The protease of herpes simplex virus type 1 is essential for functional capsid formation and viral growth.

The herpes simplex virus type 1 protease and related proteins are involved in the assembly of viral capsids. The protease encoded by the UL26 gene can process itself and its substrate ICP35, encoded by the UL26.5 gene. To better understand the functions of the protease in infected cells, we have isolated a complementing cell line (BMS-MG22) and constructed and characterized a null UL26 mutant virus, m100. The mutant virus failed to grow on Vero cells and required a complementing cell line for its propagation, confirming that the UL26 gene product is essential for viral growth. Phenotypic analysis of m100 shows that (i) normal amounts of the c and d forms of ICP35 were produced, but they failed to be processed to the cleaved forms, e and f; (ii) viral DNA replication of the mutant proceeded at near wild-type levels, but DNA was not processed to unit length or encapsidated; (iii) capsid structures were observed in thin sections of m100-infected Vero cells by electron microscopy, but assembly of VP5 into hexons of the capsid structure was conformationally altered; and (iv) nuclear localizations of the protease and ICP35 are independent of each other, and the function(s) of Na, at least in part, is to direct the catalytic domain N(o) to the nucleus.     

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)     

Phenotype of the herpes simplex virus type 1 protease substrate ICP35 mutant virus.

The herpes simplex virus type 1 ICP35 assembly protein is involved in the formation of viral capsids. ICP35 is encoded by the UL26.5 gene and is specifically processed by the herpes simplex virus type 1 protease encoded by the UL26 gene. To better understand the functions of ICP35 in infected cells, we have isolated and characterized an ICP35 mutant virus, delta ICP35. The mutant virus was propagated in complementing 35J cells, which express wild-type ICP35. Phenotypic analysis of delta ICP35 shows that (i) mutant virus growth in Vero cells was severely restricted, although small amounts of progeny virus was produced; (ii) full-length ICP35 protein was not produced, although autoproteolysis of the protease still occurred in mutant-infected nonpermissive cells; (iii) viral DNA replication of the mutant proceeded at wild-type levels, but only a very small portion of the replicated DNA was processed to unit length and encapsidated; (iv) capsid structures were observed in delta ICP35-infected Vero cells by electron microscopy and by sucrose sedimentation analysis; (v) assembly of VP5 into hexons of the capsids was conformationally altered; and (vi) ICP35 has a novel function which is involved in the nuclear transport of VP5.     

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.     

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).     

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.     

The Product of the Herpes Simplex Virus Type 1 UL25 Gene Is Required for Encapsidation but Not for Cleavage of Replicated Viral DNA

The herpes simplex virus type 1 (HSV-1) UL25 gene contains a 580-amino-acid open reading frame that codes for an essential protein. Previous studies have shown that the UL25 gene product is a virion component (M. A. Ali et al., Virology 216:278–283, 1996) involved in virus penetration and capsid assembly (C. Addison et al., Virology 138:246–259, 1984). In this study, we describe the isolation of a UL25 mutant (KUL25NS) that was constructed by insertion of an in-frame stop codon in the UL25 open reading frame and propagated on a complementing cell line. Although the mutant was capable of synthesis of viral DNA, it did not form plaques or produce infectious virus in noncomplementing cells. Antibodies specific for the UL25 protein were used to demonstrate that KUL25NS-infected Vero cells did not express the UL25 protein. Western immunoblotting showed that the UL25 protein was associated with purified, wild-type HSV A, B, and C capsids. Transmission electron microscopy indicated that the nucleus of Vero cells infected with KUL25NS contained large numbers of both A and B capsids but no C capsids. Analysis of infected cells by sucrose gradient sedimentation analysis confirmed that the ratio of A to B capsids was elevated in KUL25NS-infected Vero cells. Following restriction enzyme digestion, specific terminal fragments were observed in DNA isolated from KUL25NS-infected Vero cells, indicating that the UL25 gene was not required for cleavage of replicated viral DNA. The latter result was confirmed by pulsed-field gel electrophoresis (PFGE), which showed the presence of genome-size viral DNA in KUL25NS-infected Vero cells. DNase I treatment prior to PFGE demonstrated that monomeric HSV DNA was not packaged in the absence of the UL25 protein. Our results indicate that the product of the UL25 gene is required for packaging but not cleavage of replicated viral DNA.     

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.     

Incorporation of the Green Fluorescent Protein into the Herpes Simplex Virus Type 1 Capsid

The herpes simplex virus type 1 (HSV-1) UL35 open reading frame (ORF) encodes a 12-kDa capsid protein designated VP26. VP26 is located on the outer surface of the capsid specifically on the tips of the hexons that constitute the capsid shell. The bioluminescent jellyfish (Aequorea victoria) green fluorescent protein (GFP) was fused in frame with the UL35 ORF to generate a VP26-GFP fusion protein. This fusion protein was fluorescent and localized to distinct regions within the nuclei of transfected cells following infection with wild-type virus. The VP26-GFP marker was introduced into the HSV-1 (KOS) genome resulting in recombinant plaques that were fluorescent. A virus, designated K26GFP, was isolated and purified and was shown to grow as well as the wild-type virus in cell culture. An analysis of the intranuclear capsids formed in K26GFP-infected cells revealed that the fusion protein was incorporated into A, B, and C capsids. Furthermore, the fusion protein incorporated into the virion particle was fluorescent as judged by fluorescence-activated cell sorter (FACS) analysis of infected cells in the absence of de novo protein synthesis. Cells infected with K26GFP exhibited a punctate nuclear fluorescence at early times in the replication cycle. At later times during infection a generalized cytoplasmic and nuclear fluorescence, including fluorescence at the cell membranes, was observed, confirming visually that the fusion protein was incorporated into intranuclear capsids and mature virions.     

Packaging of genomic and amplicon DNA by the herpes simplex virus type 1 UL25-null mutant KUL25NS

The herpes simplex virus type 1 (HSV-1) mutant KUL25NS, containing a null mutation within the UL25 gene, was isolated and characterized by McNab and coworkers (A. R. McNab, P. Desai, S. Person, L. L. Roof, D. R. Thomsen, W. W. Newcomb, J. C. Brown, and F. L. Homa, J. Virol. 72:1060-1070, 1998). This mutant was able to cleave the concatemeric products of viral DNA replication into monomeric units, but in contrast to wild-type (wt) HSV-1, they were degraded by DNase treatment, indicating that they were not stably packaged into virus capsids. I have examined the packaging of the KUL25NS genome and an HSV-1 amplicon in cells infected with the mutant virus. In contrast to the previous results, a low level of KUL25NS DNA was resistant to DNase digestion, indicating that it was retained in capsids. The proportion of this packaged DNA present as full-length genomes was much lower than in cells infected by wt HSV-1, and there was a significant overrepresentation of the long terminus and underrepresentation of the short terminus. KUL25NS was less impaired in stably packaging amplicon DNA than in packaging its own genome, and the packaged molecules contained approximately equimolar amounts of the two terminal fragments. Below about 100 kbp, the packaged amplicon molecules exhibited an abundance and size distribution similar to those generated using wt HSV-1 as a helper, but the mutant was relatively impaired in packaging longer amplicon molecules. Both packaged genomic and amplicon DNAs were retained in the nuclei of KUL25NS-infected cells. These results suggest that the UL25 protein may play an important role during the later stages of the head-filling process, prior to release of capsids into 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.     

Release of the catalytic domain N(o) from the herpes simplex virus type 1 protease is required for viral growth.

The herpes simplex virus type 1 (HSV-1) protease and its substrate, ICP35, are involved in the assembly of viral capsids and required for efficient viral growth. The full-length protease (Pra) consists of 635 amino acid (aa) residues and is autoproteolytically processed at the release (R) site and the maturation (M) site, releasing the catalytic domain No (VP24), Nb (VP21), and a 25-aa peptide. To understand the biological importance of cleavage at these sites, we constructed several mutations in the cloned protease gene. Transfection assays were performed to determine the functional properties of these mutant proteins by their abilities to complement the growth of the protease deletion mutant m100. Our results indicate that (i) expression of full-length protease is not required for viral replication, since a 514-aa protease molecule lacking the M site could support viral growth; and that (ii) elimination of the R site by changing the residue Ala-247 to Ser abolished viral replication. To better understand the functions that are mediated by proteolytic processing at the R site of the protease, we engineered an HSV-1 recombinant virus containing a mutation at this site. Analysis of the mutant A247S virus demonstrated that (i) the mutant protease retained the ability to cleave at the M site and to trans process ICP35 but failed to support viral growth on Vero cells, demonstrating that release of the catalytic domain No from Pra is required for viral replication; and that (ii) only empty capsid structures were observed by electron microscopy in thin sections of A247S-infected Vero cells, indicating that viral DNA was not encapsidated. Our results demonstrate that processing of ICP35 is not sufficient to support viral replication and provide genetic evidence that the HSV-1 protease has nuclear functions other than enzymatic activity.     

Human cytomegalovirus (HCMV) smallest capsid protein identified as product of short open reading frame located between HCMV UL48 and UL49.

The capsid of cytomegalovirus contains an abundant, low-molecular-weight protein whose coding sequence within the viral genome had not been identified. We have used a combination of biochemical and immunological techniques to demonstrate that this protein, called the smallest capsid protein in human cytomegalovirus, is encoded by a previously unidentified 225-bp open reading frame (ORF) located between ORFs UL48 and UL49. This short ORF, called UL48/49, is the positional homolog of herpes simplex virus ORF UL35 (encoding capsid protein VP26) and shows partial amino acid sequence identity to positional homologs in human herpes viruses 6 and 7.     

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 pseudorabies virus VP1/2 tegument protein is required for intracellular capsid transport

Transport of capsids in cells is critical to alphaherpesvirus infection and pathogenesis; however, viral factors required for transport have yet to be identified. Here we provide a detailed examination of capsid dynamics during the egress phase of infection in Vero cells infected with pseudorabies virus. We demonstrate that the VP1/2 tegument protein is required for processive microtubule-based transport of capsids in the cytoplasm. A second tegument protein that binds to VP1/2, UL37, was necessary for wild-type transport but was not essential for this process. Both proteins were also required for efficient nuclear egress of capsids to the cytoplasm.     

The interaction between the major capsid protein and the smallest capsid protein of human cytomegalovirus is dependent on two linear sequences in the smallest capsid protein

The assembly of human cytomegalovirus (HCMV) with recombinant systems has not been accomplished. An understanding of specific interactions between individual capsid proteins could point to unique characteristics of the assembly process of HCMV capsids. Similar to its herpes simplex virus counterpart, VP26 (UL35), the HCMV smallest capsid protein (SCP; UL48/49) decorates hexons in the mature capsid. In contrast to VP26, the HCMV SCP is essential for virus assembly. In this study we have shown that the major capsid protein (MCP) and the SCP interact in the cytoplasm of transfected cells and can be coprecipitated from insect cells expressing the MCP and the SCP. Using a two-hybrid reporter assay, we demonstrated that two linear sequences within the SCP are sufficient for SCP and MCP interactions.     

Disulfide bond formation contributes to herpes simplex virus capsid stability and retention of pentons

Disulfide bonds reportedly stabilize the capsids of several viruses, including papillomavirus, polyomavirus, and simian virus 40, and have been detected in herpes simplex virus (HSV) capsids. In this study, we show that in mature HSV-1 virions, capsid proteins VP5, VP23, VP19C, UL17, and UL25 participate in covalent cross-links, and that these are susceptible to dithiothreitol (DTT). In addition, several tegument proteins were found in high-molecular-weight complexes, including VP22, UL36, and UL37. Cross-linked capsid complexes can be detected in virions isolated in the presence and absence of N-ethylmaleimide (NEM), a chemical that reacts irreversibly with free cysteines to block disulfide formation. Intracellular capsids isolated in the absence of NEM contain disulfide cross-linked species; however, intracellular capsids isolated from cells pretreated with NEM did not. Thus, the free cysteines in intracellular capsids appear to be positioned such that disulfide bond formation can occur readily if they are exposed to an oxidizing environment. These results indicate that disulfide cross-links are normally present in extracellular virions but not in intracellular capsids. Interestingly, intracellular capsids isolated in the presence of NEM are unstable; B and C capsids are converted to a novel form that resembles A capsids, indicating that scaffold and DNA are lost. Furthermore, these capsids also have lost pentons and peripentonal triplexes as visualized by cryoelectron microscopy. These data indicate that capsid stability, and especially the retention of pentons, is regulated by the formation of disulfide bonds in the capsid.     

The amino-conserved domain of human cytomegalovirus UL80a proteins is required for key interactions during early stages of capsid formation and virus production

Assembly of many spherical virus capsids is guided by an internal scaffolding protein or group of proteins that are often cleaved and eliminated in connection with maturation and incorporation of the genome. In cytomegalovirus there are at least two proteins that contribute to this scaffolding function; one is the maturational protease precursor (pUL80a), and the other is the assembly protein precursor (pUL80.5) encoded by a shorter genetic element within UL80a. Yeast GAL4 two-hybrid assays established that both proteins contain a carboxyl-conserved domain that is required for their interaction with the major capsid protein (pUL86) and an amino-conserved domain (ACD) that is required for their self-interaction and for their interaction with each other. In the work reported here, we demonstrate that when the ACD is deleted (deltaACD) or disrupted by a point mutation (L47A), the bacterially expressed mutant protein sediments as a monomer during rate-velocity centrifugation, whereas the wild-type protein sediments mainly as oligomers. We also show that the L47A mutation reduces the production of infectious virus by at least 90%, results in the formation of irregular nuclear capsids, gives rise to tube-like structures in the nucleus that resemble the capsid core in cross-section and contain UL80 proteins, slows nuclear translocation of the major capsid protein, and may slow cleavage by the maturational protease. We provide physical corroboration that mutating the ACD disrupts self-interaction of the UL80 proteins and biological support for the proposal that the ACD has a critical role in capsid assembly and production of infectious virus.     

The C-terminal 25 amino acids of the protease and its substrate ICP35 of herpes simplex virus type 1 are involved in the formation of sealed capsids.

The herpes simplex virus type 1 protease and its substrate, ICP35, are involved in the assembly of viral capsids. Both proteins are encoded by a single open reading frame from overlapping mRNAs. The protease is autoproteolytically processed at two sites. The protease cleaves itself at the C-terminal site (maturation site) and also cleaves ICP35 at an identical site, releasing a 25-amino-acid (aa) peptide from each protein. To determine whether these 25 aa play a role in capsid assembly, we constructed a mutant virus expressing only Prb, the protease without the C-terminal 25 aa. Phenotypic analysis of the Prb virus in the presence and absence of ICP35 shows the following: (i) Prb retains the functional activity of the wild-type protease which supports virus growth in the presence of ICP35; (ii) in contrast to the ICP35 null mutant delta ICP35 virus, the Prb virus fails to grow in the absence of ICP35; and (iii) trans-complementation experiments indicated that full-length ICP35 (ICP35 c,d), but not the cleaved form (ICP35 e,f), complements the growth of the Prb virus. The most striking phenotype of the Prb virus is that only unsealed aberrant capsid structures are observed by electron microscopy in mutant-infected Vero cells. Our results demonstrate that the growth of herpes simplex virus type 1 requires the C-terminal 25 aa of either the protease or its substrate, ICP35, and that the C-terminal 25 aa are involved in the formation of sealed capsids.