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.     

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.     

Electron Microscopy of Herpes Simplex Virus: I. Entry

Although capsids of herpes simplex virus were encountered within phagocytic vesicles, they were more commonly observed free within the cytoplasm. Stages in the release of virus from vesicles were not seen. There appeared to be five distinct steps in the process whereby the virus initiates infection: attachment, digestion of the viral envelope, digestion of the cell wall, passage of the capsid directly into the cytoplasm, and digestion of the capsid with release of the core. Antibody probably interferes with the first two stages.     

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.     

Diffusion of macromolecules and virus-like particles in human cervical mucus

To determine whether or not large macromolecules and viruses can diffuse through mucus, we observed the motion of proteins, microspheres, and viruses in fresh samples of human cervical mucus using fluorescent recovery after photobleaching and multiple image photography. Two capsid virus-like particles, human papilloma virus (55 nm, approximately 20,000 kDa) and Norwalk virus (38 nm, approximately 10,000 kDa), as well as most of the globular proteins tested (15-650 kDa) diffused as rapidly in mucus as in saline. Electron microscopy of cervical mucus confirmed that the mesh spacing between mucin fibers is large enough (20-200 nm) for small viruses to diffuse essentially unhindered through mucus. In contrast, herpes simplex virus (180 nm) colocalized with strands of thick mucus, suggesting that herpes simplex virus, unlike the capsid virus particles, makes low-affinity bonds with mucins. Polystyrene microspheres (59-1000 nm) bound more tightly to mucins, bundling them into thick cables. Although immunoglobulins are too small to be slowed by the mesh spacing between mucins, diffusion by IgM was slowed by mucus. Diffusion by IgM-Fc(5 mu), the Fc pentamer core of an IgM with all 10 Fab moieties removed, was comparably slowed by mucus. This suggests that the Fc moieties of antibodies make low-affinity bonds with mucins.     

Mutational pressure in genomes of alphaherpesviruses infecting human

Genomes of the herpes simplex viruses are extremely enriched with GC. Elevated G+C level in genomes of the simplex viruses is a result of their long-term evolution under the influence of the mutational pressure. We counted the rates of nucleotide substitutions from gene coding major capsid protein (MCP) (G+C = 0.68, 3GC = 0.89) of human simplex virus 1 (HSV-1) to the MCP gene (G+C = 0.70, 3GC = 0.91) of HSV-2 (the first pair of genes) and from the same MCP gene of HSV-1 to the homologous gene (G+C = 0.73, 3GC = 0.99) from cercopithecine herpes virus 16 (the second pair of genes). The rates of transitions from A-T to G-C base pairs increases 2.17-, 3.09-, and 1.27-fold in the first, second, and third codon positions, respectively, if compared those rates between the second and first pair of genes (the growth of GC-richness is only 3%). This effect is due to an approximately 90% GC-richness of the third codon positions in all those genes. Transitions caused by the strong mutational pressure (from A-T to G-C base pairs) have a low probability to occur in the third positions, but high probability to occur in the first and second positions. For MCP gene of human herpes 3, the probability of the occurrence of transition caused by mutational pressure in the third codon position is 2.36 times higher than in MCP gene of HSV1, and 3 times higher than in MCP gene of HSV2. These data could provide an explanation of rarely occurring relapses of herpes Zoster infection and frequently occurring relapses of herpes simplex infection.     

Dynamic interactions of the UL16 tegument protein with the capsid of herpes simplex virus

The UL16 tegument protein of herpes simplex virus is conserved throughout the herpesvirus family. It has been reported to be capsid associated and may be involved in budding by providing an interaction with the membrane-bound UL11 protein. UL16 has been shown to be present in all the major locations that capsids are found (i.e., the nucleus, cytoplasm, and virions), but whether it is actually capsid associated in each of these has not been reported. Therefore, capsids were purified from each compartment, and it was found that UL16 was present on cytoplasmic but not nuclear capsids. In extracellular virions, the majority of UL16 (87%) was once again not capsid associated, which suggests that the interaction is transient during egress. Because herpes simplex virus (HSV) buds into the acidic compartment of the trans-Golgi network (TGN), the effect of pH on the interaction was examined. The amount of capsid-associated UL16 dramatically increased when extracellular virions were exposed to mildly acidic medium (pH 5.0 to 5.5), and this association was fully reversible. After budding into the TGN, capsid and tegument proteins also encounter an oxidizing environment, which is conducive to disulfide bond formation. UL16 contains 20 cysteines, including five that are conserved within a putative zinc finger. Any free cysteines that are involved in the capsid interaction or release mechanism of UL16 would be expected to be modified by N-ethylmaleimide, and, consistent with this, the amount of capsid-associated UL16 dramatically increased when virions were incubated with this compound. Taken together, these data suggest a transient interaction between UL16 and capsids, possibly modified in the acidic compartment of secretory vesicles and requiring a release mechanism that involves cysteines.     

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.     

Seeing the portal in herpes simplex virus type 1 B capsids

Resolving the nonicosahedral components in large icosahedral viruses remains a technical challenge in structural virology. We have used the emerging technique of Zernike phase-contrast electron cryomicroscopy to enhance the image contrast of ice-embedded herpes simplex virus type 1 capsids. Image reconstruction enabled us to retrieve the structure of the unique portal vertex in the context of the icosahedral capsid and, for the first time, show the subunit organization of a portal in a virus infecting eukaryotes. Our map unequivocally resolves the 12-subunit portal situated beneath one of the pentameric vertices, thus removing uncertainty over the location and stoichiometry of the herpesvirus portal.     

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.     

Sequential detection of different antigens induced by Epstein-Barr virus and herpes simplex virus in the same Western blot by using dual antibody probes

A dual antibody probing technique that permitted a color-coded identification of polypeptides representing different classes of Epstein-Barr virus (EBV) antigens as well as differentiation of the polypeptides induced by different herpesviruses in the same Western blot was developed. When the nitrocellulose sheet was probed first with monoclonal antibody against EBV early antigen diffuse component (EA-D) and then stained with 4-chloro-1-naphthol, four polypeptides specific for EA-D were identified by purple bands. Subsequently, the same nitrocellulose sheet was reprobed with human serum containing antibodies against EBV early antigen, viral capsid antigen, and nuclear antigen and stained with 3,3'-diaminobenzidine. Several brown bands corresponding to early, viral capsid, and nuclear antigen polypeptides were detected. The dual antibody probing technique was used in an analysis to differentiate polypeptides resulting from either EBV or herpes simplex virus infection, either in cells infected by individual virus or in a cell line dually infected by both viruses. On the basis of different colored bands in different lanes of the same gel, 20 polypeptides with molecular weights ranging from 31,000 to 165,000 were identified as herpes simplex virus-specific proteins. These results suggested that the dual antibody probing technique may be applicable in clinical diagnosis for detecting antigens and antibodies derived from different pathogens.     

Metabolism and mode of action of (R)-9-(3,4-dihydroxybutyl)guanine in herpes simplex virus-infected vero cells

The metabolism and mode of action of the anti-herpes compound buciclovir [R)-9-(3,4-dihydroxybutyl)-guanine, BCV) has been studied in herpes simplex virus-infected and uninfected Vero cells. In uninfected cells, a low and constant concentration of intracellular BCV was found, while in herpes simplex virus-infected cells, an increasing concentration of BCV phosphates was found due to metabolic trapping. The major phosphorylation product was BCV triphosphate (BCVTP) which was 92% of the total amount of BCV phosphates. BCV phosphates were accumulated to the same extent in cells infected with either a herpes simplex virus type 1 or a herpes simplex virus type 2 strain while thymidine kinase-deficient mutants of herpes simplex virus type 1 were 10 times less efficient in accumulating BCV phosphates. In uninfected Vero cells, the concentration of the phosphorylated forms of BCV was less than 1% of that found in herpes simplex virus-infected cells. The BCVTP formed in herpes simplex virus-infected cells was highly stable, as 80% of the amount of BCVTP was still present even 17 h after removal of extracellular BCV. BCV was a good substrate for herpes simplex virus type 1- and type 2-induced thymidine kinases but not for the cellular cytosol or mitochondrial thymidine kinases. BCV monophosphate could be phosphorylated by cellular guanylate kinase to BCV diphosphate. BCVTP was a selective and competitive inhibitor to deoxyguanosine triphosphate of the purified herpes simplex virus type 1- and type 2-induced DNA polymerases. BCVTP could neither act as an alternative substrate in the herpes simplex virus type 2 or cellular DNA polymerase reactions, nor could [3H]BCV monophosphate be detected in DNA formed by herpes simplex virus type 2 DNA polymerase, or be detected in nucleic acids extracted from herpes simplex virus type 1-infected cells. These data indicate that BCVTP may inhibit the herpes simplex virus-induced DNA polymerase without being incorporated into DNA.     

Major capsid reinforcement by a minor protein in herpesviruses and phage

Herpes simplex type 1 virus (HSV-1) and bacteriophage λ capsids undergo considerable structural changes during self-assembly and DNA packaging. The initial steps of viral capsid self-assembly require weak, non-covalent interactions between the capsid subunits to ensure free energy minimization and error-free assembly. In the final stages of DNA packaging, however, the internal genome pressure dramatically increases, requiring significant capsid strength to withstand high internal genome pressures of tens of atmospheres. Our data reveal that the loosely formed capsid structure is reinforced post-assembly by the minor capsid protein UL25 in HSV-1 and gpD in bacteriophage λ. Using atomic force microscopy nano-indentation analysis, we show that the capsid becomes stiffer upon binding of UL25 and gpD due to increased structural stability. At the same time the force required to break the capsid increases by ∼70% for both herpes and phage. This demonstrates a universal and evolutionarily conserved function of the minor capsid protein: facilitating the retention of the pressurized viral genome in the capsid. Since all eight human herpesviruses have UL25 orthologs, this discovery offers new opportunities to interfere with herpes replication by disrupting the precise force balance between the encapsidated DNA and the capsid proteins crucial for viral replication.     

CD4 T-cell responses to herpes simplex virus type 2 major capsid protein VP5: comparison with responses to tegument and envelope glycoproteins

We used CD4 lymphocyte clones from herpes simplex virus type 2 (HSV-2) lesions or the cervix and molecular libraries of HSV-2 DNA to define HSV-2 major capsid protein VP5 and glycoprotein E (gE) as T-cell antigens. Responses to eight HSV-2 glycoprotein, tegument, nonstructural, or capsid antigens were compared in 19 donors. Recognition of VP5 and tegument VP22 were similar to that of gB2 and gD2, currently under study as vaccines. These prevalence data suggest that HSV capsid and tegument proteins may also be candidate vaccine antigens.     

Structural analysis of the capsid polypeptides of herpes simplex virus types 1 and 2

Capsids of herpes simplex virus (HSV) types 1 and 2 contain seven polypeptides ranging in molecular weight from 154,000 to 12,000 (termed NC-1 through NC-7 in order of descending molecular weight). Antibodies prepared to HSV-1 capsid polypeptides isolated from sodium dodecyl sulfate-polyacrylamide gels reacted in an immunofluorescence assay against HSV-1-infected KB cells. Three of the antibodies (anti-NC-1, anti-NC-2, and anti-NC-3,4) also reacted with HSV-2-infected cells. Tryptic peptide analysis showed that each of the HSV-1 capsid polypeptides had a unique methionine peptide profile, and none appeared to be derived from the major capsid polypeptide. Comparative peptide analysis of HSV-1 and HSV-2 showed that one polypeptide (NC-7, 12,000 molecular weight) had an identical methionine peptide profile and a very similar arginine peptide profile in both virus types. The arginine peptide profile of NC-7 of HSV-1 was very different from the arginine profile of KB histone H4. Although there were certain intertypic similarities in the methionine peptide profiles of the other capsid components especially in NC-1 (the major capsid protein), there was no case where the tryptic peptides were identical in the two virus types.     

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.