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.     

Herpesvirus Envelopment

The growth and envelopment processes of three representative herpesviruses, equine abortion, pseudorabies, and herpes simplex, were examined in baby hamster kidney (BHK 21/13) cells by bioassay (plaque-forming units) and electron microscopy. The envelopment process was identical for all three viruses. After assembly in the nucleus, the nucleocapsid acquired an envelope by budding from the inner nuclear membrane. This membrane was reduplicated as the enveloped particle was released so that the budding process did not result in disruption of the continuity of the nuclear membrane. That portion of the nuclear membrane which comprised the viral envelope was appreciably thicker than the remainder of the membrane and exhibited numerous projections on its surface. Once enveloped, the viral particles were seen in vesicles and vacuoles in the cell cytoplasm. These appeared to open at the cytoplasmic membrane, releasing the virus from the cell. There was no detectable difference in the size or appearance of enveloped particles in intra- or extracellular locations.     

More than one door - Budding of enveloped viruses through cellular membranes

Enveloped viruses exit their host cell by budding from a cellular membrane and thereby spread from one cell to another. Virus budding in general involves the distortion of a cellular membrane away from the cytoplasm, envelopment of the viral capsid by one or more lipid bilayers that are enriched in viral membrane glycoproteins, and a fission event that separates the enveloped virion from the cellular membrane. While it was initially thought that virus budding is always driven by viral transmembrane proteins interacting with the inner structural proteins, it is now clear that the driving force may be different depending on the virus. Research over the past years has shown that viral components specifically interact with host cell lipids and proteins, thereby adopting cellular functions and pathways to facilitate virus release. This review summarizes the current knowledge of the cellular membrane systems that serve as viral budding sites and of the viral and cellular factors involved in budding. One of the best studied cellular machineries required for virus egress is the ESCRT complex, which will be described in more detail.     

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.     

Biochemical requirements of virus wrapping by the endoplasmic reticulum: involvement of ATP and endoplasmic reticulum calcium store during envelopment of African swine fever virus

Enwrapment by membrane cisternae has emerged recently as a mechanism of envelopment for large enveloped DNA viruses, such as herpesviruses, poxviruses, and African swine fever (ASF) virus. For both ASF virus and the poxviruses, wrapping is a multistage process initiated by the recruitment of capsid proteins onto membrane cisternae of the endoplasmic reticulum (ER) or associated ER-Golgi intermediate membrane compartments. Capsid assembly induces progressive bending of membrane cisternae into the characteristic shape of viral particles, and envelopment provides virions with two membranes in one step. We have used biochemical assays for ASF virus capsid recruitment, assembly, and envelopment to define the cellular processes important for the enwrapment of viruses by membrane cisternae. Capsid assembly on the ER membrane, and envelopment by ER cisternae, were inhibited when cells were depleted of ATP or depleted of calcium by incubation with A23187 and EDTA or the ER calcium ATPase inhibitor, thapsigargin. Electron microscopy analysis showed that cells depleted of calcium were unable to assemble icosahedral particles. Instead, assembly sites contained crescent-shaped and bulbous structures and, in rare cases, empty closed five-sided particles. Interestingly, recruitment of the capsid protein from the cytosol onto the ER membrane did not require ATP or an intact ER calcium store. The results show that following recruitment of the virus capsid protein onto the ER membrane, subsequent stages of capsid assembly and enwrapment are dependent on ATP and are regulated by the calcium gradients present across the ER membrane cisternae.     

Intracellular Trafficking of the UL11 Tegument Protein of Herpes Simplex Virus Type 1

Growing evidence indicates that herpes simplex virus type 1 (HSV-1) acquires its final envelope in the trans-Golgi network (TGN). During the envelopment process, the viral nucleocapsid as well as the envelope and tegument proteins must arrive at this site in order to be incorporated into assembling virions. To gain a better understanding of how these proteins associate with cellular membranes and target to the correct compartment, we have been studying the intracellular trafficking properties of the small tegument protein encoded by the U(L)11 gene of HSV-1. This 96-amino-acid, myristylated protein accumulates on the cytoplasmic face of internal membranes, where it is thought to play a role in nucleocapsid envelopment and egress. When expressed in the absence of other HSV-1 proteins, the UL11 protein localizes to the Golgi apparatus, and previous deletion analyses have revealed that the membrane-trafficking information is contained within the first 49 amino acids. The goal of this study was to map the functional domains required for proper Golgi membrane localization. In addition to N-terminal myristylation, which allows for weak membrane binding, UL11 appears to be palmitylated on one or more of three consecutive N-terminal cysteines. Using membrane-pelleting experiments and confocal microscopy, we show that palmitylation of UL11 is required for both Golgi targeting specificity and strong membrane binding. Furthermore, we found that a conserved acidic cluster within the first half of UL11 is required for the recycling of this tegument protein from the plasma membrane to the Golgi apparatus. Taken together, our results demonstrate that UL11 has highly dynamic membrane-trafficking properties, which suggests that it may play multiple roles on the plasma membrane as well as on the nuclear and TGN membranes.     

Mono-ubiquitylated ORF45 Mediates Association of KSHV Particles with Internal Lipid Rafts for Viral Assembly and Egress

Herpesviruses acquire their envelope by budding into the lumen of cytoplasmic membrane vesicles. This process is initiated by component(s) on viral particles, which recognize the budding site where the viral glycoproteins are present and recruit cellular cargo transport and sorting machinery to the site to complete the budding process. Proteins in the tegument layer, connecting capsid and envelope, are candidates for the recognition of budding sites on vesicle membrane and induction of budding and final envelopment. We examined several outer and matrix tegument proteins of Kaposi’s sarcoma-associated herpesvirus (KSHV) and found that ORF45 associates with lipid rafts (LRs) of cellular membrane. LRs are membrane micro-domains, which have been implicated as relay stations in intracellular signaling and transport including viral entry and virion assembly. The ability of ORF45 to target LR is dependent on the mono-ubiquitylation of ORF45 at Lys297 as the mutation at Lys297 (K297R) abolished LR-association of ORF45. The K297R mutation also impairs ORF45 and viral particle co-localization with trans-Golgi network and endosomes, but facilitates ORF45 and viral particles co-localizing with lysosomes. More importantly, the recombinant KSHV carrying ORF45 K297R mutant (BAC-K297R) was found severely defective in producing mature and infectious virion particles in comparison to wild type KSHV (BAC16). Taken together, our results reveal a new function of KSHV tegument protein ORF45 in targeting LR of host cell membrane, promoting viral particles co-localization with trans-Golgi and endosome vesicles and facilitating the maturation and release of virion particles, suggesting that ORF45 plays a role in bringing KSHV particles to the budding site on cytoplasmic vesicle membrane and triggering the viral budding process for final envelopment and virion maturation.     

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.     

Pseudorabies virus UL37 gene product is involved in secondary envelopment

Herpesvirus envelopment is a two-step process which includes acquisition of a primary envelope resulting from budding of intranuclear capsids through the inner nuclear membrane. Fusion with the outer leaflet of the nuclear membrane releases nucleocapsids into the cytoplasm, which then gain their final envelope by budding into trans-Golgi vesicles. It has been shown that the UL34 gene product is required for primary envelopment of the alphaherpesvirus pseudorabies virus (PrV) (B. G. Klupp, H. Granzow, and T. C. Mettenleiter, J. Virol. 74:10063-10073, 2000). For secondary envelopment, several virus-encoded PrV proteins are necessary, including glycoproteins E, I, and M (A. R. Brack, J. M. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364-5372, 1999). We show here that the product of the UL37 gene of PrV, which is a constituent of mature virions, is involved in secondary envelopment. Replication of a UL37 deletion mutant, PrV-DeltaUL37, was impaired in normal cells; this defect could be complemented on cells stably expressing UL37. Ultrastructural analysis demonstrated that intranuclear capsid maturation and budding of capsids into and release from the perinuclear space were unimpaired. However, secondary envelopment was drastically reduced. Instead, apparently DNA-filled capsids accumulated in the cytoplasm in large aggregates similar to those observed in the absence of glycoproteins E/I and M but lacking the surrounding electron-dense tegument material. Although displaying an ordered structure, capsids did not contact each other directly. We postulate that the UL37 protein is necessary for correct addition of other tegument proteins, which are required for secondary envelopment. In the absence of the UL37 protein, capsids interact with each other through unknown components but do not acquire the electron-dense tegument which is normally found around wild-type capsids during and after secondary envelopment. Thus, apposition of the UL37 protein to cytoplasmic capsids may be crucial for the addition of other tegument proteins, which in turn are able to interact with viral glycoproteins to mediate secondary envelopment.     

Varicella-zoster viral glycoprotein envelopment: ultrastructural cytochemical localization

The periodate-thiocarbohydrazide silver proteinate (PA-TCH-SP) method was used to study the envelopment process in varicella-zoster virus-infected human melanoma cells. Viral envelopment could be seen at two sites, the nuclear membrane and at virus-induced intracytoplasmic vacuoles. Virus-associated glycoconjugates were detected by the PA-TCH-SP method at the plasmalemma and on the inner membrane of the intracytoplasmic vacuoles. Virion envelopes acquired at the nuclear membrane were PA-TCH-SP negative, whereas those acquired at intracytoplasmic vacuoles were PA-TCH-SP positive. All virions found inside these vacuoles contained periodate-reactive envelopes. Release of virions into the extracellular space, where virtually all virions were PA-TCH-SP positive, appeared to be via exocytosis. Thus, the PA-TCH-SP method identifies glycoprotein incorporation at specific cytoplasmic vacuoles distinct from nuclear envelope, endoplasmic reticulum, and Golgi lamellae. These results suggest that envelopment within the cytoplasm is a stage in the assembly of the varicella-zoster virion.     

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.     

Cytomegalovirus primary envelopment occurs at large infoldings of the inner nuclear membrane

We have investigated the morphogenesis of human and murine cytomegalovirus by transmission electron microscopy after high-pressure freezing, freeze substitution, and plastic embedding. We observed large tubular infoldings of the inner nuclear membrane that were free of lamina and active in primary envelopment and subsequent transport of capsids to the nuclear periphery. Semiquantitative determinations of the enlarged inner nuclear membrane area and the location of the primary envelopment of nucleocapsids demonstrated that this structure represents a virus-induced specialized membrane domain at which the particles are preferentially enveloped. This is a previously undescribed structural element relevant in cytomegalovirus morphogenesis.     

Herpesvirus assembly: a tale of two membranes

Herpes virions are amongst the most complex virus particles: they comprise in excess of thirty virally encoded proteins, and also contain cellular components. Capsid formation and the cleavage and encapsidation of replicated viral DNA occur in the nucleus and resemble similar processes in tailed dsDNA (double-stranded DNA) bacteriophages, which indicates they might have common ancestry. In contrast, final virion maturation takes place in the cytoplasm. Nucleocapsids gain access to this compartment by envelopment at the inner nuclear membrane, which involves the interaction between viral and cellular proteins in order to locally alter nuclear architecture. Fusion of the primary viral envelope with the outer nuclear membrane results in translocation of the nucleocapsid to the cytoplasm. Here, the majority of the tegument - a structure, composed of a multitude of different proteins, that links the capsid and the envelope - is added to nucleocapsids, which obtain their final envelope by budding into glycoprotein-containing Golgi-derived vesicles. Thus, herpesvirus morphogenesis proceeds in two different cellular compartments, involving different viral and cellular proteins.     

Herpes simplex virus 1 envelopment follows two diverse pathways

Herpesvirus envelopment is assumed to follow an uneconomical pathway including primary envelopment at the inner nuclear membrane, de-envelopment at the outer nuclear membrane, and reenvelopment at the trans-Golgi network. In contrast to the hypothesis of de-envelopment by fusion of the primary envelope with the outer nuclear membrane, virions were demonstrated to be transported from the perinuclear space to rough endoplasmic reticulum (RER) cisternae. Here we show by high-resolution microscopy that herpes simplex virus 1 envelopment follows two diverse pathways. First, nuclear envelopment includes budding of capsids at the inner nuclear membrane into the perinuclear space whereby tegument and a thick electron dense envelope are acquired. The substance responsible for the dense envelope is speculated to enable intraluminal transportation of virions via RER into Golgi cisternae. Within Golgi cisternae, virions are packaged into transport vacuoles containing one or several virions. Second, for cytoplasmic envelopment, capsids gain direct access from the nucleus to the cytoplasm via impaired nuclear pores. Cytoplasmic capsids could bud at the outer nuclear membrane, at membranes of RER, Golgi cisternae, and large vacuoles, and at banana-shaped membranous entities that were found to continue into Golgi membranes. Envelopes originating by budding at the outer nuclear membrane and RER membrane also acquire a dense substance. Budding at Golgi stacks, designated wrapping, results in single virions within small vacuoles that contain electron-dense substances between envelope and vacuolar membranes.     

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.     

Morphogenesis of a highly replicative EGFPVP22 recombinant Marek's disease virus in cell culture

Marek's disease virus (MDV) is an alphaherpesvirus for which infection is strictly cell associated in permissive cell culture systems. In contrast to most other alphaherpesviruses, no comprehensive ultrastructural study has been published to date describing the different stages of MDV morphogenesis. To circumvent problems linked to nonsynchronized infection and low infectivity titers, we generated a recombinant MDV expressing an enhanced green fluorescent protein fused to VP22, a major tegument protein that is not implicated in virion morphogenesis. Growth of this recombinant virus in cell culture was decreased threefold compared to that of the parental Bac20 virus, but this mutant was still highly replicative. The recombinant virus allowed us to select infected cells by cell-sorting cytometry at late stages of infection for subsequent transmission electron microscopy analysis. Under these conditions, all of the stages of assembly and virion morphogenesis could be observed except extracellular enveloped virions, even at the cell surface. We observed 10-fold fewer naked cytoplasmic capsids than nuclear capsids, and intracellular enveloped virions were very rare. The partial envelopment of capsids in the cytoplasm supports the hypothesis of the acquisition of the final envelope in this cellular compartment. We demonstrate for the first time that, compared to other alphaherpesviruses, MDV seems deficient in three crucial steps of viral morphogenesis, i.e., release from the nucleus, secondary envelopment, and the exocytosis process. The discrepancy between the efficiency with which this MDV mutant spreads in cell culture and the relatively inefficient process of its envelopment and virion release raises the question of the MDV cell-to-cell spreading mechanism.     

Morphogenesis of Sindbis virus in cultured Aedes albopictus cells.

Cultured mosquito cells were found to produce Sindbis virus nearly as efficiently as BHK-21 cells at 28 C. In virtually all of the cells observed in the electron microscope, virus morphogenesis was found to occur within complex vesicular structures which developed after viral infection. Viral nucleocapsids were first seen in these vesicles and appeared to be enveloped within these structures. The process of envelopment within these inclusions differed in some respects from the process previously described for the envelopment of nucleocapsids at the plasma membrane of vertebrae cells. Free nucleocapsids were only rarely seen in the cytoplasm of infected mosquito cells, and budding of virus from the cell surface was detected so infrequently that this process of virus production could not account for the amount of virus produced by the infected cells. The vast majority of extracellular virus was produced by the fusion of the virus-containing vesicles with the plasma membrane releasing mature virions and membrane nucleocapsid complexes in various stages of development.     

Analysis of Rab GTPase-activating proteins indicates that Rab1a/b and Rab43 are important for herpes simplex virus 1 secondary envelopment

Assembly of herpes simplex virus 1 (HSV-1) occurs in the cytoplasm, where the capsid and tegument bud into host cell membranes. It is at this point that the viral glycoproteins are incorporated into the virion, as they are located at the assembly site. We investigated the role of the Rab GTPases in coordinating the assembly process by overexpressing 37 human Rab GTPase-activating proteins (GAPs) and assessing infectious titers. Rab GTPases are key cellular regulators of membrane trafficking events that, by their membrane association and binding of effector proteins, ensure the appropriate fusion of membranes. We identified that TBC1D20 and RN-tre and their partner Rabs, Rab1a/b and Rab43, respectively, are important for virion assembly. In the absence of Rab1a/b, the viral glycoproteins are unable to traffic from the endoplasmic reticulum to the assembly compartment, and thus unenveloped particles build up in the cytoplasm. The defect resulting from Rab43 depletion is somewhat more complex, but it appears that the fragmentation and dispersal of the trans-Golgi network and associated membranes render these compartments unable to support secondary envelopment.     

Nuclear egress and envelopment of herpes simplex virus capsids analyzed with dual-color fluorescence HSV1(17+)

To analyze the assembly of herpes simplex virus type 1 (HSV1) by triple-label fluorescence microscopy, we generated a bacterial artificial chromosome (BAC) and inserted eukaryotic Cre recombinase, as well as β-galactosidase expression cassettes. When the BAC pHSV1(17⁺)blueLox was transfected back into eukaryotic cells, the Cre recombinase excised the BAC sequences, which had been flanked with loxP sites, from the viral genome, leading to HSV1(17⁺)blueLox. We then tagged the capsid protein VP26 and the envelope protein glycoprotein D (gD) with fluorescent protein domains to obtain HSV1(17⁺)blueLox-GFPVP26-gDRFP and -RFPVP26-gDGFP. All HSV1 BACs had variations in the a-sequences and lost the oriL but were fully infectious. The tagged proteins behaved as their corresponding wild type, and were incorporated into virions. Fluorescent gD first accumulated in cytoplasmic membranes but was later also detected in the endoplasmic reticulum and the plasma membrane. Initially, cytoplasmic capsids did not colocalize with viral glycoproteins, indicating that they were naked, cytosolic capsids. As the infection progressed, they were enveloped and colocalized with the viral membrane proteins. We then analyzed the subcellular distribution of capsids, envelope proteins, and nuclear pores during a synchronous infection. Although the nuclear pore network had changed in ca. 20% of the cells, an HSV1-induced reorganization of the nuclear pore architecture was not required for efficient nuclear egress of capsids. Our data are consistent with an HSV1 assembly model involving primary envelopment of nuclear capsids at the inner nuclear membrane and primary fusion to transfer capsids into the cytosol, followed by their secondary envelopment on cytoplasmic membranes.     

Cellular p32 recruits cytomegalovirus kinase pUL97 to redistribute the nuclear lamina

Replication of human cytomegalovirus is limited at the level of nucleocytoplasmic transport of viral capsids, a process that requires the disassembly of the nuclear lamina. Deletion of the protein kinase gene UL97 from the viral genome showed that the activity of pUL97 plays an important role for viral capsid egress. Here, we report that p32, a novel cellular interactor of the viral kinase pUL97, promotes the accumulation of pUL97 at the nuclear membrane by recruiting the p32-pUL97 complex to the lamin B receptor. Transfection of active pUL97, but not a catalytically inactive mutant, induced a redistribution of lamina components as demonstrated for recombinant lamin B receptor-green fluorescent protein and endogenous lamins A and C. Consistent with this, p32 itself and lamins were phosphorylated by pUL97. Importantly, overexpression of p32 in human cytomegalovirus-infected cells resulted in increased efficiency of viral replication and release of viral particles. Thus, it is highly suggestive that the cellular protein p32 recruits pUL97 to induce a dissolution of the nuclear lamina thereby facilitating the nuclear export of viral capsids.