A domain in the herpes simplex virus 1 triplex protein VP23 is essential for closure of capsid shells into icosahedral structures

VP23 is a key component of the triplex structure. The triplex, which is unique to herpesviruses, is a complex of three proteins, two molecules of VP23 which interact with a single molecule of VP19C. This structure is important for shell accretion and stability of the protein coat. Previous studies utilized a random transposition mutagenesis approach to identify functional domains of the triplex proteins. In this study, we expand on those findings to determine the key amino acids of VP23 that are required for triplex formation. Using alanine-scanning mutagenesis, we have made mutations in 79 of 318 residues of the VP23 polypeptide. These mutations were screened for function both in the yeast two-hybrid assay for interaction with VP19C and in a genetic complementation assay for the ability to support the replication of a VP23 null mutant virus. These assays identified a number of amino acids that, when altered, abolish VP23 function. Abrogation of virus assembly by a single-amino-acid change bodes well for future development of small-molecule inhibitors of this process. In addition, a number of mutations which localized to a C-terminal region of VP23 (amino acids 205 to 241) were still able to interact with VP19C but were lethal for virus replication when introduced into the herpes simplex virus 1 (HSV-1) KOS genome. The phenotype of many of these mutant viruses was the accumulation of large open capsid shells. This is the first demonstration of capsid shell accumulation in the presence of a lethal VP23 mutation. These data thus identify a new domain of VP23 that is required for or regulates capsid shell closure during virus assembly.     

Molecular interactions of Epstein-Barr virus capsid proteins

The capsids of herpesviruses, which comprise major and minor capsid proteins, have a common icosahedral structure with 162 capsomers. An electron microscopic study shows that Epstein-Barr virus (EBV) capsids in the nucleus are immunolabeled by anti-BDLF1 and anti-BORF1 antibodies, indicating that BDLF1 and BORF1 are the minor capsid proteins of EBV. Cross-linking and electrophoresis studies of purified BDLF1 and BORF1 revealed that these two proteins form a triplex that is similar to that formed by the minor capsid proteins, VP19C and VP23, of herpes simplex virus type 1 (HSV-1). Although the interaction between VP23, a homolog of BDLF1, and the major capsid protein VP5 could not be verified biochemically in earlier studies, the interaction between BDLF1 and the EBV major capsid protein, viral capsid antigen (VCA), can be confirmed by glutathione S-transferase (GST) pulldown assay and coimmunoprecipitation. Additionally, in HSV-1, VP5 interacts with only the middle region of VP19C; in EBV, VCA interacts with both the N-terminal and middle regions of BORF1, a homolog of VP19C, revealing that the proteins in the EBV triplex interact with the major capsid protein differently from those in HSV-1. A GST pulldown study also identifies the oligomerization domains in VCA and the dimerization domain in BDLF1. The results presented herein reveal how the EBV capsid proteins interact and thereby improve our understanding of the capsid structure of the virus.     

DNA Binding and Condensation Properties of the Herpes Simplex Virus Type 1 Triplex Protein VP19C

Herpesvirus capsids are regular icosahedrons with a diameter of a 125 nm and are made up of 162 capsomeres arranged on a T = 16 lattice. The capsomeres (VP5) interact with the triplex structure, which is a unique structural feature of herpesvirus capsid shells. The triplex is a heterotrimeric complex; one molecule of VP19C and two of VP23 form a three-pronged structure that acts to stabilize the capsid shell through interactions with adjacent capsomeres. VP19C interacts with VP23 and with the major capsid protein VP5 and is required for the nuclear localization of VP23. Mutation of VP19C results in the abrogation of capsid shell synthesis. Analysis of the sequence of VP19C showed the N-terminus of VP19C is very basic and glycine rich. It was hypothesized that this domain could potentially bind to DNA. In this study an electrophoretic mobility shift assay (EMSA) and a DNA condensation assay were performed to demonstrate that VP19C can bind DNA. Purified VP19C was able to bind to both a DNA fragment of HSV-1 origin as well as a bacterial plasmid sequence indicating that this activity is non-specific. Ultra-structural imaging of the nucleo-protein complexes revealed that VP19C condensed the DNA and forms toroidal DNA structures. Both the DNA binding and condensing properties of VP19C were mapped to the N-terminal 72 amino acids of the protein. Mutational studies revealed that the positively charged arginine residues in this N-terminal domain are required for this binding. This DNA binding activity, which resides in a non-conserved region of the protein could be required for stabilization of HSV-1 DNA association in the capsid shell.     

Assembly of the Herpes Simplex Virus Capsid: Preformed Triplexes Bind to the Nascent Capsid

The herpes simplex virus type 1 (HSV-1) capsid is a T=16 icosahedral shell that forms in the nuclei of infected cells. Capsid assembly also occurs in vitro in reaction mixtures created from insect cell extracts containing recombinant baculovirus-expressed HSV-1 capsid proteins. During capsid formation, the major capsid protein, VP5, and the scaffolding protein, pre-VP22a, condense to form structures that are extended into procapsids by addition of the triplex proteins, VP19C and VP23. We investigated whether triplex proteins bind to the major capsid-scaffold protein complexes as separate polypeptides or as preformed triplexes. Assembly products from reactions lacking one triplex protein were immunoprecipitated and examined for the presence of the other. The results showed that neither triplex protein bound unless both were present, suggesting that interaction between VP19C and VP23 is required before either protein can participate in the assembly process. Sucrose density gradient analysis was employed to determine the sedimentation coefficients of VP19C, VP23, and VP19C-VP23 complexes. The results showed that the two proteins formed a complex with a sedimentation coefficient of 7.2S, a value that is consistent with formation of a VP19C-VP23₂ heterotrimer. Furthermore, VP23 was observed to have a sedimentation coefficient of 4.9S, suggesting that this protein exists as a dimer in solution. Deletion analysis of VP19C revealed two domains that may be required for attachment of the triplex to major capsid-scaffold protein complexes; none of the deletions disrupted interaction of VP19C with VP23. We propose that preformed triplexes (VP19C-VP23₂ heterotrimers) interact with major capsid-scaffold protein complexes during assembly of the HSV-1 capsid.     

Mutational analysis of the herpes simplex virus triplex protein VP19C

Herpes simplex virus type 1 (HSV-1) capsids have an icosahedral structure with capsomers formed by the major capsid protein, VP5, linked in groups of three by distinctive structures called triplexes. Triplexes are heterotrimers formed by two proteins in a 1:2 stoichiometry. The single-copy protein is called VP19C, and the dimeric protein is VP23. We have carried out insertional and deletional mutagenesis on VP19C and have examined the effects of the mutations on virus growth and capsid assembly. Insertional mutagenesis showed that the N-terminal approximately 100 amino acids of the protein, which correspond to a region that is poorly conserved among herpesviruses, are insensitive to disruption and that insertions into the rest of the protein had various effects on virus growth. Some, but not all, severely disabled mutants were compromised in the ability to bind VP23 or VP5. Analysis of deletion mutants revealed the presence of a nuclear localization signal (NLS) near the N terminus of VP19C, and this was mapped to a 33-amino-acid region by fusion of specific sequences to a green fluorescent protein marker. By replacing the endogenous NLS with that from the simian virus 40 large T antigen, we were able to show that the first 45 amino acids of VP19C were not essential for assembly of functional capsids and infectious virus particles. However, removing the first 63 amino acids resulted in formation of aberrant capsids and prevented virus growth, suggesting that the poorly conserved N-terminal sequences have some as-yet-unidentified function.     

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.     

The N terminus of the herpes simplex virus type 1 triplex protein, VP19C, cannot be detected on the surface of the capsid shell by using an antibody (hemagglutinin) epitope tag

The herpes simplex virus (HSV) triplex is a complex of three protein subunits, VP19C and a dimer of VP23 that is essential for capsid assembly. We have derived HSV-1 recombinant viruses that contain monomeric red fluorescent protein (mRFP1), a Flu hemagglutinin (HA) epitope, and a six-histidine tag fused to the amino terminus of VP19C. These viruses were capable of growth on Vero cells, indicating that the amino terminus of VP19C could tolerate these fusions. By use of immunoelectron microscopy methods, capsids that express VP19C-mRFP but not VP19C-HA were labeled with gold particles when incubated with the corresponding antibody. Our conclusion from the data is that a large tag at the N terminus of VP19C was sufficiently exposed on the capsid surface for polyclonal antibody reactivity, while the small HA epitope was inaccessible to the antibody. These data indicate that an epitope tag at the amino terminus of VP19C is not exposed at the capsid surface for reactivity to its antibody.     

Expression of the HSV-1 capsid protein VP19C in Escherichia coli: a single amino acid change overcomes an expression block of the full-length polypeptide

The herpesvirus triplex is a key structural feature of the capsids of these viruses. It is composed of a hetero-trimer of one molecule of VP19C and two molecules of VP23. It acts to stabilize capsid shells by connecting the capsomeric subunits together. Although it has been possible to over-express in Escherichia coli and purify one component of the triplex, VP23; this has not been the case with VP19C. Because an N-terminal polypeptide of VP19C could be expressed and purified using a GST affinity tag, a directed mutagenic approach was used to determine the region of VP19C that caused the block in expression of the full-length protein. The region was mapped to reside between VP19C amino acids 145 and 150 using truncation gene fusions and subsequently a single amino acid, R146 was identified which when changed to alanine, allowed stable expression and accumulation of VP19C. This change does not affect the biological function of VP19C. Finally using this altered VP19C, co-expression of the triplex proteins in the same cell has been achieved making it now possible to purify this complex for biophysical and structural studies.     

Residues of VP26 of herpes simplex virus type 1 that are required for its interaction with capsids

VP26 is the smallest capsid protein and decorates the outer surface of the capsid shell of herpes simplex virus. It is located on the hexons at equimolar amounts with VP5. Its small size (112 amino acids) and high copy number make it an attractive molecule to use as a probe to investigate the complex pattern of capsid protein interactions. An in vitro capsid binding assay and a green fluorescent protein (GFP) localization assay were used to identify VP26 residues important for its interaction with capsids. To test for regions of VP26 that may be essential for binding to capsids, three small in-frame deletion mutations were generated in VP26, Delta18-25, Delta54-60, and Delta93-100. Their designations refer to the amino acids deleted by the mutation. The mutation at the C terminus of the molecule, which encompasses a region of highly conserved residues, abolished binding to the capsid and the localization of GFP to the nucleus in characteristic large puncta. Additional mutations revealed that a region of VP26 spanning from residue 50 to 112 was sufficient for the localization of the fused protein (VP26-GFP) to the nucleus and for it to bind to capsids. Using site-directed mutagenesis of conserved residues in VP26, two key residues for protein-protein interaction, F79 and G93, were identified as judged by the localization of GFP to nuclear puncta. When these mutations were analyzed in the capsid binding assay, they were also found to eliminate binding of VP26 to the capsid structure. Surprisingly, additional mutations that affected the ability of VP26 to bind to capsids in vitro were uncovered. Mutations at residues A58 and L64 resulted in a reduced ability of VP26 to bind to capsids. Mutation of the hydrophobic residues M78 and A80, which are adjacent to the hydrophobic residue F79, abolished VP26 capsid binding. In addition, the block of conserved amino acids in the carboxy end of the molecule had the most profound effect on the ability of VP26 to interact with capsids. Mutation of amino acid G93, L94, R95, R96, or T97 resulted in a greatly diminished ability of VP26 to bind capsids. Yet, all of these residues other than G93 were able to efficiently translocate or concentrate GFP into the nucleus, giving rise to the punctate fluorescence. Thus, the interaction of VP26 with the capsid appears to occur through at least two separate mechanisms. The initial interaction of VP26 and VP5 may occur in the cytoplasm or when VP5 is localized in the nucleus. Residues F79 and G93 are important for this bi-molecular interaction, resulting in the accumulation of VP26 in the nucleus in concentrated foci. Subsequent to this association, additional amino acids of VP26, including those in the C-terminal conserved domain, are important for interaction of VP26 with the three-dimensional capsid structure.     

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).     

Mutation of single hydrophobic residue I27, L35, F39, L58, L65, L67, or L71 in the N terminus of VP5 abolishes interaction with the scaffold protein and prevents closure of herpes simplex virus type 1 capsid shells

Protein-protein interactions drive the assembly of the herpes simplex virus type 1 (HSV-1) capsid. A key interaction occurs between the C-terminal tail of the scaffold protein (pre-22a) and the major capsid protein (VP5). Previously (Z. Hong, M. Beaudet-Miller, J. Durkin, R. Zhang, and A. D. Kwong, J. Virol. 70:533-540, 1996) it was shown that the minimal domain in the scaffold protein necessary for this interaction was composed of a hydrophobic amphipathic helix. The goal of this study was to identify the hydrophobic residues in VP5 important for this bimolecular interaction. Results from the genetic analysis of second-site revertant virus mutants identified the importance of the N terminus of VP5 for the interaction with the scaffold protein. This allowed us to focus our efforts on a small region of this large polypeptide. Twenty-four hydrophobic residues, starting at L23 and ending at F84, were mutated to alanine. All the mutants were first screened for interaction with pre-22a in the yeast two-hybrid assay. From this in vitro assay, seven residues, I27, L35, F39, L58, L65, L67, and L71, that eliminated the interaction when mutated were identified. All 24 mutants were introduced into the virus genome with a genetic marker rescue/marker transfer system. For this system, viruses and cell lines that greatly facilitated the introduction of the mutants into the genome were made. The same seven mutants that abolished interaction of VP5 with pre-22a resulted in an absolute requirement for wild-type VP5 for growth of the viruses. The viruses encoding these mutations in VP5 were capable of forming capsid shells comprised of VP5, VP19C, VP23, and VP26, but the closure of these shells into an icosahedral structure was prevented. Mutation at L75 did not affect the ability of this protein to interact with pre-22a, as judged from the in vitro assay, but this mutation specified a lethal effect for virus growth and abolished the formation of any detectable assembled structure. Thus, it appears that the L75 residue is important for another essential interaction of VP5 with the capsid shell proteins. The congruence of the data from the previous and present studies demonstrates the key roles of two regions in the N terminus of this large protein that are crucial for this bimolecular interaction. Thus, residues I27, L35, and F39 comprise the first subdomain and residues L58, L65, L67 and L71 comprise a second subdomain of VP5. These seven hydrophobic residues are important for the interaction of VP5 with the scaffold protein and consequently the formation of an icosahedral shell structure that encloses the viral genome.     

Assembly of the herpes simplex virus capsid: requirement for the carboxyl-terminal twenty-five amino acids of the proteins encoded by the UL26 and UL26.5 genes.

Herpes simplex virus type 1 (HSV-1) intermediate capsids are composed of seven proteins, VP5, VP19C, VP21, VP22a, VP23, VP24, and VP26, and the genes that encode these proteins, UL19, UL38, UL26, UL26.5, UL18, UL26, and UL35, respectively. The UL26 gene encodes a protease that cleaves itself and the product of the UL26.5 gene at a site (M site) 25 amino acids from the C terminus of these two proteins. In addition, the protease cleaves itself at a second site (R site) between amino acids 247 and 248. Cleavage of the UL26 protein gives rise to the capsid proteins VP21 and VP24, and cleavage of the UL26.5 protein gives rise to the capsid protein VP22a. Previously we described the production of HSV-1 capsids in insect cells by infecting the cells with recombinant baculoviruses expressing the six capsid genes (D. R. Thomsen, L. L. Roof, and F. L. Homa, J. Virol. 68:2442-2457, 1994). Using this system, we demonstrated that the products of the UL26 and/or UL26.5 genes are required as scaffolds for assembly of HSV-1 capsids. To better understand the functions of the UL26 and UL26.5 proteins in capsid assembly, we constructed baculoviruses that expressed altered UL26 and UL26.5 proteins. The ability of the altered UL26 and UL26.5 proteins to support HSV-1 capsid assembly was then tested in insect cells. Among the specific mutations tested were (i) deletion of the C-terminal 25 amino acids from the proteins coded for by the UL26 and UL26.5 genes; (ii) mutation of His-61 of the UL26 protein, an amino acid required for protease activity; and (iii) mutation of the R cleavage site of the UL26 protein. Analysis of the capsids formed with wild-type and mutant proteins supports the following conclusions: (i) the C-terminal 25 amino acids of the UL26 and UL26.5 proteins are required for capsid assembly; (ii) the protease activity associated with the UL26 protein is not required for assembly of morphologically normal capsids; and (iii) the uncleaved forms of the UL26 and UL26.5 proteins are employed in assembly of 125-nm-diameter capsids; cleavage of these proteins occurs during or subsequent to capsid assembly. Finally, we carried out in vitro experiments in which the major capsid protein VP5 was mixed with wild-type or truncated UL26.5 protein and then precipitated with a VP5-specific monoclonal antibody.(ABSTRACT TRUNCATED AT 400 WORDS)     

Small capsid protein pORF65 is essential for assembly of Kaposi's sarcoma-associated herpesvirus capsids

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent for KS tumors, multicentric Castleman's disease, and primary effusion lymphomas. Like other herpesvirus capsids, the KSHV capsid is an icosahedral structure composed of six proteins. The capsid shell is made up of the major capsid protein, two triplex proteins, and the small capsid protein. The scaffold protein and the protease occupy the internal space. The assembly of KSHV capsids is thought to occur in a manner similar to that determined for herpes simplex virus type 1 (HSV-1). Our goal was to assemble KSHV capsids in insect cells using the baculovirus expression vector system. Six KSHV capsid open reading frames were cloned and the proteins expressed in Sf9 cells: pORF25 (major capsid protein), pORF62 (triplex 1), pORF26 (triplex 2), pORF17 (protease), pORF17.5 (scaffold protein), and also pORF65 (small capsid protein). When insect cells were coinfected with these baculoviruses, angular capsids that contained internal core structures were readily observed by conventional electron microscopy of the infected cells. Capsids were also readily isolated from infected cells by using rate velocity sedimentation. With immuno-electron microscopy methods, these capsids were seen to be reactive to antisera to pORF65 as well as to KSHV-positive human sera, indicating the correct conformation of pORF65 in these capsids. When either virus expressing the triplex proteins was omitted from the coinfection, capsids did not assemble; similar to observations made in HSV-1-infected cells. If the virus expressing the scaffold protein was excluded, large open shells that did not attain icosahedral structure were seen in the nuclei of infected cells. The presence of pORF65 was required for capsid assembly, in that capsids did not form if this protein was absent as judged by both by ultrastructural analysis of infected cells and rate velocity sedimentation experiments. Thus, a novel outcome of this study is the finding that the small capsid protein of KSHV, like the major capsid and triplex proteins, is essential for capsid shell assembly.     

Roles of Triplex and Scaffolding Proteins in Herpes Simplex Virus Type 1 Capsid Formation Suggested by Structures of Recombinant Particles

Typical herpes simplex virus (HSV) capsids contain seven proteins that form a T=16 icosahedron of 1,250-A diameter. Infection of cells with recombinant baculoviruses expressing two of these proteins, VP5 (which forms the pentons and hexons in typical HSV capsids) and VP19C (a component of the triplexes that connect adjacent capsomeres), results in the formation of spherical particles of 880-A diameter. Electron cryomicroscopy and computer reconstruction revealed that these particles possess a T=7 icosahedral symmetry, having 12 pentons and 60 hexons. Among the characteristic structural features of the particle are the skewed appearance of the hexons and the presence of intercapsomeric mass densities connecting the middle domain of one hexon subunit to the lower domain of a subunit in the adjacent hexon. We interpret these connecting masses as being formed by VP19C. Comparison of the connecting masses with the triplexes, which occupy equivalent positions in the T=16 capsid, reveals the probable locations of the single VP19C and two VP23 molecules that make up the triplex. Their arrangement suggests that the two triplex proteins have different roles in controlling intercapsomeric interactions and capsid stability. The nature of these particles and of other aberrant forms made in the absence of scaffold demonstrates the conformational adaptability of the capsid proteins and illustrates how VP23 and the scaffolding protein modulate the nature of the VP5-VP19C network to ensure assembly of the functional T=16 capsid.     

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.     

ATP-Dependent localization of the herpes simplex virus capsid protein VP26 to sites of procapsid maturation

The herpes simplex virus type 1 (HSV-1) capsid shell is composed of four major polypeptides, VP5, VP19c, VP23, and VP26. VP26, a 12-kDa polypeptide, is associated with the tips of the capsid hexons formed by VP5. Mature capsids form upon angularization of the shell of short-lived, fragile spherical precursors termed procapsids. The cold sensitivity and short-lived nature of the procapsid have made its isolation and biochemical analysis difficult, and it remains unclear whether procapsids contain bound VP26 or whether VP26 is recruited following shell angularization. By indirect immunocytochemical analysis of virally expressed VP26 and by direct visualization of a transiently expressed VP26-green fluorescent protein fusion, we show that VP26 fails to specifically localize to intranuclear procapsids accumulated following incubation of the temperature-sensitive HSV mutant tsProt.A under nonpermissive conditions. However, following a downshift to the permissive temperature, which allows procapsid maturation to proceed, VP26 was seen to concentrate at intranuclear sites which also contained epitopes specific to mature, angularized capsids. Like the formation of these epitopes, the association of VP26 with maturing capsids was blocked in a reversible fashion by the depletion of intracellular ATP. We conclude that unlike the other major capsid shell proteins, VP26 is recruited in an ATP-dependent fashion after procapsid maturation begins.     

Use of Ar+ plasma etching to localize structural proteins in the capsid of herpes simplex virus type 1.

Partially cored herpes simplex virus type 1 (HSV-1) capsids (B capsids) were eroded in a low-energy (0.5-keV) Ar+ ion plasma under conditions in which the outermost structural proteins were expected to be degraded before more internal ones. After various periods of etching, the proteins remaining intact were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and determined quantitatively by densitometric scanning of the stained gels. The results showed that the major capsid polypeptide (VP5) and two other capsid proteins, VP19 and VP23, were degraded rapidly beginning as soon as capsids were exposed to the ion plasma. In contrast, significant lags were observed for erosion of VP21, VP22a, and VP24, suggesting that these proteins were available to accelerated ions only after other, more external structures had been damaged or eroded away. The results suggest that VP5, VP19, and VP23 are exposed on the surface of the capsid, while VP21, VP22a, and VP24 are found inside the capsid cavity. The experiments are consistent with the view that VP5 constitutes the major structural component of the hexavalent capsomers. It is proposed that VP19 and VP23 may form other surface structures such as the pentavalent capsomers, the capsid floor, or the intercapsomeric fibers.     

Isolation of herpes simplex virus procapsids from cells infected with a protease-deficient mutant virus

Herpes simplex virus type 1 (HSV-1) capsid proteins assemble in vitro into spherical procapsids that differ markedly in structure and stability from mature polyhedral capsids but can be converted to the mature form. Circumstantial evidence suggests that assembly in vivo follows a similar pathway of procapsid assembly and maturation, a pathway that resembles those of double-stranded DNA bacteriophages. We have confirmed the above pathway by isolating procapsids from HSV-1-infected cells and characterizing their morphology, thermal sensitivity, and protein composition. Experiments were carried out with an HSV-1 mutant (m100) deficient in the maturational protease for which it was expected that procapsids-normally, short-lived intermediates-would accumulate in infected cells. Particles isolated from m100-infected cells were found to share the defining properties of procapsids assembled in vitro. For example, by electron microscopy, they were found to be spherical rather than polyhedral in shape, and they disassembled at 0 degrees C, unlike mature capsids, which are stable at this temperature. A three-dimensional reconstruction computed at 18-A resolution from cryoelectron micrographs showed m100 procapsids to be structurally indistinguishable from procapsids assembled in vitro. In both cases, their predominant components are the four essential capsid proteins: the major capsid protein (VP5), the scaffolding protein (pre-VP22a), and the triplex proteins (VP19C and VP23). VP26, a small, abundant but dispensable capsid protein, was not found associated with m100 procapsids, suggesting that it binds to capsids only after they have matured into the polyhedral form. Procapsids were also isolated from cells infected at the nonpermissive temperature with the HSV-1 mutant tsProt.A (a mutant with a thermoreversible lesion in the protease), and their identity as procapsids was confirmed by cryoelectron microscopy. This analysis revealed density on the inner surface of the procapsid scaffolding core that may correspond to the location of the maturational protease. Upon incubation at the permissive temperature, tsProt.A procapsids transformed into polyhedral, mature capsids, providing further confirmation of their status as precursors.     

Herpes simplex virus type 1 capsid protein VP26 interacts with dynein light chains RP3 and Tctex1 and plays a role in retrograde cellular transport

Cytoplasmic dynein is the major molecular motor involved in minus-end-directed cellular transport along microtubules. There is increasing evidence that the retrograde transport of herpes simplex virus type 1 along sensory axons is mediated by cytoplasmic dynein, but the viral and cellular proteins involved are not known. Here we report that the herpes simplex virus outer capsid protein VP26 interacts with dynein light chains RP3 and Tctex1 and is sufficient to mediate retrograde transport of viral capsids in a cellular model. A library of herpes simplex virus capsid and tegument structural genes was constructed and tested for interactions with dynein subunits in a yeast two-hybrid system. A strong interaction was detected between VP26 and the homologous 14-kDa dynein light chains RP3 and Tctex1. In vitro pull-down assays confirmed binding of VP26 to RP3, Tctex1, and intact cytoplasmic dynein complexes. Recombinant herpes simplex virus capsids were constructed either with or without VP26. In pull-down assays VP26+ capsids bound to RP3; VP26-capsids did not. To investigate intracellular transport, the recombinant viral capsids were microinjected into living cells and incubated at 37 degrees C. After 1 h VP26+ capsids were observed to co-localize with RP3, Tctex1, and microtubules. After 2 or 4 h VP26+ capsids had moved closer to the cell nucleus, whereas VP26-capsids remained in a random distribution. We propose that VP26 mediates binding of incoming herpes simplex virus capsids to cytoplasmic dynein during cellular infection, through interactions with dynein light chains.