Common and specific properties of herpesvirus UL34/UL31 protein family members revealed by protein complementation assay

The proteins encoded by the UL34 and UL31 genes of herpes simplex virus are conserved among herpesviruses. They form a complex that is essential for the egress of the herpesvirus nucleocapsids from the nucleus. In previous work on the homologous protein complex in murine cytomegalovirus (MCMV), we defined their mutual binding domains. Here, we started to map binding domains within the UL34/UL31 proteins of alpha-, beta-, and gammaherpesviruses and to locate other functional properties. A protein complementation assay (PCA) using the TEM-1 beta-lactamase fragments fused to UL31 and UL34 protein homologues was used to study protein-protein interactions in cells. Wild-type MCMV M50 and M53 provided a strong reaction in the PCA, whereas mutants unable to form a complex did not. The homologous pairs of herpes simplex virus type 1, pseudorabies virus, human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and murine herpes virus 68 proteins also reacted, with the exception of the EBV proteins. Cross-complementation was found to be positive only within the same herpesvirus subfamily. Moreover, the HCMV homologues rescued replication-defective MCMV genomes lacking one or the other gene. We identified the binding site of M53 for M50 in the first conserved region (CR1) (M. Loetzerich, Z. Ruzsics, and U. H. Koszinowski, J. Virol. 80:73-84). Here we show that the CR1 of all tested UL31 proteins contains the UL34 binding site, and chimeric proteins carrying the subfamily-specific CR1 rescued the ability to cross-complement in the PCA.     

Human Cytomegalovirus UL50 and UL53 Recruit Viral Protein Kinase UL97, Not Protein Kinase C,for Disruption of Nuclear Lamina and Nuclear Egress in Infected Cells

Herpesvirus nucleocapsids traverse the nuclear envelope into the cytoplasm in a process called nuclear egress that includes disruption of the nuclear lamina. In several herpesviruses, a key player in nuclear egress is a complex of two proteins, whose homologs in human cytomegalovirus (HCMV) are UL50 and UL53. However, their roles in nuclear egress during HCMV infection have not been shown. Based largely on transfection studies, UL50 and UL53 have been proposed to facilitate disruption of the nuclear lamina by recruiting cellular protein kinase C (PKC), as occurs with certain other herpesviruses, and/or the viral protein kinase UL97 to phosphorylate lamins. To investigate these issues during HCMV infection, we generated viral mutants null for UL50 or UL53. Correlative light electron microscopic analysis of null mutant-infected cells showed the presence of intranuclear nucleocapsids and the absence of cytoplasmic nucleocapsids. Confocal immunofluorescence microscopy revealed that UL50 and UL53 are required for disruption of the nuclear lamina. A subpopulation of UL97 colocalized with the nuclear rim, and this was dependent on UL50 and, to a lesser extent, UL53. However, PKC was not recruited to the nuclear rim, and its localization was not affected by the absence of UL50 or UL53. Immunoprecipitation from cells infected with HCMV expressing tagged UL53 detected UL97 but not PKC. In summary, HCMV UL50 and UL53 are required for nuclear egress and disruption of nuclear lamina during HCMV infection, and they recruit UL97, not PKC, for these processes. Thus, despite the strong conservation of herpesvirus nuclear egress complexes, a key function can differ among them.     

Dominant Negative Mutants of the Murine Cytomegalovirus M53 Gene Block Nuclear Egress and Inhibit Capsid Maturation

The alphaherpesvirus proteins UL31 and UL34 and their homologues in other herpesvirus subfamilies cooperate at the nuclear membrane in the export of nascent herpesvirus capsids. We studied the respective betaherpesvirus proteins M53 and M50 in mouse cytomegalovirus (MCMV). Recently, we established a random approach to identify dominant negative (DN) mutants of essential viral genes and isolated DN mutants of M50 (B. Rupp, Z. Ruzsics, C. Buser, B. Adler, P. Walther and U. H. Koszinowski, J. Virol 81:5508-5517). Here, we report the identification and phenotypic characterization of DN alleles of its partner, M53. While mutations in the middle of the M53 open reading frame (ORF) resulted in DN mutants inhibiting MCMV replication by ∼100-fold, mutations at the C terminus resulted in up to 1,000,000-fold inhibition of virus production. C-terminal DN mutants affected nuclear distribution and steady-state levels of the nuclear egress complex and completely blocked export of viral capsids. In addition, they induced a marked maturation defect of viral capsids, resulting in the accumulation of nuclear capsids with aberrant morphology. This was associated with a two-thirds reduction in the total amount of unit length genomes, indicating an accessory role for M53 in DNA packaging.Our understanding of herpesvirus morphogenesis is mainly derived from studies of Alphaherpesvirinae, such as herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PrV). A faster replication cycle and a more productive infection in tissue culture aided genetic analysis of alphaherpesvirus morphogenesis. In addition, deletion mutants of key morphogenesis genes in alphaherpesviruses often maintain basic replication capacity, whereas the mutations of their homologues in Betaherpesvirinae or Gammaherpesvirinae mostly result in a lethal phenotype (for the UL31 and the UL34 family, see references 3, 6, 9-11, 16, 20, 21, and 42). These genes became amenable to comprehensive genetic analysis in betaherpesviruses only after their genomes were cloned as infectious bacterial artificial chromosomes (BACs), which obviated the need to generate replication-competent intermediates or complementing cell lines (3, 21, 23). BAC-based mutagenesis allowed viability screens mapping essential genes (8, 43) or even functional sites of essential genes in cytomegaloviruses (3, 21). However, these approaches cannot easily be applied to reveal the null phenotypes in the context of virus replication, as mutant viruses are not easily reconstituted. In addition, deletion of an essential viral gene can reveal the null phenotype of only the first of perhaps several essential functions during virus morphogenesis. This problem can be addressed to some extent by using dominant negative (DN) mutations (36). DN mutants are loss-of-function mutants that induce a null phenotype in the presence of the wild-type (wt) allele (14). Analysis of phenotypes induced by DN mutants proved to be extremely useful in genetics and cell biology, signaling, and biochemistry. Such inhibitory mutants of cellular proteins are often designed based on knowledge on the structural or functional role of a well-characterized protein domain. Unfortunately, we lack the structural information that would allow knowledge-based design of viral DN mutants for the majority of herpesvirus gene products. Thus, we established a random screen consisting of three steps to identify mutants of viral genes with DN potential (36): (i) a library of mutants is generated by random insertion of 5 amino acids (aa) or a stop codon into the open reading frame (ORF) of interest using transposon mutagenesis, (ii) nonfunctional mutants are identified by cis complementation of the respective deletion mutant mouse cytomegalovirus (MCMV) BAC, and (iii) nonfunctional mutants are tested for their inhibitory potential upon reconstitution of the wt BAC cloned genomes. In the last screen, mutants that have a specific inhibitory effect on the activity of the wt allele are selected. The specific phenotype obtained upon induction of the inhibitory mutants in the context of virus replication is then verified and further characterized using a tetracycline (Tet) regulon-based viral conditional expression system (36, 37).One intriguing aspect of herpesvirus morphogenesis is the transition of capsids from the nuclear to the cytoplasmic phase of virus morphogenesis. Two conserved nonstructural proteins, the homologues of the membrane protein pUL34 and its nuclear partner protein pUL31, form a nuclear egress complex (NEC) (18, 27, 42), which is required for primary envelopment and export of nuclear capsids to the cytoplasm (reviewed in references 24 and 25). Recent studies have revealed that the homologues of alphaherpesvirus pUL34 and pUL31, the M50 and the M53 gene products of the betaherpesvirus MCMV (pM50 and pM53, respectively) and the BFRF1 and the BFLF2 gene products of the gammaherpesvirus Epstein-Barr virus (EBV), apparently share the major functions of these two proteins. The lack of one or both proteins of the NEC generally results in the retention of viral capsids in the nucleus. This is lethal for beta- and gammaherpesvirus production (3, 9-11, 16, 18, 21, 27, 35, 42).The details of the mechanisms by which the NEC proteins mediate capsid export through the nuclear envelope are poorly understood. We (3, 21, 36, 38) and others (1, 19, 34) have started to dissect details of the NEC function using a genetic approach based on subtle mutagenesis of the respective genes. Analysis of the MCMV M50 gene by comprehensive mutagenesis localized two different functional sites. They were the M53 binding site within the N-terminal domain of M50, as well as the transmembrane region at its C terminus (3). Liang and Baines located the respective binding site in HSV-1 UL34 at aa 137 to 181 (19). Our approach, based on screens for DN mutants, identified a proline-rich sequence (aa 179 to 207) in the M50 gene product as an additional essential region (36). A recombinant virus expressing an M50 mutant lacking this site was defective in capsid egress from the nucleus despite the presence of the wt M50 protein. Consequently, the production of infectious particles after infection was reduced by more than 2 orders of magnitude. The UL34 homologues of alpha- and gammaherpesviruses lack a similar polyproline motif, but the result was confirmed by mutating the human cytomegalovirus (HCMV) homologue UL50 at the corresponding region, which is conserved within betaherpesviruses (36). The M50 mutants lacking the proline-rich motif still bind and colocalize to their respective NEC partner, pM53. Interestingly, Bjerke and coworkers also provided genetic evidence for the existence of at least one additional, yet-unknown, but essential functional entity in pUL34 of HSV-1, besides its known pUL31 binding activity, using a screen based on charged-cluster mutations (1). Further analysis of one of the noncomplementing charged-cluster mutants carrying the defect in the N-terminal domain of pUL34 also revealed a DN activity and suggested a new functional site involved in membrane curvature formation, together with the C-terminal domain of UL31 (34).The genetic analysis of M53 by Tn7-based linker scanning mutagenesis, followed by a cis complementation assay, localized the M50-binding site between aa 112 and 137 within the first of the four conserved regions (CRs) shared among the herpesvirus UL31 homologues (21). This analysis, together with a study we performed for further characterization of pM50/pM53 interaction, revealed that the large C-terminal part of pM53, comprising CR2 to -4, must carry at least one additional, yet-unknown, but essential functional site (21, 38).Here, we screened loss-of-function mutants of the MCMV M53 gene to retrieve M53 alleles with DN activity to localize this new functional domain. Mutants with a very strong inhibitory potential accumulated within CR4 of pM53 close to its C terminus. These CR4 mutants induced a block of capsid export from the nucleus. In addition, we could associate these mutations with the induction of a defect in capsid maturation and/or DNA packaging. These data suggested that pM53 is not only crucial for nuclear egress, but also involved in earlier steps of MCMV morphogenesis.     

Analysis of viral and cellular factors influencing herpesvirus-induced nuclear envelope breakdown

Herpesvirus nucleocapsids are translocated from their assembly site in the nucleus to the cytosol by acquisition of a primary envelope at the inner nuclear membrane which subsequently fuses with the outer nuclear membrane. This transport through the nuclear envelope requires homologs of the conserved herpesviral pUL31 and pUL34 proteins which form the nuclear egress complex (NEC). In its absence, 1,000-fold less virus progeny is produced. We isolated a UL34-negative mutant of the alphaherpesvirus pseudorabies virus (PrV), PrV-ΔUL34Pass, which regained replication competence after serial passages in cell culture by inducing nuclear envelope breakdown (NEBD) (B. G. Klupp, H. Granzow, and T. C. Mettenleiter, J. Virol. 85:8285-8292, 2011). To test whether this phenotype is unique, passaging experiments were repeated with a UL31 deletion mutant. After 60 passages, the resulting PrV-ΔUL31Pass replicated similarly to wild-type PrV. Ultrastructural analyses confirmed escape from the nucleus via NEBD, indicating an inherent genetic disposition in herpesviruses. To identify the mutated viral genes responsible for this phenotype, the genome of PrV-ΔUL34Pass was sequenced and compared to the genomes of parental PrV-Ka and PrV-ΔUL34. Targeted sequencing of PrV-ΔUL31Pass disclosed congruent mutations comprising genes encoding tegument proteins (pUL49, pUL46, pUL21, pUS2), envelope proteins (gI, pUS9), and protease pUL26. To investigate involvement of cellular pathways, different inhibitors of cellular kinases were tested. While induction of apoptosis or inhibition of caspases had no specific effect on the passaged mutants, roscovitine, a cyclin-dependent kinase inhibitor, and U0126, an inhibitor of MEK1/2, specifically impaired replication of the passaged mutants, indicating involvement of mitosis-related processes in herpesvirus-induced NEBD.     

Crystal Structure of the Human Cytomegalovirus pUL50-pUL53 Core Nuclear Egress Complex Provides Insight into a Unique Assembly Scaffold for Virus-Host Protein Interactions

Nuclear replication of cytomegalovirus relies on elaborate mechanisms of nucleocytoplasmic egress of viral particles. Thus, the role of two essential and conserved viral nuclear egress proteins, pUL50 and pUL53, is pivotal. pUL50 and pUL53 heterodimerize and form a core nuclear egress complex (NEC), which is anchored to the inner nuclear membrane and provides a scaffold for the assembly of a multimeric viral-cellular NEC. Here, we report the crystal structure of the pUL50-pUL53 heterodimer (amino acids 1–175 and 50–292, respectively) at 2.44 Å resolution. Both proteins adopt a globular fold with mixed α and β secondary structure elements. pUL53-specific features include a zinc-binding site and a hook-like N-terminal extension, the latter representing a hallmark element of the pUL50-pUL53 interaction. The hook-like extension (amino acids 59–87) embraces pUL50 and contributes 1510 Ų to the total interface area (1880 Ų). The pUL50 structure overall resembles the recently published NMR structure of the murine cytomegalovirus homolog pM50 but reveals a considerable repositioning of the very C-terminal α-helix of pUL50 upon pUL53 binding. pUL53 shows structural resemblance with the GHKL domain of bacterial sensory histidine kinases. A close examination of the crystal structure indicates partial assembly of pUL50-pUL53 heterodimers to hexameric ring-like structures possibly providing additional scaffolding opportunities for NEC. In combination, the structural information on pUL50-pUL53 considerably improves our understanding of the mechanism of HCMV nuclear egress. It may also accelerate the validation of the NEC as a unique target for developing a novel type of antiviral drug and improved options of broad-spectrum antiherpesviral therapy.     

Structural basis of membrane budding by the nuclear egress complex of herpesviruses

During nuclear egress, herpesvirus capsids bud at the inner nuclear membrane forming perinuclear viral particles that subsequently fuse with the outer nuclear membrane, releasing capsids into the cytoplasm. This unusual budding process is mediated by the nuclear egress complex (NEC) composed of two conserved viral proteins, UL31 and UL34. Earlier, we discovered that the herpesvirus nuclear egress complex (NEC) could bud synthetic membranes in vitro without the help of other proteins by forming a coat‐like hexagonal scaffold inside the budding membrane. To understand the structural basis of NEC‐mediated membrane budding, we determined the crystal structures of the NEC from two herpesviruses. The hexagonal lattice observed in the NEC crystals recapitulates the honeycomb coats within the budded vesicles. Perturbation of the oligomeric interfaces through mutagenesis blocks budding in vitro confirming that NEC oligomerization into a honeycomb lattice drives budding. The structure represents the first atomic‐level view of an oligomeric array formed by a membrane‐deforming protein, making possible the dissection of its unique budding mechanism and the design of inhibitors to block it.     

Unexpected features and mechanism of heterodimer formation of a herpesvirus nuclear egress complex

Herpesvirus nucleocapsids escape from the nucleus in a process orchestrated by a highly conserved, viral nuclear egress complex. In human cytomegalovirus, the complex consists of two proteins, UL50 and UL53. We solved structures of versions of UL53 and the complex by X‐ray crystallography. The UL53 structures, determined at 1.93 and 3.0 Å resolution, contained unexpected features including a Bergerat fold resembling that found in certain nucleotide‐binding proteins, and a Cys₃His zinc finger. Substitutions of zinc‐coordinating residues decreased UL50–UL53 co‐localization in transfected cells, and, when incorporated into the HCMV genome, ablated viral replication. The structure of the complex, determined at 2.47 Å resolution, revealed a mechanism of heterodimerization in which UL50 clamps onto helices of UL53 like a vise. Substitutions of particular residues on the interaction interface disrupted UL50–UL53 co‐localization in transfected cells and abolished virus production. The structures and the identification of contacts can be harnessed toward the rational design of novel and highly specific antiviral drugs and will aid in the detailed understanding of nuclear egress.     

Structural determinants for nuclear envelope localization and function of pseudorabies virus pUL34

Herpesvirus proteins pUL34 and pUL31 form a complex at the inner nuclear membrane (INM) which is necessary for efficient nuclear egress. Pseudorabies virus (PrV) pUL34 is a type II membrane protein of 262 amino acids (aa). The transmembrane region (TM) is predicted to be located between aa 245 and 261, leaving only one amino acid in the C terminus that probably extends into the perinuclear space. It is targeted to the nuclear envelope in the absence of other viral proteins, pointing to intrinsic localization motifs, and shows structural similarity to cellular INM proteins like lamina-associated polypeptide (Lap) 2ß and Emerin. To investigate which domains of pUL34 are relevant for localization and function, we constructed chimeric proteins by replacing parts of pUL34 with regions of cellular INM proteins. First the 18 C-terminal amino acids encompassing the TM were exchanged with TM regions and C-terminal domains of Lap2ß and Emerin or with the first TM region of the polytopic lamin B receptor (LBR), including the nine following amino acids. All resulting chimeric proteins complemented the replication defect of PrV-ΔUL34, demonstrating that the substitution of the TM and the extension of the C-terminal domain does not interfere with the function of pUL34. Complementation was reduced but not abolished when the C-terminal 50 aa were replaced by corresponding Lap2ß sequences (pUL34-LapCT50). However, replacing the C-terminal 100 aa (pUL34-LapCT100) resulted in a nonfunctional protein despite continuing pUL31 binding, pointing to an important functional role of this region. The replacement of the N-terminal 100 aa (pUL34-LapNT100) had no effect on nuclear envelope localization but abrogated pUL31 binding and function.     

pUL21 regulation of pUs3 kinase activity influences the nature of nuclear envelope deformation by the HSV-2 nuclear egress complex

It is well established that the herpesvirus nuclear egress complex (NEC) has an intrinsic ability to deform membranes. During viral infection, the membrane-deformation activity of the NEC must be precisely regulated to ensure efficient nuclear egress of capsids. One viral protein known to regulate herpes simplex virus type 2 (HSV-2) NEC activity is the tegument protein pUL21. Cells infected with an HSV-2 mutant lacking pUL21 (ΔUL21) produced a slower migrating species of the viral serine/threonine kinase pUs3 that was shown to be a hyperphosphorylated form of the enzyme. Investigation of the pUs3 substrate profile in ΔUL21-infected cells revealed a prominent band with a molecular weight consistent with that of the NEC components pUL31 and pUL34. Phosphatase sensitivity and retarded mobility in phos-tag SDS-PAGE confirmed that both pUL31 and pUL34 were hyperphosphorylated by pUs3 in the absence of pUL21. To gain insight into the consequences of increased phosphorylation of NEC components, the architecture of the nuclear envelope in cells producing the HSV-2 NEC in the presence or absence of pUs3 was examined. In cells with robust NEC production, invaginations of the inner nuclear membrane were observed that contained budded vesicles of uniform size. By contrast, nuclear envelope deformations protruding outwards from the nucleus, were observed when pUs3 was included in transfections with the HSV-2 NEC. Finally, when pUL21 was included in transfections with the HSV-2 NEC and pUs3, decreased phosphorylation of NEC components was observed in comparison to transfections lacking pUL21. These results demonstrate that pUL21 influences the phosphorylation status of pUs3 and the HSV-2 NEC and that this has consequences for the architecture of the nuclear envelope.     

Identification of a 709-amino-acid internal nonessential region within the essential conserved tegument protein (p)UL36 of pseudorabies virus

Tegument proteins homologous to the essential herpes simplex virus type 1 UL36 gene product (p)UL36 are conserved throughout the Herpesviridae and constitute the largest herpesvirus-encoded proteins. So far, only limited information is available on their functions, which include complex formation with the (p)UL37 homologs via an N-terminal domain and a deubiquitinating activity in the extreme N terminus. For further analysis we constructed deletion mutants lacking 437, 784, 926, 1,046, 1,217, or 1,557 amino acids (aa) from the C terminus. While none of them supported replication of a pseudorabies virus (PrV) UL36 deletion mutant, a mutant polypeptide with an internal deletion from aa 2087 to 2795, which comprises a proline/alanine-rich region, fully complemented the lethal replication defect. Thus, our data indicate that the extreme C terminus of (p)UL36 fulfills an essential role in PrV replication, while a large internal portion of the C-terminal half of the protein is dispensable for replication in cell culture.     

Deletion of Epstein-Barr virus BFLF2 leads to impaired viral DNA packaging and primary egress as well as to the production of defective viral particles

Previous genetic and biochemical studies performed with several members of the Alphaherpesvirus subfamily have shown that the UL31 and UL34 proteins are essential components of the molecular machinery that mediates the primary egress of newly assembled capsids across the nuclear membrane. Further, there is substantial evidence that BFLF2 and BFRF1, the respective positional homologs of UL31 and UL34 in the Epstein-Barr virus (EBV) genome, are also their functional homologs, i.e., that the UL31/UL34 pathway is common to distant herpesviruses. However, the low degree of protein sequence identity between UL31 and BFLF2 would argue against such a hypothesis. To further clarify this issue, we have constructed a recombinant EBV strain devoid of BFLF2 (DeltaBFLF2) and show that BFLF2 is crucial for efficient virus production but not for lytic DNA replication or B-cell transformation. This defective phenotype could be efficiently restored by trans complementation with a BFLF2 expression plasmid. Detailed analysis of replicating cells by electron microscopy revealed that, as expected, DeltaBFLF2 viruses not only failed to egress from the nucleus but also showed defective DNA packaging. Nonfunctional primary egress did not, however, impair the production and extracellular release of enveloped but empty viral particles that comprised L particles containing tegument-like structures and a few virus-like particles carrying empty capsids. The DeltaBFLF2 and DeltaUL31 phenotypes therefore only partly overlap, from which we infer that BFLF2 and UL31 have substantially diverged during evolution to fulfil related but distinct functions.     

Ultrastructural localization of the herpes simplex virus type 1 UL31, UL34, and US3 proteins suggests specific roles in primary envelopment and egress of nucleocapsids

The wild-type UL31, UL34, and US3 proteins localized on nuclear membranes and perinuclear virions; the US3 protein was also on cytoplasmic membranes and extranuclear virions. The UL31 and UL34 proteins were not detected in extracellular virions. US3 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 US3 protein plays an important, but nonessential, role in the egress pathway.     

Specific residues of a conserved domain in the N terminus of the human cytomegalovirus pUL50 protein determine its intranuclear interaction with pUL53

Herpesviral capsids are assembled in the host cell nucleus and are subsequently translocated to the cytoplasm. During this process it has been demonstrated that the human cytomegalovirus proteins pUL50 and pUL53 interact and form, together with other viral and cellular proteins, the nuclear egress complex at the nuclear envelope. In this study we provide evidence that specific residues of a conserved N-terminal region of pUL50 determine its intranuclear interaction with pUL53. In silico evaluation and biophysical analyses suggested that the conserved region forms a regular secondary structure adopting a globular fold. Importantly, site-directed replacement of individual amino acids by alanine indicated a strong functional influence of specific residues inside this globular domain. In particular, mutation of the widely conserved residues Glu-56 or Tyr-57 led to a loss of interaction with pUL53. Consistent with the loss of binding properties, mutants E56A and Y57A showed a defective function in the recruitment of pUL53 to the nuclear envelope in expression plasmid-transfected and human cytomegalovirus-infected cells. In addition, in silico analysis suggested that residues 3-20 form an amphipathic α-helix that appears to be conserved among Herpesviridae. Point mutants revealed a structural role of this N-terminal α-helix for pUL50 stability rather than a direct role in the binding of pUL53. In contrast, the central part of the globular domain including Glu-56 and Tyr-57 is directly responsible for the functional interaction with pUL53 and thus determines formation of the basic nuclear egress complex.     

Proteomic Analysis of the Multimeric Nuclear Egress Complex of Human Cytomegalovirus

Herpesviral capsids are assembled in the host cell nucleus before being translocated into the cytoplasm for further maturation. The crossing of the nuclear envelope represents a major event that requires the formation of the nuclear egress complex (NEC). Previous studies demonstrated that human cytomegalovirus (HCMV) proteins pUL50 and pUL53, as well as their homologs in all members of Herpesviridae, interact with each other at the nuclear envelope and form the heterodimeric core of the NEC. In order to characterize further the viral and cellular protein content of the multimeric NEC, the native complex was isolated from HCMV-infected human primary fibroblasts at various time points and analyzed using quantitative proteomics. Previously postulated components of the HCMV-specific NEC, as well as novel potential NEC-associated proteins such as emerin, were identified. In this regard, interaction and colocalization between emerin and pUL50 were confirmed by coimmunoprecipitation and confocal microscopy analyses, respectively. A functional validation of viral and cellular NEC constituents was achieved through siRNA-mediated knockdown experiments. The important role of emerin in NEC functionality was demonstrated by a reduction of viral replication when emerin expression was down-regulated. Moreover, under such conditions, reduced production of viral proteins and deregulation of viral late cytoplasmic maturation were observed. Combined, these data prove the functional importance of emerin as an NEC component, associated with pUL50, pUL53, pUL97, p32/gC1qR, and further regulatory proteins. Summarized, our findings provide the first proteomics-based characterization and functional validation of the HCMV-specific multimeric NEC.Viruses are tightly linked to the regulatory processes governing the metabolic state of their host cells. This regulatory linkage is reflected by viral activation or silencing of gene expression and productive replication in response to cellular changes in signaling, cell cycle, apoptosis, differentiation, and other parameters. Viruses also tend to exert a strong influence on regulatory cellular pathways and the developmental fate of virus-infected tissues (1, 2). These examples of virus-cell interregulation have been studied in detail, but in many cases the essential molecular mechanisms are still poorly understood. In the field of herpesviruses, profound efforts in molecular research have been undertaken to characterize those direct protein–protein interactions that regulate cross-talk between the virus and its host. Multi-protein complexes composed of both viral and cellular constituents were identified in several stages of herpesviral lytic replication. In particular, detailed studies on the replication of human cytomegalovirus (HCMV)¹ in primary fibroblasts and other permissive cell types have provided very interesting insights into the nature of chimeric multi-protein complexes. These examples were described for viral entry, viral response to intrinsic immunity, intracellular transport of viral products, nucleocytoplasmic egress of viral capsids, and other processes (3–6). In classical approaches, protein–protein interaction was studied by means of approved methods including yeast two-hybrid, coimmunoprecipitation (CoIP), and pulldown analyses with purified proteins. More recently, very sensitive methods have been introduced into this field, such as proteomic analysis using tandem mass spectrometry (MS/MS), confocal imaging techniques, surface plasmon resonance analysis, and others.During HCMV replication, the translocation of genome-containing viral capsids from the nucleus to the cytoplasm (nuclear egress) is one of the most crucial steps. In this process, the nuclear envelope represents a barrier consisting of three distinct elements: nuclear membranes, nuclear pores, and the proteinaceous network of the nuclear lamina. The viral capsids traverse the nuclear envelope by budding through nuclear membranes. Importantly, HCMV capsids access the inner nuclear membrane by overcoming the proteinaceous network of the nuclear lamina. To regulate the serial steps in this procedure, a multimeric protein complex is formed, termed the nuclear egress complex (NEC) (4, 7). One of the main tasks of the NEC is the distortion of the nuclear lamina. Our recent studies identified the formation of lamina-depleted areas that result from the recruitment of sophisticated enzymatic activities to these specific sites at the lamina (8). Viral and cellular effectors, such as protein kinases, a proline cis/trans isomerase, and possibly further regulatory proteins, are involved in this process (4). It is commonly accepted that the core NEC is composed of two viral proteins, namely, pUL50 and pUL53 (9–13). Moreover, the association of pUL50–pUL53 with a number of viral and cellular proteins supports the concept of a multimeric NEC that may include the viral protein kinase pUL97, multi-ligand binding protein p32/gC1qR, lamin B receptor, and protein kinase C (PKC) (14).In this work, we first confirmed the major role played by pUL50 and pUL53 in NEC formation. The pUL50–pUL53 core NEC was then used as bait for the identification of other NEC components at different time points post-infection. Quantitative MS-based proteomics confirmed known members of the multimeric NEC and also identified the cellular inner nuclear membrane protein emerin as a novel NEC constituent. Importantly, colocalization of emerin with the HCMV-specific NEC and its interaction with pUL50 were demonstrated for the first time. Knockdown experiments provided functional validation of the importance of emerin and other NEC proteins for HCMV replication. Together, these data provide an extended mechanistic model for the composition and function of the HCMV-specific NEC.     

Analysis of a Charge Cluster Mutation of Herpes Simplex Virus Type 1 UL34 and Its Extragenic Suppressor Suggests a Novel Interaction between pUL34 and pUL31 That Is Necessary for Membrane Curvature around Capsids

Interaction between pUL34 and pUL31 is essential for targeting both proteins to the inner nuclear membrane (INM). Sequences mediating the targeting interaction have been mapped by others with both proteins. We have previously reported identification of charge cluster mutants of herpes simplex virus type 1 UL34 that localize properly to the inner nuclear membrane, indicating interaction with UL31, but fail to complement a UL34 deletion. We have characterized one mutation (CL04) that alters a charge cluster near the N terminus of pUL34 and observed the following. (i) The CL04 mutant has a dominant-negative effect on pUL34 function, indicating disruption of some critical interaction. (ii) In infections with CL04 pUL34, capsids accumulate in close association with the INM, but no perinuclear enveloped viruses, cytoplasmic capsids, or virions or cell surface virions were observed, suggesting that CL04 UL34 does not support INM curvature around the capsid. (iii) Passage of UL34-null virus on a stable cell line that expresses CL04 resulted in selection of extragenic suppressor mutants that grew efficiently using the mutant pUL34. (iv) All extragenic suppressors contained an R229→L mutation in pUL31 that was sufficient to suppress the CL04 phenotype. (v) Immunolocalization and coimmunoprecipitation experiments with truncated forms of pUL34 and pUL31 confirm that N-terminal sequences of pUL34 and a C-terminal domain of pUL31 mediate interaction but not nuclear membrane targeting. pUL34 and pUL31 may make two essential interactions—one for the targeting of the complex to the nuclear envelope and another for nuclear membrane curvature around capsids.Egress of herpesvirus capsids from the nucleus occurs by envelopment of capsids at the inner nuclear membrane (INM) and is followed by de-envelopment at the outer nuclear membrane (ONM). This process can be broken down into a pathway of discrete steps that begin with recruitment of the viral envelopment apparatus to the INM. Herpes simplex virus type 1 (HSV-1) UL34 and UL31 and their homologs in other herpesviruses are required for efficient envelopment at the INM (7, 13, 22, 23, 29). HSV-1 pUL31 and pUL34 are targeted specifically to the INM by a mechanism that requires their interaction with each other (27, 28), and this mutual dependence is a conserved feature of herpesvirus envelopment (9, 14, 27, 28, 32, 33, 39). Localization of these two proteins at the INM results in the recruitment of other proteins, including protein kinase C delta and pUS3, to the nuclear membrane (22, 24, 30). The sequences in HSV-1 pUL34 that mediate interaction with UL31 and that lead to nuclear envelope targeting were mapped to amino acids (aa) 137 to 181 (16). The sequences in the murine cytomegalovirus (MCMV) homolog of UL31, M53, that mediate the nuclear envelope targeting interaction with the UL34 homolog, M50, were mapped to the N-terminal third of the protein in the first of four conserved regions (17), and Schnee et al. subsequently showed that this same region of pUL31 homologs from other families of herpesviruses mediates interaction with the corresponding pUL34 homologs (33).After the targeting of the pUL34/pUL31 complex to the INM, subsequent steps in nuclear egress include, it is thought, (i) local disruption of the nuclear lamina to allow capsid access to the INM, (ii) recognition and docking of capsids by the envelopment apparatus at the INM, (iii) curvature of the inner and outer nuclear membranes around the capsid, (iv) scission of the INM to create an enveloped virion in the space between the INM and ONM, (v) fusion of the virion envelope with the outer nuclear membrane, and (vi) capsid release into the cytoplasm.At least some of the viral and cellular factors critical for nuclear lamina disruption and for de-envelopment fusion have been identified. pUL34, pUL31, and pUS3 of HSV-1 have all been implicated in changes in localization, interaction, and phosphorylation of nuclear lamina components, including lamins A/C and B and the lamina-associated protein, emerin (3, 15, 19, 20, 24, 26, 34, 35). pUS3, pUL31, and glycoproteins B and H have been implicated in de-envelopment of primary virions at the ONM (8, 21, 28, 30, 38).pUL34 and pUL31 are thought to be involved in steps between lamina disruption and de-envelopment, but genetic evidence in infected cells has so far been lacking. Klupp et al. have shown that overexpression of alphaherpesvirus pUL31 and pUL34 in the absence of other viral proteins can induce formation of small vesicles derived from the INM, suggesting a role for these two proteins in membrane curvature around the capsid (12). Tight membrane curvature is an energetically unfavorable event and is thought to be accomplished by coupling curvature to energetically favorable interactions between membrane-bound proteins or protein complexes (reviewed in reference 40). The data of Klupp et al. suggest the possibility that upon recognition of a capsid, pUL31 and pUL34 may interact in a way that induces tight curvature of the INM. Here we present data in support of this hypothesis, showing that a specific point mutation in UL34 induces accumulation of docked capsids at the INM, extragenic suppression of the mutant phenotype is associated with a mutation in UL31, and pUL31 and pUL34 can interact via sequences that are not involved in their INM targeting interaction.We previously published a characterization of a library of 19 charge cluster mutants of pUL34. In each of these mutants, one charge cluster (defined as a group of five consecutive amino acids in which two or more of the residues have charged side chains) was mutated such that the charged residues were replaced by alanine. Six of the 19 charge cluster mutants tested failed to complement replication of UL34-null virus, indicating that they disrupt essential functions of pUL34. Interestingly, five of the six noncomplementing mutants were synthesized at levels comparable to that of wild-type UL34 and localized normally to the nuclear envelope, suggesting that they were unimpaired in their ability to make a nuclear envelope targeting interaction with UL31. In order to identify essential functions of pUL34 downstream of nuclear envelope targeting, we have undertaken a detailed study of the behavior and interactions of these mutants.     

Identification of Conserved Amino Acids in the Herpes Simplex Virus Type 1 UL8 Protein Required for DNA Synthesis and UL52 Primase Interaction in the Virus Replisome

We have used oriS-dependent transient replication assays to search for species-specific interactions within the herpes simplex virus replisome. Hybrid replisomes derived from herpes simplex virus type 1 (HSV-1) and equine herpesvirus type 1 (EHV-1) failed to support DNA replication in cells. Moreover, the replisomes showed a preference for their cognate origin of replication. The results demonstrate that the herpesvirus replisome behaves as a molecular machine relying on functionally important interactions. We then searched for functional interactions in the replisome context by subjecting HSV-1 UL8 protein to extensive mutagenesis. 52 mutants were made by replacing single or clustered charged amino acids with alanines. Four mutants showed severe replication defects. Mutant A23 exhibited a lethal phenotype, and mutants A49, A52 and A53 had temperature-sensitive phenotypes. Mutants A49 and A53 did not interact with UL52 primase as determined by co-immunoprecipitation experiments. Using GFP-tagged UL8, we demonstrate that all mutants were unable to support formation of ICP8-containing nuclear replication foci. Extended mutagenesis suggested that a highly conserved motif corresponding to mutant A49 serves an important role for establishing a physical contact between UL8 and UL52. The replication-defective mutations affected conserved amino acids, and similar phenotypes were observed when the corresponding mutations were introduced into EHV-1 UL8.     

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.     

Comprehensive mutational analysis of a herpesvirus gene in the viral genome context reveals a region essential for virus replication

Essential viral proteins perform vital functions during morphogenesis via a complex interaction with other viral and cellular gene products. Here, we present a novel approach to comprehensive mutagenesis of essential cytomegalovirus genes and biological analysis in the 230-kbp-genome context. A random Tn7-based mutagenesis procedure at the single-gene level was combined with site-specific recombination via the FLP/FLP recognition target site system for viral genome reconstitution. We show the function of more than 100 mutants from a larger library of M50/p35, a protein involved in capsid egress from the nucleus. This protein recruits other viral proteins and cellular enzymes to the inner nuclear membrane. Our approach enabled us to rapidly discriminate between essential and nonessential regions within the coding sequence. Based on the prediction of the screen, we were able to map a site essential for viral protein-protein interaction at the amino acid level.     

Random screening for dominant-negative mutants of the cytomegalovirus nuclear egress protein M50

Inactivation of gene products by dominant-negative (DN) mutants is a powerful tool to assign functions to proteins. Here, we present a two-step procedure to establish a random screen for DN alleles, using the essential murine cytomegalovirus gene M50 as an example. First, loss-of-function mutants from a linker-scanning library were tested for inhibition of virus reconstitution with the help of FLP-mediated ectopic insertion of the mutants into the viral genome. Second, DN candidates were confirmed by conditional expression of the inhibitory proteins in the virus context. This allowed the quantification of the inhibitory effect, the identification of the morphogenesis block, and the construction of DN mutants with improved activity. Based on these observations a DN mutant of the homologous gene (UL50) in human cytomegalovirus was predicted and constructed. Our data suggest that a proline-rich sequence motif in the variable region of M50/UL50 represents a new functional site which is essential for nuclear egress of cytomegalovirus capsids.     

Cytomegalovirus pUL96 is critical for the stability of pp150-associated nucleocapsids

Maturation of human cytomegalovirus (HCMV) initiates with nucleocapsids that egress from the nucleus and associate with a juxtanuclear cytoplasmic assembly compartment, where virion envelopment and release are orchestrated. Betaherpesvirus conserved proteins pp150 (encoded by UL32) and pUL96 are critical for HCMV growth in cell culture. pp150 is a capsid-proximal tegument protein that preserves the integrity of nucleocapsids during maturation. pUL96, although expressed as an early protein, acts late during virus maturation, similar to pp150, based on the comparable antigen distribution in UL96, UL32, or UL96/UL32 dual mutant virus-infected cells. pp150 associates with nuclear capsids prior to DNA encapsidation, whereas both pp150 and pUL96 associate with extracellular virus, suggesting that pUL96 is added after pp150. In the absence of pUL96, capsid egress from the nucleus continues; however, unlike wild-type virus infection, pp150 accumulates in the nuclear, as well as in the cytoplasmic, compartment. Ultrastructural evaluation of a UL96 conditional mutant revealed intact nuclear stages but aberrant nucleocapsids accumulating in the cytoplasm comparable to the known phenotype of UL32 mutant virus. In summary, pUL96 preserves the integrity of pp150-associated nucleocapsids during translocation from the nucleus to the cytoplasm.