Allosteric signaling and a nuclear exit strategy: binding of UL25/UL17 heterodimers to DNA-Filled HSV-1 capsids.
ABSTRACT: UL25 and UL17 are two essential minor capsid proteins of HSV-1, implicated in DNA packaging and capsid maturation. We used cryo-electron microscopy to examine their binding to capsids, whose architecture observes T = 16 icosahedral geometry. C-capsids (mature DNA-filled capsids) have an elongated two-domain molecule present at a unique, vertex-adjacent site that is not seen at other quasiequivalent sites or on unfilled capsids. Using SDS-PAGE and mass spectrometry to analyze wild-type capsids, UL25 null capsids, and denaturant-extracted capsids, we conclude that (1) the C-capsid-specific component is a heterodimer of UL25 and UL17, and (2) capsids have additional populations of UL25 and UL17 that are invisible in reconstructions because of sparsity and/or disorder. We infer that binding of the ordered population reflects structural changes induced on the outer surface as pressure builds up inside the capsid during DNA packaging. Its binding may signal that the C-capsid is ready to exit the nucleus.
Project description:The herpes simplex virus type 1 UL25 protein is one of seven viral proteins that are required for DNA cleavage and packaging. Together with UL17, UL25 forms part of an elongated molecule referred to as the C-capsid-specific component (CCSC). Five copies of the CCSC are located at each of the capsid vertices on DNA-containing capsids. To study the conformation of UL25 as it is folded on the capsid surface, we identified the sequence recognized by a UL25-specific monoclonal antibody and localized the epitope on the capsid surface by immunogold electron microscopy. The epitope mapped to amino acids 99-111 adjacent to the region of the protein (amino acids 1-50) that is required for capsid binding. In addition, cryo-EM reconstructions of C-capsids in which the green fluorescent protein (GFP) was fused within the N-terminus of UL25 localized the point of contact between UL25 and GFP. The result confirmed the modeled location of the UL25 protein in the CCSC density as the region that is distal to the penton with the N-terminus of UL25 making contact with the triplex one removed from the penton. Immunofluorescence experiments at early times during infection demonstrated that UL25-GFP was present on capsids located within the cytoplasm and adjacent to the nucleus. These results support the view that UL25 is present on incoming capsids with the capsid-binding domain of UL25 located on the surface of the mature DNA-containing capsid.
Project description: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.
Project description: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.
Project description: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.
Project description:VP26 is a herpes simplex virus 1 (HSV-1) small capsomere-interacting protein. In this study, we investigated the function of VP26 in HSV-1-infected cells with the following results. (i) The VP26 null mutation significantly impaired incorporation of minor capsid protein UL25 into nucleocapsids (type C capsids) in the nucleus. (ii) The VP26 mutation caused improper localization of UL25 in discrete punctate domains containing multiple capsid proteins (e.g., the VP5 major capsid protein) in the nucleus; these domains corresponded to capsid aggregates. (iii) The VP26 mutation significantly impaired packaging of replicated viral DNA genomes into capsids but had no effect on viral DNA concatemer cleavage. (iv) The VP26 mutation reduced the frequency of type C capsids, which contain viral DNA but not scaffolding proteins, and produced an accumulation of type A capsids, which lack both viral DNA and scaffold proteins, and had no effect on accumulation of type B capsids, which lack viral DNA but retain cleaved scaffold proteins. Collectively, these results indicated that VP26 was required for efficient viral DNA packaging and proper localization of nuclear capsids. The phenotype of the VP26 null mutation was similar to that reported previously of the UL25 null mutation and of UL25 mutations that preclude UL25 binding to capsids. Thus, VP26 appeared to regulate nucleocapsid maturation by promoting incorporation of UL25 into capsids, which is likely to be required for proper capsid nuclear localization.IMPORTANCE HSV-1 VP26 has been reported to be important for viral replication and virulence in cell cultures and/or mouse models. However, little is known about the function of VP26 during HSV-1 replication, in particular, in viral nucleocapsid maturation although HSV-1 nucleocapsids are estimated to contain 900 copies of VP26. In this study, we present data suggesting that VP26 promoted packaging of HSV-1 DNA genomes into capsids by regulating incorporation of capsid protein UL25 into capsids, which was reported to increase stability of the capsid structure. We also showed that VP26 was required for proper localization of capsids in the infected cell nucleus. This is the first report showing that HSV-1 VP26 is a regulator for nucleocapsid maturation.
Project description: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.
Project description:Homologs of the UL25 gene product of herpes simplex virus 1 (HSV-1) are highly conserved among the Herpesviridae. However, their exact function during viral replication is unknown. Current evidence suggests that in the alphaherpesvirus pseudorabies virus (PrV) the capsid-associated pUL25 plays a role in primary envelopment of DNA-containing mature capsids at the inner nuclear membrane. In the absence of pUL25, capsids were found in close association with the inner nuclear membrane, but nuclear egress was not observed (B. G. Klupp, H. Granzow, G. M. Keil, and T. C. Mettenleiter, J. Virol. 80:6235-6246, 2006). In contrast, HSV-1 pUL25 has been assigned a role in stable packaging of viral genomes (N. Stow, J. Virol. 75:10755-10765, 2001). Despite these apparently divergent functions, we wanted to assess whether the high sequence homology translates into functional homology. Therefore, we first analyzed a newly constructed HSV-1 UL25 deletion mutant in our assay system and observed a similar phenotype as in PrV. In the nuclei of infected cells, numerous electron-dense C capsids were detected, whereas primary envelopment of these capsids did not ensue. In agreement with results from PrV, vesicles were observed in the perinuclear space. Since these data indicated functional homology, we analyzed the ability of pUL25 of HSV-1 to complement a PrV UL25 deletion mutant and vice versa. Whereas a HSV-1 pUL25-expressing cell line partially complemented the pUL25 defect in PrV, reciprocal complementation of a HSV-1 UL25 deletion mutant by PrV pUL25 was not observed. Thus, our data demonstrate overlapping, although not identical functions of these two conserved herpesvirus proteins, and point to a conserved functional role in herpes virion formation.
Project description:Herpesviruses replicate their double stranded DNA genomes as high-molecular-weight concatemers which are subsequently cleaved into unit-length genomes by a complex mechanism that is tightly coupled to DNA insertion into a preformed capsid structure, the procapsid. The herpes simplex virus type 1 UL25 protein is incorporated into the capsid during DNA packaging, and previous studies of a null mutant have demonstrated that its function is essential at the late stages of the head-filling process, either to allow packaging to proceed to completion or for retention of the viral genome within the capsid. We have expressed and purified an N-terminally truncated form of the 580-residue UL25 protein and have determined the crystallographic structure of the region corresponding to amino acids 134 to 580 at 2.1-Angstroms resolution. This structure, the first for any herpesvirus protein involved in processing and packaging of viral DNA, reveals a novel fold, a distinctive electrostatic distribution, and a unique "flexible" architecture in which numerous flexible loops emanate from a stable core. Evolutionary trace analysis of UL25 and its homologues in other herpesviruses was used to locate potentially important amino acids on the surface of the protein, leading to the identification of four putative docking regions for protein partners.
Project description: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.
Project description:During DNA encapsidation, herpes simplex virus 1 (HSV-1) procapsids are converted to DNA-containing capsids by a process involving activation of the viral protease, expulsion of the scaffold proteins, and the uptake of viral DNA. Encapsidation requires six minor capsid proteins (UL6, UL15, UL17, UL25, UL28, and UL33) and one viral protein, UL32, not found to be associated with capsids. Although functions have been assigned to each of the minor capsid proteins, the role of UL32 in encapsidation has remained a mystery. Using an HSV-1 variant containing a functional hemagglutinin-tagged UL32, we demonstrated that UL32 was synthesized with true late kinetics and that it exhibited a previously unrecognized localization pattern. At 6 to 9 h postinfection (hpi), UL32 accumulated in viral replication compartments in the nucleus of the host cell, while at 24 hpi, it was additionally found in the cytoplasm. A newly generated UL32-null mutant was used to confirm that although B capsids containing wild-type levels of capsid proteins were synthesized, these procapsids were unable to initiate the encapsidation process. Furthermore, we showed that UL32 is redox sensitive and identified two highly conserved oxidoreductase-like C-X-X-C motifs that are essential for protein function. In addition, the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, were altered in the absence of UL32, suggesting that UL32 may act to modulate disulfide bond formation during procapsid assembly and maturation.Although functions have been assigned to six of the seven required packaging proteins of HSV, the role of UL32 in encapsidation has remained a mystery. UL32 is a cysteine-rich viral protein that contains C-X-X-C motifs reminiscent of those in proteins that participate in the regulation of disulfide bond formation. We have previously demonstrated that disulfide bonds are required for the formation and stability of the viral capsids and are also important for the formation and stability of the UL6 portal ring. In this report, we demonstrate that the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, are altered in cells infected with a newly isolated UL32-null mutant virus, suggesting that UL32 acts as a chaperone capable of modulating disulfide bond formation. Furthermore, these results suggest that proper regulation of disulfide bonds is essential for initiating encapsidation.