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.     

The UL25 protein of pseudorabies virus associates with capsids and localizes to the nucleus and to microtubules

The UL25 gene of pseudorabies virus (PrV) can encode a protein of about 57 kDa which is well conserved among herpesviruses. The UL25 protein of herpes simplex virus type 1 is a capsid constituent involved in virus penetration and capsid maturation. To identify and characterize the UL25 gene product of PrV, polyclonal mouse anti-UL25 antibodies were raised to a bacterially expressed fusion protein. In immunoblotting and immunoprecipitation assays of PrV-infected cell lysates, these anti-UL25 antisera specifically recognized a protein of the expected size with late expression kinetics. This 57-kDa product was also present in purified virions and was found to be associated with all types of capsids. Synthesis of a protein migrating at the same size point was directed from the eukaryotic expression plasmid pCG-UL25. To determine the subcellular localization of UL25, immunofluorescence studies with anti-UL25 antisera were performed on Nonidet P-40-extracted COS-7 cells infected with PrV or transfected with pCG-UL25. In PrV-infected cells, newly synthesized UL25 is directed mainly to distinct nuclear compartments, whereas UL25 expressed in the absence of other viral proteins is distributed more uniformly in the nucleus and colocalizes also with microtubules. To study the fate of UL25 at very early stages of infection, immunofluorescence experiments were performed on invading PrV particles in the presence or absence of drugs that specifically depolymerize components of the cytoskeleton. We found that the incoming nucleocapsids colocalize with microtubules during their transport to the nucleus and that UL25 remains associated with nucleocapsids during this transport.     

The herpes simplex virus UL37 protein is phosphorylated in infected cells.

The herpes simplex virus type 1 (HSV-1) UL37 open reading frame encodes a 120-kDa late (gamma 1), nonstructural protein in infected cells. Recent studies in our laboratory have demonstrated that the UL37 protein interacts in the cytoplasm of infected cells with ICP8, the major HSV-1 DNA-binding protein. As a result of this interaction, the UL37 protein is transported to the nucleus and can be coeluted with ICP8 from single-stranded DNA columns. Pulse-labeling and pulse-chase studies of HSV-1-infected cells with [35S]methionine and 32Pi demonstrated that UL37 was a phosphoprotein which did not have a detectable rate of turnover. The protein was phosphorylated soon after translation and remained phosphorylated throughout the viral replicative cycle. UL37 protein expressed from a vaccinia virus recombinant was also phosphorylated during infection, suggesting that the UL37 protein was phosphorylated by a cellular kinase and that interaction with the ICP8 protein was not a prerequisite for UL37 phosphorylation.     

The herpes simplex virus 1 UL17 protein is the second constituent of the capsid vertex-specific component required for DNA packaging and retention

The herpes simplex virus (HSV) UL17 and UL25 minor capsid proteins are essential for DNA packaging. They are thought to comprise a molecule arrayed in five copies around each of the capsid vertices. This molecule was initially termed the "C-capsid-specific component" (CCSC) (B. L. Trus et al., Mol. Cell 26:479-489, 2007), but as we have subsequently observed this feature on reconstructions of A, B, and C capsids, we now refer to it more generally as the "capsid vertex-specific component" (CVSC) (S. K. Cockrell et al., J. Virol. 85:4875-4887, 2011). We previously confirmed that UL25 occupies the vertex-distal region of the CVSC density by visualizing a large UL25-specific tag in reconstructions calculated from cryo-electron microscopy (cryo-EM) images. We have pursued the same strategy to determine the capsid location of the UL17 protein. Recombinant viruses were generated that contained either a small tandem affinity purification (TAP) tag or the green fluorescent protein (GFP) attached to the C terminus of UL17. Purification of the TAP-tagged UL17 or a similarly TAP-tagged UL25 protein clearly demonstrated that the two proteins interact. A cryo-EM reconstruction of capsids containing the UL17-GFP protein reveals that UL17 is the second component of the CVSC and suggests that UL17 interfaces with the other CVSC component, UL25, through its C terminus. The portion of UL17 nearest the vertex appears to be poorly constrained, which may provide flexibility in interacting with tegument proteins or the DNA-packaging machinery at the portal vertex. The exposed locations of the UL17 and UL25 proteins on the HSV-1 capsid exterior suggest that they may be attractive targets for highly specific antivirals.     

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

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

Proteins associated with purified human cytomegalovirus particles.

Virion-associated proteins isolated from purified human cytomegalovirus particles (strain AD169) were used as substrates for chemical sequence analysis. Extracellular virions, noninfectious enveloped particles, and dense bodies were purified by negative viscosity-positive density gradient centrifugation, and their component proteins were separated by denaturing polyacrylamide gel electrophoresis. The deduced amino acid sequence of individual protein bands was used to identify six corresponding viral genes whose products have not previously been identified as virion constituents: UL47, UL25, UL88, UL85, UL26, and UL48.5. In addition, a 45-kDa cellular protein was identified, and the protein fragments sequenced have a high degree of amino acid identity with actin. However, antiactin monoclonal and polyclonal antibodies did not react with a specific protein in the virus preparations, suggesting that this 45-kDa protein is an immunologically distinct isoform of actin. The newly identified viral and cellular proteins were resistant to protease treatment of purified virions, suggesting that they are unlikely to be contaminants of the viral preparations.     

Composition of pseudorabies virus particles lacking tegument protein US3, UL47, or UL49 or envelope glycoprotein E

Proteins located in the tegument layer of herpesvirus particles play important roles in the replicative cycle at both early and late times after infection. As major constituents of the virion, they execute important functions in particular during formation of progeny virions. These functions have mostly been elucidated by construction and analysis of mutant viruses deleted in single or multiple tegument protein-encoding genes (reviewed in the work of T. C. Mettenleiter, Virus Res. 106:167-180, 2004). However, since tegument proteins have been shown to be involved in numerous protein-protein interactions, the impact of single protein deletions on the composition of the virus particle is unknown, but they could impair correct interpretation of the results. To analyze how the absence of single virion constituents influences virion composition, we established a procedure to assay relative amounts of virion structural proteins in deletion mutants of the alphaherpesvirus Pseudorabies virus (PrV) in comparison to wild-type particles. The assay is based on the mass spectrometric quantitation of virion protein-derived peptides carrying stable isotope mass tags. After deletion of the US3, UL47, UL49, or glycoprotein E gene, relative amounts of a capsid protein (UL38), a capsid-associated protein (UL25), several tegument proteins (UL36 and UL47, if present), and glycoprotein H were unaffected, whereas the content of other tegument proteins (UL46, UL48, and UL49, if present) varied significantly. In the case of the UL48 gene product, a specific increase in incorporation of a smaller isoform was observed after deletion of the UL47 or UL49 gene, whereas a larger isoform remained unaffected. The cellular protein actin was enriched in virions of mutants deficient in any of the tegument proteins UL47, UL49, or US3. By two-dimensional gel electrophoresis multiple isoforms of host cell-derived heat shock protein 70 and annexins A1 and A2 were also identified as structural components of PrV virions.     

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.     

Host Cell Protein C9orf9 Promotes Viral Proliferation via Interaction with HSV-1 UL25 Protein

In light of the scarcity of reports on the interaction between HSV-1 nucleocapsid protein UL25 and its host cell proteins,the purpose of this study is to use yeast two-hybrid screening to search for cellular proteins that can interact with the UL25 protein.C9orf69,a protein of unknown function was identified.The interaction between the two proteins under physiological conditions was also confirmed by biological experiments including co-localization by fluorescence and immunoprecipitation.A preliminary study...     

Herpes simplex virus capsid structure: DNA packaging protein UL25 is located on the external surface of the capsid near the vertices

UL25 is one of seven herpes simplex virus-encoded proteins involved specifically in DNA encapsidation. Its role appears to be to stabilize the capsid so that DNA is prevented from escaping once it has entered. To clarify the function of UL25, we have examined capsids with the goal of defining where it is located. Analysis of trypsin-treated capsids showed that UL25 is sensitive to cleavage like other proteins such as the major capsid and portal proteins that are exposed on the capsid surface. Internal proteins such as the scaffolding protein and protease were not affected under the same experimental conditions. Capsids were also examined by electron microscopy after staining with gold-labeled antibody specific for UL25. Images of stained capsids demonstrated that most labeled sites (71% in C capsids) were at capsid vertices, and most stained C capsids had label at more than one vertex. A quantitative immunoblotting method showed that the capsid contents of UL25 were 56, 20, and 75 copies per capsid in A, B, and C capsids, respectively. Finally, soluble UL25 protein was found to bind in vitro to purified capsids lacking it. The amount of bound UL25 corresponded to the amount present in B capsids, and bound UL25 was found by immunoelectron microscopy to be located predominantly at the capsid vertices. The results are interpreted to suggest that five UL25 molecules are found at or near each of the capsid vertices, where they are exposed on the capsid surface. Exposure on the surface is consistent with the view that UL25 is added to the capsid as DNA is packaged or during late stages of the packaging process.     

The herpes simplex virus type 1 DNA packaging protein UL17 is a virion protein that is present in both the capsid and the tegument compartments

The UL17 protein of herpes simplex virus type 1 is essential for packaging the viral genome into the procapsid, a spherical assembly intermediate, and is present in the mature virus particle. We have examined the distribution of UL17 in various assembly products and virions to determine which component of the virus particle UL17 is associated with and at what stage in capsid assembly UL17 is required. UL17 was present in the procapsid, in the DNA-containing angularized C capsid, and in two other angularized capsid forms, A and B, that lack DNA and are thought to be dead-end products. The results suggest that UL17 is a minor capsid protein which is incorporated into the procapsid during assembly of the particle. UL17 was also found in virions and in noninfectious structures known as light (L) particles, which possess a tegument and envelope but lack a capsid. The level of UL17 in these particles was much greater than the amount that could be attributed to capsid contamination of the purified L-particle preparation, suggesting that UL17 is also a tegument protein. The finding that virions contain approximately twofold more UL17 than do C capsids provided further support for the idea that UL17 is present in two different structural components within the mature virion. The UL25 packaging protein, which is also present in virions, was not found in significant amounts in L particles, indicating that it is associated only with the capsid. UL6, the third virion-associated packaging protein, was present in slightly increased levels in L particles.     

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

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

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.     

Cytomegalovirus immediate early gene UL37 encodes a novel MHC-like protein

The cytomegalovirus (CMV) genome encodes four clusters of genes expressed immediately after infection--i.e.: UL36-38, UL122-123, TRS1-IRS1, and US3. The general function of these genes is associated with inhibition of cellular mechanisms of antiviral response. Although several biological processes have been mapped onto specific gene products, the knowledge of the molecular mechanism of their activity remains fragmentary. Here, we report the application of protein structure prediction methods in assigning the function to a glycosylated domain encoded by UL37 of CMV (gpUL37, UL37x3). The discerned similarity clearly points out that this domain represents a novel type of a major histocompatibility complex (MHC)-like protein, and consequently may play a central role in an additional mechanism of escape from antiviral response.     

Binding partners for the UL11 tegument protein of herpes simplex virus type 1

The product of the U(L)11 gene of herpes simplex virus type 1 (HSV-1) is a 96-amino-acid tegument protein that accumulates on the cytoplasmic face of internal membranes. Although it is thought to be important for nucleocapsid envelopment and egress, the actual function of this protein is unknown. Previous studies focused on the characterization of sequence elements within the UL11 protein that function in membrane binding and trafficking to the Golgi apparatus. Binding was found to be mediated by two fatty acyl groups (myristate and palmitate), while an acidic cluster and a dileucine motif were identified as being important for the recycling of UL11 from the plasma membrane to the Golgi apparatus. The goal of the experiments described here was to identify and characterize binding partners (viral or cellular) of UL11. Using both immunoprecipitation and glutathione S-transferase (GST) pull-down assays, we identified a 40-kDa protein that specifically associates with UL11 from infected Vero cells. Mutational analyses revealed that the acidic cluster and the dileucine motif are required for this association, whereas the entire second half of UL11 is not. In addition, UL11 homologs from pseudorabies and Marek's disease herpesviruses were also found to be capable of binding to the 40-kDa protein from HSV-1-infected cells, suggesting that the interaction is conserved among alphaherpesviruses. Purification and analysis of the 40-kDa protein by mass spectrometry revealed that it is the product of the U(L)16 gene, a virion protein reported to be involved in nucleocapsid assembly. Cells transfected with a UL16-green fluorescent protein expression vector produced a protein that was of the expected size, could be pulled down with GST-UL11, and accumulated in a Golgi-like compartment only when coexpressed with UL11, indicating that the interaction does not require any other viral products. These data represent the first steps toward elucidating the network of tegument proteins that UL11 links to membranes.     

Functional and physical interactions of the herpes simplex virus type 1 UL20 membrane protein with glycoprotein K

Herpes simplex virus type 1 glycoprotein K (gK) and the UL20 protein (UL20p) are coordinately transported to the trans-Golgi network (TGN) and cell surfaces and are required for cytoplasmic virion envelopment at the TGN. In addition, cell surface expression of gK and UL20p is required for virus-induced cell fusion. Previously, confocal microscopy colocalization and intracellular transport experiments strongly suggested direct protein-protein interactions between gK and UL20p. Direct protein-protein interactions between gK and UL20p were demonstrated through reciprocal coimmunoprecipitation experiments, as well as with glutathione S-transferase (GST) pull-down experiments. A fusion protein consisting of the amino-terminal 66 amino acids of UL20p fused in-frame with GST was expressed in Escherichia coli and purified via glutathione column chromatography. Precipitation of GST-UL20p from mixtures of GST-UL20p fusion protein with cellular extracts containing gK specifically coprecipitated gK but not other viral glycoproteins. The purified UL20p-GST fusion protein reacted with all gK-associated protein species. It was concluded that the amino terminus of UL20p, most likely, interacted with gK domain III, which is predicted to lie intracellularly. UL20p-gK domain-specific interactions must serve important functions in the coordinate transport of UL20p and gK to the TGN, because retention of UL20p in the endoplasmic reticulum (ER) via the addition of an ER retention signal at the carboxyl terminus of UL20p forced the ER retention of gK and drastically inhibited intracellular virion envelopment and virus-induced cell fusion.     

The human cytomegalovirus UL44 protein is a substrate for the UL97 protein kinase

The human cytomegalovirus UL97 protein is an unusual protein kinase that is able to autophosphorylate and to phosphorylate certain exogenous substrates, including nucleoside analogs such as ganciclovir. However, no natural substrate of UL97 in infected cells has been identified. We report here that recombinant UL44 protein became radiolabeled when incubated with recombinant UL97 and [(32)P]ATP and that both proteins could be coimmunoprecipitated by an antibody that recognizes either protein. Subsequent studies showed that highly purified, recombinant UL97 phosphorylated purified, recombinant UL44. This phosphorylation occurred on serine and threonine residues and was sensitive to inhibition by maribavir and to a mutation that inactivates UL97 catalytic activity. Two-dimensional gel electrophoresis revealed the absence of specific phosphorylated forms of UL44 in immunoprecipitates from lysates of cells infected with a UL97 null mutant virus or with wild-type virus in the presence of maribavir. The results indicate that UL97 is sufficient to phosphorylate UL44 in vitro and is necessary for the normal phosphorylation of UL44 in infected cells. This strongly suggests that UL44 is a natural substrate of UL97.