viral replication is strongly impaired but not totally abolished. We used the residual infectivity of a pUL34-deleted mutant of the alphaherpesvirus pseudorabies virus (PrV) for reversion analysis. To this end, PrV-ΔUL34 was serially passaged in rabbit kidney cells until final titers of the mutant virus PrV-ΔUL34Pass were comparable to those of wild-type PrV. PrV-ΔUL34Pass produced infectious progeny independently of the pUL34/pUL31 nuclear egress complex and the pUS3 protein kinase. Ultrastructural analyses demonstrated that this effect was due to virus-induced disintegration of the nuclear envelope, thereby releasing immature and mature capsids into the cytosol for secondary envelopment. Our data indicate that nuclear egress primarily serves to transfer capsids through the intact nuclear envelope. Immature and mature intranuclear capsids are competent for further virion maturation once they reach the cytoplasm. However, nuclear egress exhibits a strong bias for nucleocapsids, thereby also functioning as a quality control checkpoint which is abolished by herpesvirus-induced nuclear envelope breakdown.     

Primary envelopment of pseudorabies virus at the nuclear membrane requires the UL34 gene product

Primary envelopment of several herpesviruses has been shown to occur by budding of intranuclear capsids through the inner nuclear membrane. By subsequent fusion of the primary envelope with the outer nuclear membrane, capsids are released into the cytoplasm and gain their final envelope by budding into vesicles in the trans-Golgi area. We show here that the product of the UL34 gene of pseudorabies virus, an alphaherpesvirus of swine, is localized in transfected and infected cells in the nuclear membrane. It is also detected in the envelope of virions in the perinuclear space but is undetectable in intracytoplasmic and extracellular enveloped virus particles. Conversely, the tegument protein UL49 is present in mature virus particles and absent from perinuclear virions. In the absence of the UL34 protein, acquisition of the primary envelope is blocked and neither virus particles in the perinuclear space nor intracytoplasmic capsids or virions are observed. However, light particles which label with the anti-UL49 serum are formed in the cytoplasm. We conclude that the UL34 protein is required for primary envelopment, that the primary envelope is biochemically different from the final envelope in that it contains the UL34 protein, and that perinuclear virions lack the tegument protein UL49, which is present in mature virions. Thus, we provide additional evidence for a two-step envelopment process in herpesviruses.     

Herpes simplex virus type 1 capsids transit by the trans-Golgi network, where viral glycoproteins accumulate independently of capsid egress

Egress of herpes capsids from the nucleus to the plasma membrane is a complex multistep transport event that is poorly understood. The current model proposes an initial envelopment at the inner nuclear membrane of capsids newly assembled in the nucleus. The capsids are then released in cytosol by fusion with the outer nuclear membrane. They are finally reenveloped at a downstream organelle before traveling to the plasma membrane for their extracellular release. Although the trans-Golgi network (TGN) is often cited as a potential site of reenvelopment, other organelles have also been proposed, including the Golgi, endoplasmic reticulum-Golgi intermediate compartment, aggresomes, tegusomes, and early or late endosomes. To clarify this important issue, we followed herpes simplex virus type 1 egress by immunofluorescence under conditions that slowed intracellular transport and promoted the accumulation of the otherwise transient reenvelopment intermediate. The data show that the capsids transit by the TGN and point to this compartment as the main reenvelopment site, although a contribution by endosomes cannot formally be excluded. Given that viral glycoproteins are expected to accumulate where capsids acquire their envelope, we examined this prediction and found that all tested could indeed be detected at the TGN. Moreover, this accumulation occurred independently of capsid egress. Surprisingly, capsids were often found immediately adjacent to the viral glycoproteins at the TGN.     

Ultrastructural analysis of the replication cycle of pseudorabies virus in cell culture: a reassessment.

We reinvestigated major steps in the replicative cycle of pseudorabies virus (PrV) by electron microscopy of infected cultured cells. Virions attached to the cell surface were found in two distinct stages, with a distance of 12 to 14 nm or 6 to 8 nm between virion envelope and cell surface, respectively. After fusion of virion envelope and cell membrane, immunogold labeling using a monoclonal antibody against the envelope glycoprotein gE demonstrated a rapid drift of gE from the fusion site, indicating significant lateral movement of viral glycoproteins during or immediately after the fusion event. Naked nucleocapsids in the cytoplasm frequently appeared close to microtubules prior to transport to nuclear pores. At the nuclear pore, nucleocapsids invariably were oriented with one vertex pointing to the central granulum at a distance of about 40 nm and viral DNA appeared to be released via the vertex region into the nucleoplasm. Intranuclear maturation followed the typical herpesvirus nucleocapsid morphogenesis pathway. Regarding egress, our observations indicate that primary envelopment of nucleocapsids occurred at the inner leaflet of the nuclear membrane by budding into the perinuclear cisterna. This nuclear membrane-derived envelope exhibited a smooth surface which contrasts the envelope obtained by putative reenvelopment at tubular vesicles in the Golgi area which is characterized by distinct surface projections. Loss of the primary envelope and release of the nucleocapsid into the cytoplasm appeared to occur by fusion of envelope and outer leaflet of the nuclear membrane. Nucleocapsids were also found engulfed by both lamella of the nuclear membrane. This vesiculation process released nucleocapsids surrounded by two membranes into the cytoplasm. Our data also indicate that fusion between the two membranes then leads to release of naked nucleocapsids in the Golgi area. Egress of virions appeared to occur via transport vesicles containing one or more virus particles by fusion of vesicle and cell membrane. Our data thus support biochemical data and mutant virus studies of (i) two steps of attachment, (ii) the involvement of microtubules in the transport of nucleocapsids to the nuclear pore, and (iii) secondary envelopment in the trans-Golgi area in PrV infection.     

A cellular function is required for pseudorabies virus envelope glycoprotein processing and virus egress.

The mouse L-cell mutant gro29 is defective for egress of herpes simplex virus type 1 (HSV-1) virions and is significantly reduced in HSV-1 glycoprotein export (B. W. Banfield and F. Tufaro, J. Virol. 64:5716-5729, 1990). In this report, we demonstrate that pseudorabies virus (PRV), a distantly related alphaherpesvirus, shows a distinctive set of defects after infection of gro29 cells. Specifically, we identify defects in the rate and extent of viral glycoprotein export, infectious particle formation, plaque formation, and virus egress. The initial rate of viral glycoprotein synthesis was unaffected in gro29 cells, but the extent of export from the endoplasmic reticulum to the Golgi apparatus was impaired and export through the Golgi apparatus became essentially blocked late in infection. Moreover, by using a secreted variant of a viral membrane protein, we found that export from the Golgi apparatus out of the cell was also defective in gro29 cells. PRV does not form plaques on gro29 monolayers. A low level of infectious virus is formed and released early after infection, but further virus egress is blocked. Taken together, these observations suggest that the gro29 phenotype involves either multiple proteins or a single protein used at multiple steps in viral glycoprotein export and virus egress from cells. Moreover, this host cell protein is required by both HSV and PRV for efficient propagation in infected cells.     

Herpes simplex virus glycoproteins gB and gD function in a redundant fashion to promote secondary envelopment

Egress of herpes simplex virus (HSV) and other herpesviruses from cells involves extensive modification of cellular membranes and sequential envelopment and deenvelopment steps. HSV glycoproteins are important in these processes, and frequently two or more glycoproteins can largely suffice in any step. Capsids in the nucleus undergo primary envelopment at the inner nuclear membrane (INM), and then enveloped virus particles undergo deenvelopment by fusing with the outer nuclear membrane (ONM). Capsids delivered into the cytoplasm then undergo secondary envelopment, involving trans-Golgi network (TGN) membranes. The deenvelopment step involves HSV glycoproteins gB and gH/gL acting in a redundant fashion. This fusion has features common to the fusion that occurs between the virion envelope and cellular membranes when HSV enters cells, a process requiring gB, gD, and gH/gL. Whether HSV gD also participates (in a redundant fashion with gB or gH/gL) in deenvelopment has not been characterized. Secondary envelopment in the cytoplasm is known to involve HSV gD and gE/gI, also acting in a redundant fashion. Whether gB might also contribute to secondary envelopment, collaborating with gD and gE/gI, is also not clear. To address these questions, we constructed an HSV double mutant lacking gB and gD. The HSV gB(-)/gD(-) mutant exhibited no substantial defects in nuclear egress. In contrast, secondary envelopment was markedly reduced, and there were numerous unenveloped capsids that accumulated in the cytoplasm, as well as increased numbers of partially enveloped capsids and morphologically aberrant enveloped particles with thicker, oblong tegument layers. These defects were different from those observed with HSV gD(-)/gE(-)/gI(-) mutants, which accumulated capsids in large, aggregated masses in the cytoplasm. Our results suggest that HSV gB functions in secondary envelopment, apparently acting downstream of gE/gI.     

A herpes simplex virus gD-YFP fusion glycoprotein is transported separately from viral capsids in neuronal axons

Two models describing how alphaherpesviruses exit neurons differ with respect to whether nucleocapsids and envelope glycoproteins travel toward axon termini separately or as assembled enveloped virions. Recently, a pseudorabies virus glycoprotein D (gD)-green fluorescent protein fusion was found to colocalize with viral capsids, supporting anterograde transport of enveloped virions. Previous antibody staining experiments demonstrated that herpes simplex virus (HSV) glycoproteins and capsids are separately transported in axons. Here, we generated an HSV expressing a gD-yellow fluorescent protein (YFP) fusion and found that gD-YFP and capsids were transported separately in neuronal axons. Anti-gD antibodies colocalized with gD-YFP, indicating that gD-YFP behaves like wild-type HSV gD.     

Effects of brefeldin A on the processing of viral envelope glycoproteins in murine erythroleukemia cells.

This paper documents the effects of brefeldin A (BFA) on the processing and transport of viral envelope glycoproteins in a retrovirus-transformed murine erythroleukemia (MEL) cell line. BFA is a fungal metabolite that disrupts intracellular membrane traffic at the endoplasmic reticulum (ER)-Golgi complex junction. In MEL cells, BFA inhibited the processing of the newly synthesized precursor, gPr90env, of the murine leukemia virus envelope protein, gp70, and curtailed the budding of virions into the culture medium by blocking the transport of this protein out of the ER. The block resulted in the intracellular accumulation of gPr90env and two putative products of its processing (78 and 66 kDa). The results of endoglycosidase (endo) H and D digestion of the viral glycoproteins in the presence and absence of BFA indicated that (i) there was no glycoprotein processing during the first approximately 2 h of the BFA block; (ii) active Golgi enzymes relocated to the ER in approximately 2 h during BFA treatment, resulting in the production of partially endo H-resistant forms of the spleen focus-forming virus glycoprotein, gp55 (in controls, this glycoprotein was generally retained in the ER as an endo H-sensitive entity); and (iii) proteolytic processing of gPr90env to gp70 occurred prior to the acquisition of endo H resistance and at approximately the same time as endo D sensitivity (i.e. in a cis Golgi compartment). In control cells, the spleen focus-forming virus glycoprotein, gp55, underwent turnover with a half-life of approximately 5 h. In contrast, its turnover was considerably slower during BFA treatment (t 1/2 = approximately 20 h), suggesting that transport of gp55 out of the ER was required for its degradation or that BFA afforded it protection from proteolysis within the ER.     

Herpes simplex virus capsids are transported in neuronal axons without an envelope containing the viral glycoproteins

Electron micrographic studies of neuronal axons have produced contradictory conclusions on how alphaherpesviruses are transported from neuron cell bodies to axon termini. Some reports have described unenveloped capsids transported on axonal microtubules with separate transport of viral glycoproteins within membrane vesicles. Others have observed enveloped virions in proximal and distal axons. We characterized transport of herpes simplex virus (HSV) in human and rat neurons by staining permeabilized neurons with capsid- and glycoprotein-specific antibodies. Deconvolution microscopy was used to view 200-nm sections of axons. HSV glycoproteins were very rarely associated with capsids (3 to 5%) and vice versa. Instances of glycoprotein/capsid overlap frequently involved nonconcentric puncta and regions of axons with dense viral protein concentrations. Similarly, HSV capsids expressing a VP26-green fluorescent protein fusion protein (VP26/GFP) did not stain with antiglycoprotein antibodies. Live-cell imaging experiments with VP26/GFP-labeled capsids demonstrated that capsids moved in a saltatory fashion, and very few stalled for more than 1 to 2 min. To determine if capsids could be transported down axons without glycoproteins, neurons were treated with brefeldin A (BFA). However, BFA blocked both capsid and glycoprotein transport. Glycoproteins were transported into and down axons normally when neurons were infected with an HSV mutant that produces immature capsids that are retained in the nucleus. We concluded that HSV capsids are transported in axons without an envelope containing viral glycoproteins, with glycoproteins transported separately and assembling with capsids at axon termini.     

The herpes simplex virus UL20 protein compensates for the differential disruption of exocytosis of virions and viral membrane glycoproteins associated with fragmentation of the Golgi apparatus.

The Golgi apparatus is fragmented and dispersed in Vero cells but not in human 143TK- cells infected with wild-type herpes simplex virus 1. Moreover, a recombinant virus lacking the gene encoding the membrane protein UL20 (UL20- virus) accumulates in the space between the inner and outer nuclear membranes of Vero cells but is exported and spreads from cell to cell in 143TK- cell cultures. Here we report that in Vero cells infected with UL20- virus, the virion envelope glycoproteins were of the immature type, whereas the viral glycoproteins associated with cell membranes were fully processed up to the addition of sialic acid, a trans-Golgi function. Moreover, the amounts of viral glycoproteins accumulating in the plasma membranes were considerably smaller than those detected on the surface of Vero cells infected with wild-type virus. In contrast, the amounts of viral glycoproteins present on the plasma membranes of 143TK- cells infected with wild-type or UL20- virus were nearly identical. We conclude that (i) in Vero cells infected with UL20- virus the block in the export of virions is at the entry into the exocytic pathway, and a second block in the exocytosis of viral glycoproteins associated with cytoplasmic membranes is due to an impairment of transport beyond Golgi fragments containing trans-Golgi enzymes and not to a failure of the Golgi oligosaccharide-processing functions; (ii) these defects are manifested in cells in which the Golgi apparatus is fragmented; and (iii) the UL20 protein compensates for these defects by enabling transport to and from the fragmented Golgi apparatus.     

Monensin inhibits the processing of herpes simplex virus glycoproteins, their transport to the cell surface, and the egress of virions from infected cells.

HEp-2 cells or Vero cells infected with herpes simplex virus type 1 were exposed to the ionophore monensin, which is thought to block the transit of membrane vesicles from the Golgi apparatus to the cell surface. We found that yields of extracellular virus were reduced to less than 0.5% of control values by 0.2 microM monensin under conditions that permitted accumulation of cell-associated infectious virus at about 20% of control values. Viral protein synthesis was not inhibited by monensin, whereas late stages in the post-translational processing of the viral glycoproteins were blocked. The transport of viral glycoproteins to the cell surface was also blocked by monensin. Although the assembly of nucleocapsids appeared to be somewhat inhibited in monensin-treated cells, electron microscopy revealed that nucleocapsids were enveloped to yield virions, and electrophoretic analyses showed that the isolated virions contained immature forms of the envelope glycoproteins. Most of the virions which were assembled in monensin-treated cells accumulated in large intracytoplasmic vacuoles, whereas most of the virions produced by and associated with untreated cells were found attached to the cell surface. Our results implicate the Golgi apparatus in the egress of herpes simplex virus from infected cells and also suggest that complete processing of the viral envelope glycoproteins is not essential for nucleocapsid envelopment or for virion infectivity.     

Herpes simplex virus nucleocapsids mature to progeny virions by an envelopment --> deenvelopment --> reenvelopment pathway

Herpes simplex virus (HSV) nucleocapsids acquire an envelope by budding through the inner nuclear membrane, but it is uncertain whether this envelope is retained during virus maturation and egress or whether mature progeny virions are derived by deenvelopment at the outer nuclear membrane followed by reenvelopment in a cytoplasmic compartment. To resolve this issue, we used immunogold electron microscopy to examine the distribution of glycoprotein D (gD) in cells infected with HSV-1 encoding a wild-type gD or a gD which is retrieved to the endoplasmic reticulum (ER). In cells infected with wild-type HSV-1, extracellular virions and virions in the perinuclear space bound approximately equal amounts of gD antibody. In cells infected with HSV-1 encoding an ER-retrieved gD, the inner and outer nuclear membranes were heavily gold labeled, as were perinuclear enveloped virions. Extracellular virions exhibited very little gold decoration (10- to 30-fold less than perinuclear virions). We conclude that the envelope of perinuclear virions must be lost during maturation and egress and that mature progeny virions must acquire an envelope from a post-ER cytoplasmic compartment. We noted also that gD appears to be excluded from the plasma membrane in cells infected with wild-type virus.     

In rat dorsal root ganglion neurons,herpes simplex virus type 1 tegument forms in the cytoplasm of the cell body

The herpes simplex virus type 1 (HSV-1) tegument is the least understood component of the virion, and the mechanism of tegument assembly and incorporation into virions during viral egress has not yet been elucidated. In the present study, the addition of tegument proteins (VP13/14, VP16, VP22, and US9) and envelope glycoproteins (gD and gH) to herpes simplex virions in the cell body of rat dorsal root ganglion neurons was examined by immunoelectron microscopy. All tegument proteins were detected diffusely spread in the nucleus within 10 to 12 h and, at these times, nucleocapsids were observed budding from the nucleus. The majority (96%) of these nucleocapsids had no detectable label for tegument and glycoproteins despite the presence of tegument proteins in the nucleus and glycoproteins adjacent to the nuclear membrane. Immunolabeling for tegument proteins and glycoproteins was found abundantly in the cytoplasm of the cell body in multiple discrete vesicular areas: on unenveloped, enveloped, or partially enveloped capsids adjacent to these vesicles and in extracellular virions. These vesicles and intracytoplasmic and extracellular virions also labeled with Golgi markers, giantin, mannosidase II, and TGN38. Treatment with brefeldin A from 2 to 24 h postinfection markedly inhibited incorporation into virions of VP22 and US9 but to a lesser degree with VP16 and VP13/14. These results suggest that, in the cell body of neurons, most tegument proteins are incorporated into unenveloped nucleocapsids prior to envelopment in the Golgi and the trans-Golgi network. These findings give further support to the deenvelopment-reenvelopment hypothesis for viral egress. Finally, the addition of tegument proteins to unenveloped nucleocapsids in the cell body allows access to these unenveloped nucleocapsids to one of two pathways: egress through the cell body or transport into the axon.     

Brefeldin A inhibits pestivirus release from infected cells, without affecting its assembly and infectivity

The enveloped bovine viral diarrhea virus (BVDV) is a member of the Pestivirus genus within the Flaviviridae family. While considerable information has been gathered on virus entry into the host cell, genome structure and protein function, little is known about pestivirus morphogenesis and release from cells. Here, we analyzed the intracellular localization, N-glycan processing and secretion of BVDV using brefeldin A (BFA), which blocks protein export from the endoplasmic reticulum (ER) and causes disruption of the Golgi complex with subsequent fusion of its cis and medial cisternae with the ER. BFA treatment of infected cells resulted in complete inhibition of BVDV secretion and increased co-localization of the envelope glycoproteins with the cis-Golgi marker GM 130. Processing of the N-linked glycans was affected by BFA, however, virus assembly was not perturbed and intracellular virions were fully infectious, suggesting that trafficking beyond the cis-Golgi is not a prerequisite for pestivirus infectivity.     

Redistribution of cellular and herpes simplex virus proteins from the trans-golgi network to cell junctions without enveloped capsids

Herpes simplex virus (HSV) and other alphaherpesviruses assemble enveloped virions in the trans-Golgi network (TGN) or endosomes. Enveloped particles are formed when capsids bud into TGN/endosomes and virus particles are subsequently ferried to the plasma membrane in TGN-derived vesicles. Little is known about the last stages of virus egress from the TGN/endosomes to cell surfaces except that the HSV directs transport of nascent virions to specific cell surface domains, i.e., epithelial cell junctions. Previously, we showed that HSV glycoprotein gE/gI accumulates extensively in the TGN at early times after infection and also when expressed without other viral proteins. At late times of infection, gE/gI and a cellular membrane protein, TGN46, were redistributed from the TGN to epithelial cell junctions. We show here that gE/gI and a second glycoprotein, gB, TGN46, and another cellular protein, carboxypeptidase D, all moved to cell junctions after infection with an HSV mutant unable to produce cytoplasmic capsids. This redistribution did not involve L particles. In contrast to TGN membrane proteins, several cellular proteins that normally adhere to the cytoplasmic face of TGN, Golgi, and endosomal membranes remained primarily dispersed throughout the cytoplasm. Therefore, cellular and viral membrane TGN proteins move to cell junctions at late times of HSV infection when the production of enveloped particles is blocked. This is consistent with the hypothesis that there are late HSV proteins that reorganize or redistribute TGN/endosomal compartments to promote virus egress and cell-to-cell spread.     

Role of cytoplasmic vacuoles in varicella-zoster virus glycoprotein trafficking and virion envelopment.

Varicella-zoster virus (VZV) encodes several glycoproteins which are present on both mature viral envelopes and the surfaces of infected cell membranes. Mechanisms of VZV glycoprotein transport and virion envelopment were investigated by both continuous radiolabeling and pulse-chase analyses with tritiated fucose in VZV-infected cells. We studied in detail the large cytoplasmic vacuoles which were present in infected cells but absent from uninfected cells. The specific activity in each subcellular compartment was defined by quantitative electron microscope autoradiography, using a cross-fire probability matrix analysis to more accurately assess the individual compartment demarcated by the silver grains. By these techniques, we documented a progression of activity originating in the Golgi apparatus and traveling through the post-Golgi region into virus-induced cytoplasmic vacuoles and finally to areas of the cellular membrane associated with the egress of viral particles. Significant amounts of radiolabel were not observed in the nucleus, and only low levels of radiolabel were associated with the cellular membrane not involved with the egress of viral particles. In addition, immunolabeling of Lowicryl-embedded VZV-infected cells demonstrated the presence of VZV glycoproteins within cytoplasmic vacuole membranes as well as on virion envelopes. These observations suggested that cytoplasmic vacuoles harbored VZV-specified glycoproteins and were also the predominant site of VZV virion envelopment within the infected cell. Neither enveloped nor unenveloped viral particles were observed within the Golgi apparatus itself.     

Glycoprotein M of herpes simplex virus 1 is incorporated into virions during budding at the inner nuclear membrane

It is widely accepted that nucleocapsids of herpesviruses bud through the inner nuclear membrane (INM), but few studies have been undertaken to characterize the composition of these nascent virions. Such knowledge would shed light on the budding reaction at the INM and subsequent steps in the egress pathway. The present study focuses on glycoprotein M (gM), a type III integral membrane protein of herpes simplex virus 1 (HSV-1) that likely contains eight transmembrane domains. The results indicated that gM localized primarily at the perinuclear region, with especially bright staining near the nuclear membrane (NM). Immunogold electron microscopic analysis indicated that, like gB and gD (M. R. Torrisi et al., J. Virol. 66:554-561, 1992), gM localized within both leaflets of the NM, the envelopes of nascent virions that accumulate in the perinuclear space, and the envelopes of cytoplasmic and mature extracellular virus particles. Indirect immunofluorescence studies revealed that gM colocalized almost completely with a marker of the Golgi apparatus and partially with a marker of the trans-Golgi network (TGN), whether or not these markers were displaced to the perinuclear region during infection. gM was also located in punctate extensions and invaginations of the NM induced by the absence of a viral kinase encoded by HSV-1 U(S)3 and within virions located in these extensions. Our findings therefore support the proposition that gM, like gB and gD, becomes incorporated into the virion envelope upon budding through the INM. The localization of viral glycoproteins and Golgi and TGN markers to a perinuclear region may represent a mechanism to facilitate the production of infectious nascent virions, thereby increasing the amount of infectivity released upon cellular lysis.