The capsid-associated UL25 protein of the alphaherpesvirus pseudorabies virus is nonessential for cleavage and encapsidation of genomic DNA but is required for nuclear egress of capsids

Homologs of the UL25 gene product of herpes simplex virus (HSV) have been identified in all three subfamilies of the Herpesviridae. However, their exact function during viral replication is not yet known. Whereas earlier studies indicated that the UL25 protein of HSV-1 is not required for cleavage of newly replicated viral DNA but is necessary for stable encapsidation (A. R. McNab, P. Desai, S. Person, L. Roof, D. R. Thompson, W. W. Newcomb, J. C. Brown, and F. L. Homa, J. Virol. 72:1060-1070, 1998), viral DNA packaging has recently been demonstrated to occur in the absence of UL25, although at significantly decreased levels compared to wild-type HSV-1 (N. Stow, J. Virol. 75:10755-10765 2001). To clarify the functional role of UL25 we analyzed the homologous protein of the alphaherpesvirus pseudorabies virus (PrV). PrV UL25 was found to be essential for viral replication, as a mutant virus lacking the UL25 protein required UL25-expressing cells for productive propagation. In the absence of the UL25 protein, newly replicated PrV DNA was cleaved and DNA-containing C-type capsids were detected in infected cell nuclei. However, although capsids were frequently found in close association with the inner nuclear membrane, nuclear egress was not observed. Consequently, no capsids were found in the cytoplasm, resulting in an inhibition of virion morphogenesis. In contrast, the formation of capsidless enveloped tegument structures (L particles) in the cytoplasm was readily observed. Thus, our data demonstrate that the PrV UL25 protein is not essential for cleavage and encapsidation of viral genomes, although both processes occur more efficiently in the presence of the protein. However, the presence of the PrV UL25 protein is a prerequisite for nuclear egress. By immunoelectron microscopy, we detected UL25-specific label on DNA-containing C capsids but not on other intranuclear immature or defective capsid forms. Thus, the PrV UL25 protein may represent the hitherto missing trigger that allows primary envelopment preferably of DNA-filled C capsids.     

The essential human cytomegalovirus gene UL52 is required for cleavage-packaging of the viral genome

Replication of human cytomegalovirus (HCMV) produces large DNA concatemers of head-to-tail-linked viral genomes that upon packaging into capsids are cut into unit-length genomes. The mechanisms underlying cleavage-packaging and the subsequent steps prior to nuclear egress of DNA-filled capsids are incompletely understood. The hitherto uncharacterized product of the essential HCMV UL52 gene was proposed to participate in these processes. To investigate the function of pUL52, we constructed a ΔUL52 mutant as well as a complementing cell line. We found that replication of viral DNA was not impaired in noncomplementing cells infected with the ΔUL52 virus, but viral concatemers remained uncleaved. Since the subnuclear localization of the known cleavage-packaging proteins pUL56, pUL89, and pUL104 was unchanged in ΔUL52-infected fibroblasts, pUL52 does not seem to act via these proteins. Electron microscopy studies revealed only B capsids in the nuclei of ΔUL52-infected cells, indicating that the mutant virus has a defect in encapsidation of viral DNA. Generation of recombinant HCMV genomes encoding epitope-tagged pUL52 versions showed that only the N-terminally tagged pUL52 supported viral growth, suggesting that the C terminus is crucial for its function. pUL52 was expressed as a 75-kDa protein with true late kinetics. It localized preferentially to the nuclei of infected cells and was found to enclose the replication compartments. Taken together, our results demonstrate an essential role for pUL52 in cleavage-packaging of HCMV DNA. Given its unique subnuclear localization, the function of pUL52 might be distinct from that of other cleavage-packaging proteins.     

The pseudorabies virus UL28 protein enters the nucleus after coexpression with the herpes simplex virus UL15 protein.

Herpesvirus DNA is packaged into capsids in the nuclei of infected cells in a process requiring at least six viral proteins. Of the proteins required for encapsidation of viral DNA, UL15 and UL28 are the most conserved among herpes simplex virus type 1 (HSV), varicella-zoster virus, and equine herpesvirus 1. The subcellular distribution of the pseudorabies virus (PRV) UL28 protein was examined by in situ immunofluorescence. UL28 was present in the nuclei of infected cells; however, UL28 was limited to the cytoplasm in the absence of other viral proteins. When cells expressing variant forms of UL28 were infected with a PRV UL28-null mutant, UL28 entered the nucleus, provided the carboxyl-terminal 155 amino acids were present. Additionally, PRV UL28 entered the nucleus in cells infected with HSV. Two HSV packaging proteins were tested for the ability to affect the subcellular distribution of UL28. Coexpression of HSV UL15 enabled PRV UL28 to enter the nucleus in a manner that required the carboxyl-terminal 155 amino acids of UL28. Coexpression of HSV UL25 did not affect the distribution of UL28. We propose that an interaction between UL15 and UL28 facilitates the transport of a UL15-UL28 complex to the infected-cell nucleus.     

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.     

The UL25 gene product of herpes simplex virus type 1 is involved in uncoating of the viral genome

Studies on the herpes simplex virus type 1 UL25-null mutant KUL25NS have shown that the capsid-associated UL25 protein is required at a late stage in the encapsidation of viral DNA. Our previous work on UL25 with the UL25 temperature-sensitive (ts) mutant ts1204 also implicated UL25 in a role at very early times in the virus growth cycle, possibly at the stage of penetration of the host cell. We have reexamined this mutant and discovered that it had an additional ts mutation elsewhere in the genome. The ts1204 UL25 mutation was transferred into wild-type (wt) virus DNA, and the UL25 mutant ts1249 was isolated and characterized to clarify the function of UL25 at the initial stages of virus infection. Indirect immunofluorescence assays and in situ hybridization analysis of virus-infected cells revealed that the mutant ts1249 was not impaired in penetration of the host cell but had an uncoating defect at the nonpermissive temperature. When ts1249-infected cells were incubated initially at the permissive temperature to allow uncoating of the viral genome and subsequently transferred to the restrictive temperature, a DNA-packaging defect was evident. The results suggested that ts1249, like KUL25NS, had a block at a late stage of DNA packaging and that the packaged genome was shorter than the full-length genome. Examination of ts1249 capsids produced at the nonpermissive temperature revealed that, in comparison with wt capsids, they contained reduced amounts of UL25 protein, thereby providing a possible explanation for the failure of ts1249 to package full-length viral DNA.     

Betaherpesvirus-conserved cytomegalovirus tegument protein ppUL32 (pp150) controls cytoplasmic events during virion maturation

The UL32 gene of human cytomegalovirus (CMV) encodes a prominent betaherpesvirus-conserved virion tegument protein, called pp150 (basic phosphoprotein/ppUL32), that accumulates within a cytoplasmic inclusion adjacent to the nucleus at late times during infection. Using a UL32 deletion mutant (DeltaUL32-BAC) (where BAC is bacterial artificial chromosome), we demonstrate that pp150 is critical for virion maturation in the cytoplasmic compartment. Cotransfection of a pp150 expression plasmid with DeltaUL32-BAC DNA led to complementation of the replication defect with focus formation due to secondary spread. Deletion of the amino terminus of pp150 or disruption of the betaherpesvirus conserved regions, CR1 and CR2, revealed these regions to be critical for replication. In contrast, deletion of the carboxyl terminus only partially compromised maturation while disruption of glycosylation sites had no effect. An African green monkey CMV UL32 homolog complemented DeltaUL32-BAC replication but murine CMV M32 failed to complement, consistent with evolutionary divergence of rodent and primate cytomegaloviruses. Infection with DeltaUL32-BAC showed normal expression of all kinetic classes of viral genes and replication of viral DNA, with accumulation of viral DNA-containing particles in the cytoplasm; however, mutant virus did not spread to adjacent cells. In contrast to this block in virion infectivity, cell-to-cell transfer of pp65-containing particles was observed, suggesting that release of dense bodies continued in the absence of pp150. These observations demonstrate that pp150 is critical for virion egress, possibly at the stage of final envelopment.     

A Mutation Deleting Sequences Encoding the Amino Terminus of Human Cytomegalovirus UL84 Impairs Interaction with UL44 and Capsid Localization

Protein-protein interactions are required for many biological functions. Previous work has demonstrated an interaction between the human cytomegalovirus DNA polymerase subunit UL44 and the viral replication factor UL84. In this study, glutathione S-transferase pulldown assays indicated that residues 1 to 68 of UL84 are both necessary and sufficient for efficient interaction of UL84 with UL44 in vitro. We created a mutant virus in which sequences encoding these residues were deleted. This mutant displayed decreased virus replication compared to wild-type virus. Immunoprecipitation assays showed that the mutation decreased but did not abrogate association of UL84 with UL44 in infected cell lysate, suggesting that the association in the infected cell can involve other protein-protein interactions. Further immunoprecipitation assays indicated that IRS1, TRS1, and nucleolin are candidates for such interactions in infected cells. Quantitative real-time PCR analysis of viral DNA indicated that the absence of the UL84 amino terminus does not notably affect viral DNA synthesis. Western blotting experiments and pulse labeling of infected cells with [(35)S]methionine demonstrated a rather modest downregulation of levels of multiple proteins and particularly decreased levels of the minor capsid protein UL85. Electron microscopy demonstrated that viral capsids assemble but are mislocalized in nuclei of cells infected with the mutant virus, with fewer cytoplasmic capsids detected. In sum, deletion of the sequences encoding the amino terminus of UL84 affects interaction with UL44 and virus replication unexpectedly, not viral DNA synthesis. Mislocalization of viral capsids in infected cell nuclei likely contributes to the observed decrease in virus replication.     

The UL20 gene product of pseudorabies virus functions in virus egress.

The UL20 open reading frame is positionally conserved in different alphaherpesvirus genomes and is predicted to encode an integral membrane protein. A previously described UL20- mutant of herpes simplex virus type 1 (HSV-1) exhibited a defect in egress correlating with retention of virions in the perinuclear space (J. D. Baines, P. L. Ward, G. Campadelli-Fiume, and B. Roizman, J. Virol. 65:6414-6424, 1991). To analyze UL20 function in a related but different herpesvirus, we constructed a UL20- pseudorabies virus (PrV) mutant by insertional mutagenesis. Similar to HSV-1, UL20- PrV was found to be severely impaired in both cell-to-cell spread and release from cultured cells. The severity of this defect appeared to be cell type dependent, being more prominent in Vero than in human 143TK- cells. Surprisingly, electron microscopy revealed the retention of enveloped virus particles in cytoplasmic vesicles of Vero cells infected with UL20- PrV. This contrasts with the situation in the UL20- HSV-1 mutant, which accumulated virions in the perinuclear cisterna of Vero cells. Therefore, the UL20 gene products of PrV and HSV-1 appear to be involved in distinct steps of viral egress, acting in different intracellular compartments. This might be caused either by different functions of the UL20 proteins themselves or by generally different egress pathways of PrV and HSV-1 mediated by other viral gene products.     

Human cytomegalovirus UL97 kinase and nonkinase functions mediate viral cytoplasmic secondary envelopment

Previous studies have revealed critical roles for the human cytomegalovirus (HCMV) UL97 kinase in viral nuclear maturation events. We have shown recently that UL97 affects the morphology of the viral cytoplasmic assembly compartment (AC) (M. Azzeh, A. Honigman, A. Taraboulos, A. Rouvinski, and D. G. Wolf, Virology 354:69-79, 2006). Here, we employed a comprehensive ultrastructural analysis to dissect the impact of UL97 on cytoplasmic steps of HCMV assembly. Using UL97 deletion (ΔUL97) and kinase-null (K355M) mutants, as well as the UL97 kinase inhibitor NGIC-I, we demonstrated that the loss of UL97 kinase activity resulted in a unique combination of cytoplasmic features: (i) the formation of pp65-rich aberrant cytoplasmic tegument aggregates, (ii) distorted intracytoplasmic membranes, which replaced the normal architecture of the AC, and (iv) a paucity of cytoplasmic tegumented capsids and dense bodies (DBs). We further showed that these abnormal assembly intermediates did not result from impaired nuclear capsid maturation and egress per se by using 2-bromo-5,6-dichloro-1-(β-d-ribofuranosyl) benzimidizole (BDCRB) to induce the artificial inhibition of nuclear maturation and the nucleocytoplasmic translocation of capsids. The specific abrogation of UL97 kinase activity under low-multiplicity-of-infection conditions resulted in the improved release of extracellular virus compared to that of ΔUL97, despite similar rates of viral DNA accumulation and similar effects on nuclear capsid maturation and egress. The only ultrastructural correlate of the growth difference was a higher number of cytoplasmic DBs, tegumented capsids, and clustered viral particles observed upon the specific abrogation of UL97 kinase activity compared to that of ΔUL97. These combined findings reveal a novel role for UL97 in HCMV cytoplasmic secondary envelopment steps, with a further distinction of kinase-mediated function in the formation of the virus-induced AC and a nonkinase function enhancing the efficacy of viral tegumentation and release.     

Defective infectious particles and rare packaged genomes produced by cells carrying terminal-repeat-negative epstein-barr virus

The Epstein-Barr virus (EBV) lytic program includes lytic viral DNA replication and the production of a viral particle into which the replicated viral DNA is packaged. The terminal repeats (TRs) located at the end of the linear viral DNA have been identified as the packaging signals. A TR-negative (TR(-)) mutant therefore provides an appropriate tool to analyze the relationships between EBV DNA packaging and virus production. Here, we show that supernatants from lytically induced 293 cells carrying TR mutant EBV genomes (293/TR(-)) contain large amounts of viral particles devoid of viral DNA which are nevertheless able to bind to EBV target cells. This shows that viral DNA packaging is not a prerequisite for virion formation and egress. Rather surprisingly, supernatants from lytically induced 293/TR(-) cells also contained rare infectious viruses carrying the viral mutant DNA. This observation indicates that the TRs are important but not absolutely essential for virus encapsidation.     

A null mutation in the gene encoding the herpes simplex virus type 1 UL37 polypeptide abrogates virus maturation

The tegument is an integral and essential structural component of the herpes simplex virus type 1 (HSV-1) virion. The UL37 open reading frame of HSV-1 encodes a 120-kDa virion polypeptide which is a resident of the tegument. To analyze the function of the UL37-encoded polypeptide a null mutation was generated in the gene encoding this protein. In order to propagate this mutant virus, transformed cell lines that express the UL37 gene product in trans were produced. The null mutation was transferred into the virus genome using these complementing cell lines. A mutant virus designated KDeltaUL37 was isolated based on its ability to form plaques on the complementing cell line but not on nonpermissive (noncomplementing) Vero cells. This virus was unable to grow in Vero cells; therefore, UL37 encodes an essential function of the virus. The mutant virus KDeltaUL37 produced capsids containing DNA as judged by sedimentation analysis of extracts derived from infected Vero cells. Therefore, the UL37 gene product is not required for DNA cleavage or packaging. The UL37 mutant capsids were tagged with the smallest capsid protein, VP26, fused to green fluorescent protein. This fusion protein decorates the capsid shell and consequently the location of the capsid and the virus particle can be visualized in living cells. Late in infection, KDeltaUL37 capsids were observed to accumulate at the periphery of the nucleus as judged by the concentration of fluorescence around this organelle. Fluorescence was also observed in the cytoplasm in large puncta. Fluorescence at the plasma membrane, which indicated maturation and egress of virions, was observed in wild-type-infected cells but was absent in KDeltaUL37-infected cells. Ultrastructural analysis of thin sections of infected cells revealed clusters of DNA-containing capsids in the proximity of the inner nuclear membrane. Occasionally enveloped capsids were observed between the inner and outer nuclear membranes. Clusters of unenveloped capsids were also observed in the cytoplasm of KDeltaUL37-infected cells. Enveloped virions, which were observed in the cytoplasm of wild-type-infected cells, were never detected in the cytoplasm of KDeltaUL37-infected cells. Crude cell fractionation of infected cells using detergent lysis demonstrated that two-thirds of the UL37 mutant particles were associated with the nuclear fraction, unlike wild-type particles, which were predominantly in the cytoplasmic fraction. These data suggest that in the absence of UL37, the exit of capsids from the nucleus is slowed. UL37 mutant particles can participate in the initial envelopment at the nuclear membrane, although this process may be impaired in the absence of UL37. Furthermore, the naked capsids deposited in the cytoplasm are unable to progress further in the morphogenesis pathway, which suggests that UL37 is also required for egress and reenvelopment. Therefore, the UL37 gene product plays a key role in the early stages of the maturation pathway that give rise to an infectious virion.     

Varicella zoster virus ORF25 gene product: an essential hub protein linking encapsidation proteins and the nuclear egress complex

Varicella zoster virus (VZV) ORF25 is a 156 amino acid protein belonging to the approximately 40 core proteins that are conserved throughout the Herpesviridae. By analogy to its functional orthologue UL33 in Herpes simplex virus 1 (HSV-1), ORF25 is thought to be a component of the terminase complex. To investigate how cleavage and encapsidation of viral DNA links to the nuclear egress of mature capsids in VZV, we tested 10 VZV proteins that are predicted to be involved in either of the two processes for protein interactions against each other using three independent protein-protein interaction (PPI) detection systems: the yeast-two-hybrid (Y2H) system, a luminescence based MBP pull-down interaction screening assay (LuMPIS), and a bioluminescence resonance energy transfer (BRET) assay. A set of 20 interactions was consistently detected by at least 2 methods and resulted in a dense interaction network between proteins associated in encapsidation and nuclear egress. The results indicate that the terminase complex in VZV consists of ORF25, ORF30, and ORF45/42 and support a model in which both processes are closely linked to each other. Consistent with its role as a central hub for protein interactions, ORF25 is shown to be essential for VZV replication.     

A null mutation in the UL36 gene of herpes simplex virus type 1 results in accumulation of unenveloped DNA-filled capsids in the cytoplasm of infected cells

The UL36 open reading frame (ORF) encodes the largest herpes simplex virus type 1 (HSV-1) protein, a 270-kDa polypeptide designated VP1/2, which is also a component of the virion tegument. A null mutation was generated in the UL36 gene to elucidate its role in the virus life cycle. Since the UL36 gene specifies an essential function, complementing cell lines transformed for sequences encoding the UL36 ORF were made. A mutant virus, designated KDeltaUL36, that encodes a null mutation in the UL36 gene was isolated and propagated in these cell lines. When noncomplementing cells infected with KDeltaUL36 were analyzed, both terminal genomic DNA fragments and DNA-containing capsids (C capsids) were detected; therefore, UL36 is not required for cleavage or packaging of DNA. Sedimentation analysis of lysates from mutant-infected cells revealed the presence of particles that have the physical characteristics of C capsids. In agreement with this, polypeptide profiles of the mutant particles revealed an absence of the major envelope and tegument components. Ultrastructural analysis revealed the presence of numerous unenveloped DNA containing capsids in the cytoplasm of KDeltaUL36-infected cells. The UL36 mutant particles were tagged with the VP26-green fluorescent protein marker, and their movement was monitored in living cells. In KDeltaUL36-infected cells, extensive particulate fluorescence corresponding to the capsid particles was observed throughout the cytosol. Accumulation of fluorescence at the plasma membrane which indicated maturation and egress of virions was observed in wild-type-infected cells but was absent in KDeltaUL36-infected cells. In the absence of UL36 function, DNA-filled capsids are produced; these capsids enter the cytosol after traversing the nuclear envelope and do not mature into enveloped virus. The maturation and egress of the UL36 mutant particles are abrogated, possibly due to a late function of this complex polypeptide, i.e., to target capsids to the correct maturation pathway.     

Requirement for Uracil-DNA Glycosylase during the Transition to Late-Phase Cytomegalovirus DNA Replication

Cytomegalovirus gene UL114, a homolog of mammalian uracil-DNA glycosylase (UNG), is required for efficient viral DNA replication. In quiescent fibroblasts, UNG mutant virus replication is delayed for 48 h and follows the virus-induced expression of cellular UNG. In contrast, mutant virus replication proceeds without delay in actively growing fibroblasts that express host cell UNG. In the absence of viral or host cell UNG expression, mutant virus fails to proceed to late-phase DNA replication, characterized by rapid DNA amplification. The data suggest that uracil incorporated early during wild-type viral DNA replication must be removed by virus or host UNG prior to late-phase amplification and encapsidation into progeny virions. The process of uracil incorporation and excision may introduce strand breaks to facilitate the transition from early-phase replication to late-phase amplification.     

The UL14 tegument protein of herpes simplex virus type 1 is required for efficient nuclear transport of the alpha transinducing factor VP16 and viral capsids

The protein encoded by the UL14 gene of herpes simplex virus type 1 (HSV-1) and HSV-2 is expressed late in infection and is a minor component of the virion tegument. An UL14-deficient HSV-1 mutant (UL14D) forms small plaques and exhibits an extended growth cycle at low multiplicities of infection (MOI) compared to wild-type virus. Although UL14 is likely to be involved in the process of viral maturation and egress, its precise role in viral replication is still enigmatic. In this study, we found that immediate-early viral mRNA expression was decreased in UL14D-infected cells. Transient coexpression of UL14 and VP16 in the absence of infection stimulated the nuclear accumulation of both proteins. We intended to visualize the fate of VP16 released from the infected virion and constructed UL14-null (14D-VP16G) and rescued (14R-VP16G) viruses that expressed a VP16-green fluorescent protein (GFP) fusion protein. Synchronous high-multiplicity infection of the viruses was performed at 4°C in the absence of de novo protein synthesis. We found that the presence of UL14 in the virion had an enhancing effect on the nuclear accumulation of VP16-GFP. The lack of UL14 did not significantly alter virus internalization but affected incoming capsid transport to the nuclear pore. These observations suggested that UL14 (i) enhanced VP16 nuclear localization at the immediately early phase, thus indirectly regulating the expression of immediate-early genes, and (ii) was associated with efficient nuclear targeting of capsids. The tegument protein UL14 could be part of the machinery that regulates HSV-1 replication.     

Disulfide bond formation in the herpes simplex virus 1 UL6 protein is required for portal ring formation and genome encapsidation

The herpes simplex virus 1 (HSV-1) UL6 portal protein forms a 12-subunit ring structure at a unique capsid vertex which functions as a conduit for the encapsidation of the viral genome. We have demonstrated previously that the leucine zipper region of UL6 is important for intersubunit interactions and stable ring formation (J. K. Nellissery, R. Szczepaniak, C. Lamberti, and S. K. Weller, J. Virol. 81:8868-8877, 2007). We now demonstrate that intersubunit disulfide bonds exist between monomeric subunits and contribute to portal ring formation and/or stability. Intersubunit disulfide bonds were detected in purified portal rings by SDS-PAGE under nonreducing conditions. Furthermore, the treatment of purified portal rings with dithiothreitol (DTT) resulted in the disruption of the rings, suggesting that disulfide bonds confer stability to this complex structure. The UL6 protein contains nine cysteines that were individually mutated to alanine. Two of these mutants, C166A and C254A, failed to complement a UL6 null mutant in a transient complementation assay. Furthermore, viral mutants bearing the C166A and C254A mutations failed to produce infectious progeny and were unable to cleave or package viral DNA. In cells infected with C166A or C254A, B capsids were produced which contained UL6 at reduced levels compared to those seen in wild-type capsids. In addition, C166A and C254A mutant proteins expressed in insect cells infected with recombinant baculovirus failed to form ring structures. Cysteines at positions 166 and 254 thus appear to be required for intersubunit disulfide bond formation. Taken together, these results indicate that disulfide bond formation is required for portal ring formation and/or stability and for the production of procapsids that are capable of encapsidation.     

A putative leucine zipper within the herpes simplex virus type 1 UL6 protein is required for portal ring formation

The herpes simplex virus type 1 UL6 protein forms a 12-subunit ring structure at a unique capsid vertex which functions as a conduit for encapsidation of the viral genome. To characterize UL6 protein domains that are involved in intersubunit interactions and interactions with other capsid proteins, we engineered a set of deletion mutants spanning the entire gene. Three deletion constructs, D-5 (Delta 198-295), D-6 (Delta 322-416), and D-LZ (Delta 409-473, in which a putative leucine zipper was removed), were introduced into the viral genome. All three mutant viruses produced only B capsids, indicating a defect in encapsidation. Western blot analysis showed that the UL6 protein was present in the capsids isolated from two mutants, D-6 and D-LZ. The protein encoded by D-5, on the other hand, was not associated with capsids and was instead localized in the cytoplasm of the infected cells, indicating that this deletion affected the nuclear transport of the portal protein. The UL6 protein from the KOS strain (wild type) and the D-6 mutant were purified from insect cells infected with recombinant baculoviruses and shown to form ring structures as assessed by sucrose gradient centrifugation and electron microscopy. In contrast, the D-LZ mutant protein formed aggregates that sedimented throughout the sucrose gradient as a heterogeneous mixture and did not yield stable ring structures. A mutant (L429E L436E) in which two of the heptad leucines of the putative zipper were replaced with glutamate residues also failed to form stable rings. Our results suggest that the integrity of the leucine zipper region is important for oligomer interactions and stable ring formation, which in turn are required for genome encapsidation.