Assembly of herpes simplex virus (HSV) intermediate capsids in insect cells infected with recombinant baculoviruses expressing HSV capsid proteins.

The capsid of herpes simplex virus type 1 (HSV-1) is composed of seven proteins, VP5, VP19C, VP21, VP22a, VP23, VP24, and VP26, which are the products of six HSV-1 genes. Recombinant baculoviruses were used to express the six capsid genes (UL18, UL19, UL26, UL26.5, UL35, and UL38) in insect cells. All constructs expressed the appropriate-size HSV proteins, and insect cells infected with a mixture of the six recombinant baculoviruses contained large numbers of HSV-like capsids. Capsids were purified by sucrose gradient centrifugation, and electron microscopy showed that the capsids made in Sf9 cells had the same size and appearance as authentic HSV B capsids. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that the protein composition of these capsids was nearly identical to that of B capsids isolated from HSV-infected Vero cells. Electron microscopy of thin sections clearly demonstrated that the capsids made in insect cells contained the inner electron-translucent core associated with HSV B capsids. In infections in which single capsid genes were left out, it was found that the UL18 (VP23), UL19 (VP5), UL38 (VP19C), and either the UL26 (VP21 and VP24) or the UL26.5 (VP22a) genes were required for assembly of 100-nm capsids. VP22a was shown to form the inner core of the B capsid, since in infections in which the UL26.5 gene was omitted the 100-nm capsids that formed lacked the inner core. The UL35 (VP26) gene was not required for assembly of 100-nm capsids, although assembly of B capsids was more efficient when it was present. These and other observations indicate that (i) the products of the UL18, UL19, UL35, and UL38 genes self-assemble into structures that form the outer surface (icosahedral shell) of the capsid, (ii) the products of the UL26 and/or UL26.5 genes are required (as scaffolds) for assembly of 100-nm capsids, and (iii) the interaction of the outer surface of the capsid with the scaffolding proteins requires the product of the UL18 gene (VP23).     

Cell-free assembly of the herpes simplex virus capsid.

Herpes simplex virus type 1 (HSV-1) capsids were found to assemble spontaneously in a cell-free system consisting of extracts prepared from insect cells that had been infected with recombinant baculoviruses coding for HSV-1 capsid proteins. The capsids formed in this system resembled native HSV-1 capsids in morphology as judged by electron microscopy, in sedimentation rate on sucrose density gradients, in protein composition, and in their ability to react with antibodies specific for the HSV-1 major capsid protein, VP5. Optimal capsid assembly required the presence of extracts containing capsid proteins VP5, VP19, VP23, VP22a, and the maturational protease (product of the UL26 gene). Assembly was more efficient at 27 degrees C than at 4 degrees C. The availability of a cell-free assay for HSV-1 capsid formation will be of help in identifying the morphogenetic steps that occur during capsid assembly in vivo and in evaluating candidate antiherpes therapeutics directed at capsid assembly.     

Assembly of herpes simplex virus capsids using the human cytomegalovirus scaffold protein: critical role of the C terminus.

An essential step in assembly of herpes simplex virus (HSV) type 1 capsids involves interaction of the major capsid protein (VP5) with the C terminus of the scaffolding protein (encoded by the UL26.5 gene). The final 12 residues of the HSV scaffolding protein contains an A-X-X-F-V/A-X-Q-M-M-X-X-R motif which is conserved between scaffolding proteins found in other alphaherpesviruses but not in members of the beta- or gamma-herpesviruses. Previous studies have shown that the bovine herpesvirus 1 (alphaherpesvirus) UL26.5 homolog will functionally substitute for the HSV UL26.5 gene (E. J. Haanes et al., J. Virol. 69:7375-7379, 1995). The homolog of the UL26.5 gene in the human cytomegalovirus (HCMV) genome is the UL80.5 gene. In these studies, we tested whether the HCMV UL80.5 gene would substitute for the HSV UL26.5 gene in a baculovirus capsid assembly system that we have previously described (D. R. Thomsen et al., J. Virol. 68:2442-2457, 1994). The results demonstrate that (i) no intact capsids were assembled when the full-length or a truncated (missing the C-terminal 65 amino acids) UL80.5 protein was tested; (ii) when the C-terminal 65 amino acids of the UL80.5 protein were replaced with the C-terminal 25 amino acids of the UL26.5 protein, intact capsids were made and direct interaction of the UL80.5 protein with VP5 was detected; (iii) assembly of intact capsids was demonstrated when the sequence of the last 12 amino acids of the UL80.5 protein was changed from RRIFVA ALNKLE to RRIFVAAMMKLE; (iv) self-interaction of the scaffold proteins is mediated by sequences N terminal to the maturation cleavage site; and (v) the UL26.5 and UL80.5 proteins will not coassemble into scaffold structures. The results suggest that the UL26.5 and UL80.5 proteins form a scaffold by self-interaction via sequences in the N termini of the proteins and emphasize the importance of the C terminus for interaction of scaffold with the proteins that form the capsid shell.     

Amino acids 143 to 150 of the herpes simplex virus type 1 scaffold protein are required for the formation of portal-containing capsids

The herpes simplex virus type 1 (HSV-1) portal is composed of a dodecamer of UL6 protein molecules whose incorporation into the capsid is mediated by interaction with the HSV-1 UL26.5 scaffold protein. Previous results with an in vitro capsid assembly assay demonstrated that nine amino acids (amino acids 143 to 151) of the UL26.5 protein are required for its interaction with UL6 and for incorporation of the portal complex into capsids. In the present study an HSV-1 mutant, bvFH411, was isolated and contained a deletion that removed the codons for UL26.5 amino acids 143 to 150. The mutant virus failed to produce infectious virus in noncomplementing cells, and only B capsids that contained only minor amounts of portal protein were made. These data corroborate our previous in vitro studies and demonstrate that amino acids 143 to 150 of UL26.5 are required for the formation of portal-containing HSV-1 capsids.     

The bovine herpesvirus 1 maturational proteinase and scaffold proteins can substitute for the homologous herpes simplex virus type 1 proteins in the formation of hybrid type B capsids.

We determined the nucleotide sequence of a 3.5-kb region of the bovine herpesvirus 1 (BHV-1) genome which contained the complete BHV-1 homologs of the herpes simplex virus type 1 (HSV-1) UL26 and UL26.5 genes. In HSV-1, the UL26 and UL26.5 open reading frames encode scaffold proteins upon which viral capsids are assembled. The UL26-encoded protein is also a proteinase and specifically cleaves both itself and the UL26.5-encoded protein. The overall BHV-1-encoded amino acid sequence showed only 41% identity to the HSV-1 sequences and was most divergent in the regions defined to be involved in the scaffolding function. We substituted the proteins encoded by the BHV-1 homologs of the UL26 and UL26.5 open reading frames, expressed in baculovirus, for the corresponding HSV-1 proteins in an in vitro HSV-1 capsid assembly system. The proteins expressed from the BHV-1 UL26 and UL26.5 homologs facilitated the formation of hybrid type B capsids indistinguishable from those formed entirely with HSV-1-encoded proteins.     

The capsid and tegument of the alphaherpesviruses are linked by an interaction between the UL25 and VP1/2 proteins

How alphaherpesvirus capsids acquire tegument proteins remains a key question in viral assembly. Using pseudorabies virus (PRV), we have previously shown that the 62 carboxy-terminal amino acids of the VP1/2 large tegument protein are essential for viral propagation and when transiently expressed as a fusion to green fluorescent protein relocalize to nuclear capsid assemblons following viral infection. Here, we show that localization of the VP1/2 capsid-binding domain (VP1/2cbd) into assemblons is conserved in herpes simplex virus type 1 (HSV-1) and that this recruitment is specifically on capsids. Using a mutant virus screen, we find that the protein product of the UL25 gene is essential for VP1/2cbd association with capsids. An interaction between UL25 and VP1/2 was corroborated by coimmunoprecipitation from cells transiently expressing either HSV-1 or PRV proteins. Taken together, these findings suggest that the essential function of the VP1/2 carboxy terminus is to anchor the VP1/2 tegument protein to capsids. Furthermore, UL25 encodes a multifunctional capsid protein involved in not only encapsidation, as previously described, but also tegumentation.     

The size and symmetry of B capsids of herpes simplex virus type 1 are determined by the gene products of the UL26 open reading frame.

Herpes simplex virus type 1 (HSV-1) B capsids are composed of seven proteins, designated VP5, VP19C, 21, 22a, VP23, VP24, and VP26 in order of decreasing molecular weight. Three proteins (21, 22a, and VP24) are encoded by a single open reading frame (ORF), UL26, and include a protease whose structure and function have been studied extensively by other investigators. The protease encoded by this ORF generates VP24 (amino acids 1 to 247), a structural component of the capsid and mature virions, and 21 (residues 248 to 635). The protease also cleaves C-terminal residues 611 to 635 of 21 and 22a, during capsid maturation. Protease activity has been localized to the N-terminal 247 residues. Protein 22a and probably the less abundant protein 21 occupy the internal volume of capsids but are not present in virions; therefore, they may form a scaffold that is used for B capsid assembly. The objective of the present study was to isolate and characterize a mutant virus with a null mutation in UL26. Vero cells were transformed with plasmid DNA that encoded ORF UL25 through UL28 and screened for their ability to support the growth of a mutant virus with a null mutation in UL27 (K082). Four of five transformants that supported the growth of the UL27 mutant also supported the growth of a UL27-UL28 double mutant. One of these transformants (F3) was used to isolate a mutant with a null mutation in UL26. The UL26 null mutation was constructed by replacement of DNA sequences specifying codons 41 through 593 with a lacZ reporter cassette. Permissive cells were cotransfected with plasmid and wild-type virus DNA, and progeny viruses were screened for their ability to grow on F3 but not Vero cells. A virus with these growth characteristics, designated KUL26 delta Z, that did not express 21, 22a, or VP24 during infection of Vero cells was isolated. Radiolabeled nuclear lysates from infected nonpermissive cells were layered onto sucrose gradients and subjected to velocity sedimentation. A peak of radioactivity for KUL26 delta Z that sedimented more rapidly than B capsids from wild-type-infected cells was observed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the gradient fractions showed that the peak fractions contained VP5, VP19C, VP23, and VP26. Analysis of sectioned cells and of the peak fractions of the gradients by electron microscopy revealed sheet and spiral structures that appear to be capsid shells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of a region in the herpes simplex virus scaffolding protein required for interaction with the portal

The herpes simplex virus type 1 capsid is a protective shell that acts as a container for the genetic material of the virus. After assembly of the capsid, the viral DNA is translocated into the capsid interior through a channel formed by the portal. The portal is composed of a dodecamer of UL6 molecules which form a ring-like structure found at a single vertex within the icosahedron. Formation of portal-containing capsids minimally requires the four structural proteins (VP5, VP19C, VP23, and UL6) and a scaffolding protein (UL26.5). Recently, an interaction between UL26.5 and the portal has been identified, suggesting the scaffold functions by delivering the portal to the growing capsid shell. The aim of this study was to identify regions within UL26.5 required for its interaction with the portal. A specific region was identified by mutational analysis. Deletion of scaffold amino acids (aa) 143 to 151 was found to be sufficient to inhibit formation of the scaffold-portal complex as assayed in vitro. The aa 143 to 151 contain the sequence YYPGE, which is highly conserved among alpha herpesviruses. Although it did not bind to the portal, the Delta143-151 mutant was found to retain the ability to support assembly of morphologically normal capsids in vitro. Such capsids, however, did not contain the portal. The results suggest assembly of portal-containing capsids requires formation of a scaffold-portal complex in which intermolecular contact is dependent on scaffold aa 143 to 151.     

Role of the UL25 gene product in packaging DNA into the herpes simplex virus capsid: location of UL25 product in the capsid and demonstration that it binds DNA

Recent studies have suggested that the herpes simplex type 1 (HSV-1) UL25 gene product, a minor capsid protein, is required for encapsidation but not cleavage of replicated viral DNA. This study set out to investigate the potential interactions of UL25 protein with other virus proteins and determine what properties it has for playing a role in DNA encapsidation. The UL25 protein is found in 42 +/- 17 copies per B capsid and is present in both pentons and hexons. We introduced green fluorescent protein (GFP) as a fluorescent tag into the N terminus of UL25 protein to identify its location in HSV-1-infected cells and demonstrated the relocation of UL25 protein from the cytoplasm into the nucleus at the late stage of HSV-1 infection. To clarify the cause of this relocation, we analyzed the interactions of UL25 protein with other virus proteins. The UL25 protein associates with VP5 and VP19C of virus capsids, especially of the penton structures, and the association with VP19C causes its relocation into the nucleus. Gel mobility shift analysis shows that UL25 protein has the potential to bind DNA. Moreover, the amino-terminal one-third of the UL25 protein is particularly important in DNA binding and forms a homo-oligomer. In conclusion, the UL25 gene product forms a tight connection with the capsid being linked with VP5 and VP19C, and it may play a role in anchoring the genomic DNA.     

Residues of the UL25 protein of herpes simplex virus that are required for its stable interaction with capsids

The herpes simplex virus 1 (HSV-1) UL25 gene product is a minor capsid component that is required for encapsidation, but not cleavage, of replicated viral DNA. UL25 is located on the capsid surface in a proposed heterodimer with UL17, where five copies of the heterodimer are found at each of the capsid vertices. Previously, we demonstrated that amino acids 1 to 50 of UL25 are essential for its stable interaction with capsids. To further define the UL25 capsid binding domain, we generated recombinant viruses with either small truncations or amino acid substitutions in the UL25 N terminus. Studies of these mutants demonstrated that there are two important regions within the capsid binding domain. The first 27 amino acids are essential for capsid binding of UL25, while residues 26 to 39, which are highly conserved in the UL25 homologues of other alphaherpesviruses, were found to be critical for stable capsid binding. Cryo-electron microscopy reconstructions of capsids containing either a small tag on the N terminus of UL25 or the green fluorescent protein (GFP) fused between amino acids 50 and 51 of UL25 demonstrate that residues 1 to 27 of UL25 contact the hexon adjacent to the penton. A second region, most likely centered on amino acids 26 to 39, contacts the triplex that is one removed from the penton. Importantly, both of these UL25 capsid binding regions are essential for the stable packaging of full-length viral genomes.     

Autoproteolysis of herpes simplex virus type 1 protease releases an active catalytic domain found in intermediate capsid particles.

The UL26 gene of herpes simplex virus type 1 (HSV-1) encodes a 635-amino-acid protease that cleaves itself and the HSV-1 assembly protein ICP35cd (F. Liu and B. Roizman, J. Virol. 65:5149-5156, 1991). We previously examined the HSV protease by using an Escherichia coli expression system (I. C. Deckman, M. Hagen, and P. J. McCann III, J. Virol. 66:7362-7367, 1992) and identified two autoproteolytic cleavage sites between residues 247 and 248 and residues 610 and 611 of UL26 (C. L. DiIanni, D. A. Drier, I. C. Deckman, P. J. McCann III, F. Liu, B. Roizman, R. J. Colonno, and M. G. Cordingley, J. Biol. Chem. 268:2048-2051, 1993). In this study, a series of C-terminal truncations of the UL26 open reading frame was tested for cleavage activity in E. coli. Our results delimit the catalytic domain of the protease to the N-terminal 247 amino acids of UL26 corresponding to No, the amino-terminal product of protease autoprocessing. Autoprocessing of the full-length protease was found to be unnecessary for catalysis, since elimination of either or both cleavage sites by site-directed mutagenesis fails to prevent cleavage of ICP35cd or an unaltered protease autoprocessing site. Catalytic activity of the 247-amino-acid protease domain was confirmed in vitro by using a glutathione-S-transferase fusion protein. The fusion protease was induced to high levels of expression, affinity purified, and used to cleave purified ICP35cd in vitro, indicating that no other proteins are required. By using a set of domain-specific antisera, all of the HSV-1 protease cleavage products predicted from studies in E. coli were identified in HSV-1-infected cells. At least two protease autoprocessing products, in addition to fully processed ICP35cd (ICP35ef), were associated with intermediate B capsids in the nucleus of infected cells, suggesting a key role for proteolytic maturation of the protease and ICP35cd in HSV-1 capsid assembly.     

Role of the UL25 protein in herpes simplex virus DNA encapsidation

The herpes simplex virus protein UL25 attaches to the external vertices of herpes simplex virus type 1 capsids and is required for the stable packaging of viral DNA. To define regions of the protein important for viral replication and capsid attachment, the 580-amino-acid UL25 open reading frame was disrupted by transposon mutagenesis. The UL25 mutants were assayed for complementation of a UL25 deletion virus, and in vitro-synthesized protein was tested for binding to UL25-deficient capsids. Of the 11 mutants analyzed, 4 did not complement growth of the UL25 deletion mutant, and analysis of these and additional mutants in the capsid-binding assay demonstrated that UL25 amino acids 1 to 50 were sufficient for capsid binding. Several UL25 mutations were transferred into recombinant viruses to analyze the effect of the mutations on UL25 capsid binding and on DNA cleavage and packaging. Studies of these mutants demonstrated that amino acids 1 to 50 of UL25 are essential for its stable interaction with capsids and that the C terminus is essential for DNA packaging and the production of infectious virus through its interactions with other viral packaging or tegument proteins. Analysis of viral DNA cleavage demonstrated that in the absence of a functional UL25 protein, aberrant cleavage takes place at the unique short end of the viral genome, resulting in truncated viral genomes that are not retained in capsids. Based on these observations, we propose a model where UL25 is required for the formation of DNA-containing capsids by acting to stabilize capsids that contain full-length viral genomes.     

Separate functional domains of the herpes simplex virus type 1 protease: evidence for cleavage inside capsids.

The herpes simplex virus type 1 (HSV-1) protease (Pra) and related proteins are involved in the assembly of viral capsids and virion maturation. Pra is a serine protease, and the active-site residue has been mapped to amino acid (aa) 129 (Ser). This 635-aa protease, encoded by the UL26 gene, is autoproteolytically processed at two sites, the release (R) site between amino acid residues 247 and 248 and the maturation (M) site between residues 610 and 611. When the protease cleaves itself at both sites, it releases Nb, the catalytic domain (N0), and the C-terminal 25 aa. ICP35, a substrate of the HSV-1 protease, is the product of the UL26.5 gene. As it is translated from a Met codon within the UL26 gene, ICP35 cd are identical to the C-terminal 329-aa sequence of the protease and are trans cleaved at an identical C-terminal site to generate ICP35 e,f and a 25-aa peptide. Only fully processed Pra (N0 and Nb) and ICP35 (ICP35 e,f) are present in B capsids, which are believed to be precursors of mature virions. Using an R-site mutant A247S virus, we have recently shown that this mutant protease retains enzymatic activity but fails to support viral growth, suggesting that the release of N0 is required for viral replication. Here we report that another mutant protease, with an amino acid substitution (Ser to Cys) at the active site, can complement the A247S mutant but not a protease deletion mutant. Cell lines expressing the active-site mutant protease were isolated and shown to complement the A247S mutant at the levels of capsid assembly, DNA packaging, and viral growth. Therefore, the complementation between the R-site mutant and the active-site mutant reconstituted wild-type Pra function. One feature of this intragenic complementation is that following sedimentation of infected-cell lysates on sucrose gradients, both N-terminally unprocessed and processed proteases were isolated from the fractions where normal B capsids sediment, suggesting that proteolytic processing occurs inside capsids. Our results demonstrate that the HSV-1 protease has distinct functional domains and some of these functions can complement in trans.     

The herpes simplex virus 1 gene encoding a protease also contains within its coding domain the gene encoding the more abundant substrate.

The herpes simplex virus 1 open reading frames UL26 and UL26.5 are 3' coterminal. The larger, UL26 open reading frame encodes a protein approximately 80,000 in apparent molecular weight and contains the promoter and coding sequence of the UL26.5 gene, which specifies a capsid protein designated infected cell protein 35. The larger product contains in its entirety the amino acid sequence of the smaller protein. We report that the UL26 gene encodes a protease which catalyzes its own cleavage and that of the more abundant product of UL26.5. By inserting the coding sequence of an epitope to a cytomegalovirus monoclonal antibody and homologs of the immunoglobulin G binding domain of staphylococcal protein A into the 3' termini of the coding domains of the two open reading frames, we identified both products of the cleavage and determined that the cleavage site is approximately 20 amino acids from the carboxyl termini of both proteins.     

Labeling and Localization of the Herpes Simplex Virus Capsid Protein UL25 and Its Interaction with the Two Triplexes Closest to the Penton

The herpes simplex virus type 1 UL25 protein is one of seven viral proteins that are required for DNA cleavage and packaging. Together with UL17, UL25 forms part of an elongated molecule referred to as the C-capsid-specific component (CCSC). Five copies of the CCSC are located at each of the capsid vertices on DNA-containing capsids. To study the conformation of UL25 as it is folded on the capsid surface, we identified the sequence recognized by a UL25-specific monoclonal antibody and localized the epitope on the capsid surface by immunogold electron microscopy. The epitope mapped to amino acids 99-111 adjacent to the region of the protein (amino acids 1-50) that is required for capsid binding. In addition, cryo-EM reconstructions of C-capsids in which the green fluorescent protein (GFP) was fused within the N-terminus of UL25 localized the point of contact between UL25 and GFP. The result confirmed the modeled location of the UL25 protein in the CCSC density as the region that is distal to the penton with the N-terminus of UL25 making contact with the triplex one removed from the penton. Immunofluorescence experiments at early times during infection demonstrated that UL25-GFP was present on capsids located within the cytoplasm and adjacent to the nucleus. These results support the view that UL25 is present on incoming capsids with the capsid-binding domain of UL25 located on the surface of the mature DNA-containing capsid.     

Herpes Simplex Virus Type 1 Cleavage and Packaging Proteins UL15 and UL28 Are Associated with B but Not C Capsids during Packaging

At least seven viral genes encode proteins (UL6, UL15, UL17, UL25, UL28, UL32, and UL33) that are required for DNA cleavage and packaging of herpes simplex virus type 1 (HSV-1) DNA. Sequence analysis reveals that UL15 shares homology with gp17, the large catalytic subunit of the bacteriophage T4 terminase. Thus, UL15 may play a direct role in the cleavage of viral DNA replication intermediates into monomers. In this study, we asked whether UL15 and other cleavage and packaging proteins could be detected in capsids isolated from infected cells. Consistent with previous studies showing that UL6 and UL25 are minor protein constituents of the capsids, we detected these proteins in both B and C capsids. In contrast, the previously identified full-length version (81 kDa) of UL15 was found predominantly in B capsids and in much smaller amounts in C capsids. In addition, the UL28 protein was found predominantly in B but not C capsids in a distribution similar to that of the 81-kDa version of UL15. These results suggest that UL28 and the 81-kDa form of UL15 are transiently associated with capsid intermediates during the packaging process. Surprisingly, however, a previously unidentified 87-kDa form of UL15 was found in the B and C capsids and in virions. Analysis of cells infected with mutants individually lacking UL6, UL15, UL25, UL28, or UL32 demonstrates that the lack of one cleavage and packaging protein does not affect the expression of the others. Furthermore, this analysis, together with guanidine HCl extraction analysis of purified capsids, indicates that UL6, UL25, and UL28 are able to associate with B capsids in the absence of other DNA cleavage and packaging proteins. On the other hand, the two UL15-related proteins (81 and 87 kDa) do not associate efficiently with B capsids in cells infected with UL6 and UL28 mutants. These results suggest that the ability of the UL15-related proteins to bind to B capsids may be mediated through interactions with UL6 and UL28.     

The Herpes Simplex Virus Type 1 Cleavage/Packaging Protein,UL32, Is Involved in Efficient Localization of Capsids to Replication Compartments

Six genes, including UL32, have been implicated in the cleavage and packaging of herpesvirus DNA into preassembled capsids. We have isolated a UL32 insertion mutant which is capable of near-wild-type levels of viral DNA synthesis; however, the mutant virus is unable to cleave and package viral DNA, consistent with the phenotype of a previously isolated temperature-sensitive herpes simplex virus type 1 mutant, tsN20 (P. A. Schaffer, G. M. Aron, N. Biswal, and M. Benyesh-Melnick, Virology 52:57–71, 1973). A polyclonal antibody which recognizes UL32 was previously used by Chang et al. (Y. E. Chang, A. P. Poon, and B. Roizman, J. Virol. 70:3938–3946, 1996) to demonstrate that UL32 accumulates predominantly in the cytoplasm of infected cells. In this report, a functional epitope-tagged version of UL32 showed that while UL32 is predominantly cytoplasmic, some nuclear staining which colocalizes with the major DNA binding protein (ICP8, UL29) in replication compartments can be detected. We have also used a monoclonal antibody (5C) specific for the hexon form of major capsid protein VP5 to study the distribution of capsids during infection. In cells infected with wild-type KOS (6 and 8 h postinfection), 5C staining patterns indicate that capsids are present in nuclei within replication compartments. These results suggest that cleavage and packaging occur in replication compartments at least at 6 and 8 h postinfection. Cells infected with the UL32 mutant exhibit a hexon staining pattern which is more diffusely distributed throughout the nucleus and which is not restricted to replication compartments. We propose that UL32 may play a role in “bringing” preassembled capsids to the sites of DNA packaging and that the failure to localize to replication compartments may explain the cleavage/packaging defect exhibited by this mutant. These results suggest that the UL32 protein is required at a step distinct from those at which other cleavage and packaging proteins are required and may be involved in the correct localization of capsids within infected cells.During infection of cells with herpes simplex virus type 1 (HSV-1), the large concatemeric products of DNA replication are cleaved to unit length and packaged into preassembled capsids. Capsids are icosahedral structures composed of 150 hexons and 12 pentons. Three types of capsids (A, B, and C) can be isolated from infected cells by velocity centrifugation (20). C capsids contain the viral DNA genome; B capsids contain the scaffolding protein; and A capsids contain neither DNA nor the scaffolding protein. Pulse chase experiments with another alphaherpesvirus, equine herpesvirus 1, indicate that at least some B capsids can package DNA and mature into infectious virions, while A capsids cannot (46). By analogy with the bacteriophages, these results suggest that B capsids represent procapsids which are intermediates in the packaging process. However, a new intermediate in the assembly process has recently been identified (41, 62). These newly identified capsid forms observed in in vitro assembly extracts have the same protein content as B capsids but are more spherical; these capsids are unstable and adopt the more angular form characteristic of B capsids after prolonged incubation in vitro. These results suggest that the unstable spherical forms may represent the true procapsid intermediate (41, 62).In many bacteriophages, the procapsid contains at least three essential components: an icosahedrally arranged protein shell, an internal scaffold, and a dodecameric ring called the portal vertex through or around which the phage DNA is taken up (8, 11, 18). For HSV-1, the outer shell is composed of four proteins: the major capsid protein, VP5; a small protein bound to hexons, VP26; and a triplex structure made up of heterotrimers of VP19C and VP23 (reviewed in reference 56). VP24, VP21, and VP22a are found in the interior of the capsid and are encoded by overlapping genes UL26 and UL26.5; VP21 and VP22a are present in B but not A or C capsids and are considered to make up the internal scaffold (reviewed in reference 56). Although bacteriophages contain a portal vertex, no such structure has been observed in HSV-1 capsids. Whether the herpesviruses have a unique portal vertex through which viral DNA is taken up is unclear; it is possible that this type of unique vertex is only needed in viruses which have a tail. Capsids indistinguishable from those isolated from HSV-1-infected cells have been observed in extracts from insect cells infected with recombinant baculoviruses bearing HSV-1 capsid genes (42, 60). Therefore, it is clear that these proteins are sufficient for capsid assembly in vitro; however, it is not known whether capsids formed in vitro are competent for DNA uptake. It is possible that minor components of capsids play important roles in genome encapsidation.In addition to the capsid proteins, at least six genes are essential for the encapsidation of viral DNA: the UL6, UL15, UL25, UL28, UL32, and UL33 genes. Temperature-sensitive (ts) strains with mutations in these genes have similar phenotypes, in that viral DNA can be replicated but not cleaved and packaged (1, 2, 4, 6, 48, 51, 54, 55, 66). Strains with null mutations in the UL6, UL15, UL25, UL28, and UL33 genes have been isolated and characterized, thereby confirming the roles of these genes in cleavage and packaging (5, 27, 37, 45, 59, 68). Despite the identification of these required genes, the mechanism by which viral DNA is cleaved and packaged is not understood, nor has the role of any of the gene products been determined. The UL6 and UL25 proteins have been detected in A, B, and C capsids as well as in virions (3, 28, 37, 44); however, the precise role of these two proteins in capsids remains to be determined.A ts UL32 mutant, tsN20, defective in cleavage and packaging, has been reported previously (51). Because mutants with lesions resulting in temperature sensitivity are often prone to problems associated with incomplete penetrance at the nonpermissive temperature, we isolated a UL32 insertion mutant, hr64. Characterization of hr64 confirms that UL32 is essential for cleavage and packaging. Previous studies demonstrated that UL32 localizes to the cytoplasm of infected cells (13). We have used a functional epitope-tagged version of UL32 to confirm that in infected cells, this protein is mainly cytoplasmic, although some nuclear staining was observed.HSV-1 DNA replication occurs in globular nuclear domains termed “replication compartments” initially identified by ICP8 (UL29) staining patterns in an immunofluorescence assay (49). All seven replication proteins have now been localized within replication compartments (10, 24, 29–31, 43) as has regulatory protein ICP4 (26, 50). Ward et al. have recently reported that at late times after infection (18 h), capsids accumulate in the nucleus in regions distinct from replication compartments (64). These authors suggest that these regions represent assembly stations in which DNA is packaged. We report herein, however, that at 6 and 8 h postinfection, capsids colocalize with ICP8 in replication compartments. This suggests that at these early times, cleavage and packaging occur within replication compartments. Furthermore, we report that in cells infected with the UL32 mutant virus, capsids are distributed throughout the nucleus, accumulating in regions outside the replication compartments. This suggests that UL32 may play a role in the efficient localization of capsids in infected cells.     

Regions of the Herpes Simplex Virus Scaffolding Protein That Are Important for Intermolecular Self-Interaction

The herpes simplex virus type 1 (HSV-1) scaffolding protein encoded by gene UL26.5 promotes the formation of the icosahedral capsid shell through its association with the major capsid protein VP5 and through intermolecular interactions with itself. Inside the capsid shell, the UL26.5 product together with the maturational protease, a minor protein, form a spherical structure which is broken down and released from the capsid during packaging of the viral genome. Selected residues from four internal regions of the HSV-1 scaffolding protein that have significant conservation of amino acids within the scaffolding proteins of alphaherpesviruses were mutated, and the properties of the proteins were examined. Only the HSV-1 scaffolding protein with mutations in the conserved N-terminal domain showed reduced interaction with the varicella-zoster virus homologue in a cell-based immunofluorescence assay, providing the first evidence that this domain in the HSV-1 protein is likely to be involved in intermolecular self-interaction. Scaffolding protein with mutations in this domain or in either of two other domains failed to assemble into scaffold-like particles but retained the ability to self-interact, although the aggregates were significant smaller than most of the aggregates formed by the wild-type protein. These results suggest that there are multiple domains involved in the intermolecular self-association of the HSV-1 scaffolding protein that can act independently of one another. This conclusion was supported by the observation that none of the mutant proteins with lesions in an individual domain, including a protein with mutations in a central region previously implicated in self-interaction (A. Pelletier, F. D?, J. J. Brisebois, L. Lagacé, and M. G. Cordingley, J. Virol. 71:5197-5208, 1997), interfered with capsid assembly in a baculovirus expression system. A protein mutated in the central region and another conserved domain, both of which had been predicted to form coiled coils, was impaired for capsid formation but still retained the capacity to interact with VP5.