The tegument of herpesviruses is a highly complex structural layer between the nucleocapsid and the envelope of virions. Tegument proteins play both structural and regulatory functions during replication and spread, but the interactions and functions of many of these proteins are poorly understood. Here we focus on two tegument proteins from herpes simplex virus 1 (HSV-1), pUL7 and pUL51, which have homologues in all other herpesviruses. We have now identified that HSV-1 pUL7 and pUL51 form a stable and direct protein-protein interaction, their expression levels rely on the presence of each other, and they function as a complex in infected cells. We demonstrate that expression of the pUL7-pUL51 complex is important for efficient HSV-1 assembly and plaque formation. Furthermore, we also discovered that the pUL7-pUL51 complex localizes to focal adhesions at the plasma membrane in both infected cells and in the absence of other viral proteins. The expression of pUL7-pUL51 is important to stabilize focal adhesions and maintain cell morphology in infected cells and cells infected with viruses lacking pUL7 and/or pUL51 round up more rapidly than cells infected with wild-type HSV-1. Our data suggest that, in addition to the previously reported functions in virus assembly and spread for pUL51, the pUL7-pUL51 complex is important for maintaining the attachment of infected cells to their surroundings through modulating the activity of focal adhesion complexes.IMPORTANCE Herpesviridae is a large family of highly successful human and animal pathogens. Virions of these viruses are composed of many different proteins, most of which are contained within the tegument, a complex structural layer between the nucleocapsid and the envelope within virus particles. Tegument proteins have important roles in assembling virus particles as well as modifying host cells to promote virus replication and spread. However, little is known about the function of many tegument proteins during virus replication. Our study focuses on two tegument proteins from herpes simplex virus 1 that are conserved in all herpesviruses: pUL7 and pUL51. We demonstrate that these proteins directly interact and form a functional complex that is important for both virus assembly and modulation of host cell morphology. Further, we identify for the first time that these conserved herpesvirus tegument proteins localize to focal adhesions in addition to cytoplasmic juxtanuclear membranes within infected cells.
Upon reactivation from latency and during lytic infections in neurons, alphaherpesviruses assemble cytosolic capsids, capsids associated with enveloping membranes, and transport vesicles harboring fully enveloped capsids. It is debated whether capsid envelopment of herpes simplex virus (HSV) is completed in the soma prior to axonal targeting or later, and whether the mechanisms are the same in neurons derived from embryos or from adult hosts. We used HSV mutants impaired in capsid envelopment to test whether the inner tegument proteins pUL36 or pUL37 necessary for microtubule-mediated capsid transport were sufficient for axonal capsid targeting in neurons derived from the dorsal root ganglia of adult mice. Such neurons were infected with HSV1-ΔUL20 whose capsids recruited pUL36 and pUL37, with HSV1-ΔUL37 whose capsids associate only with pUL36, or with HSV1-ΔUL36 that assembles capsids lacking both proteins. While capsids of HSV1-ΔUL20 were actively transported along microtubules in epithelial cells and in the somata of neurons, those of HSV1-ΔUL36 and -ΔUL37 could only diffuse in the cytoplasm. Employing a novel image analysis algorithm to quantify capsid targeting to axons, we show that only a few capsids of HSV1-ΔUL20 entered axons, while vesicles transporting gD utilized axonal transport efficiently and independently of pUL36, pUL37, or pUL20. Our data indicate that capsid motility in the somata of neurons mediated by pUL36 and pUL37 does not suffice for targeting capsids to axons, and suggest that capsid envelopment needs to be completed in the soma prior to targeting of herpes simplex virus to the axons, and to spreading from neurons to neighboring cells.
Herpesviruses acquire their membrane envelopes in the cytoplasm of infected cells via a molecular mechanism that remains unclear. Herpes simplex virus (HSV)−1 proteins pUL7 and pUL51 form a complex required for efficient virus envelopment. We show that interaction between homologues of pUL7 and pUL51 is conserved across human herpesviruses, as is their association with trans-Golgi membranes. We characterized the HSV-1 pUL7:pUL51 complex by solution scattering and chemical crosslinking, revealing a 1:2 complex that can form higher-order oligomers in solution, and we solved the crystal structure of the core pUL7:pUL51 heterodimer. While pUL7 adopts a previously-unseen compact fold, the helix-turn-helix conformation of pUL51 resembles the cellular endosomal complex required for transport (ESCRT)-III component CHMP4B and pUL51 forms ESCRT-III–like filaments, suggesting a direct role for pUL51 in promoting membrane scission during virus assembly. Our results provide a structural framework for understanding the role of the conserved pUL7:pUL51 complex in herpesvirus assembly.
Herpes simplex virus (HSV) replicates in the skin and mucous membranes, and initiates lytic or latent infections in sensory neurons. Assembly of progeny virions depends on the essential large tegument protein pUL36 of 3,164 amino acid residues that links the capsids to the tegument proteins pUL37 and VP16. Of the 32 tryptophans of HSV-1-pUL36, the tryptophan-acidic motifs 1766 WD 1767 and 1862 WE 1863 are conserved in all HSV-1 and HSV-2 isolates. Here, we characterized the role of these motifs in the HSV life cycle since the rare tryptophans often have unique roles in protein function due to their large hydrophobic surface. The infectivity of the mutants HSV-1(17 ؉ )Lox-pUL36-WD/AA-WE/AA and HSV-1(17 ؉ )Lox-CheVP26-pUL36-WD/AA-WE/AA, in which the capsid has been tagged with the fluorescent protein Cherry, was significantly reduced. Quantitative electron microscopy shows that there were a larger number of cytosolic capsids and fewer enveloped virions compared to their respective parental strains, indicating a severe impairment in secondary capsid envelopment. The capsids of the mutant viruses accumulated in the perinuclear region around the microtubule-organizing center and were not dispersed to the cell periphery but still acquired the inner tegument proteins pUL36 and pUL37. Furthermore, cytoplasmic capsids colocalized with tegument protein VP16 and, to some extent, with tegument protein VP22 but not with the envelope glycoprotein gD. These results indicate that the unique conserved tryptophan-acidic motifs in the central region of pUL36 are required for efficient targeting of progeny capsids to the membranes of secondary capsid envelopment and for efficient virion assembly. IMPORTANCEHerpesvirus infections give rise to severe animal and human diseases, especially in young, immunocompromised, and elderly individuals. The structural hallmark of herpesvirus virions is the tegument, which contains evolutionarily conserved proteins that are essential for several stages of the herpesvirus life cycle. Here we characterized two conserved tryptophan-acidic motifs in the central region of the large tegument protein pUL36 of herpes simplex virus. When we mutated these motifs, secondary envelopment of cytosolic capsids and the production of infectious particles were severely impaired. Our data suggest that pUL36 and its homologs in other herpesviruses, and in particular such tryptophan-acidic motifs, could provide attractive targets for the development of novel drugs to prevent herpesvirus assembly and spread. T he diversity of clinical herpesvirus isolates is shaped to a large extent by the long coevolution between virus and host as well as by homologous recombination among different strains (1-4). The subfamilies of the Herpesviridae are characterized by similar properties: for example, fast replication of members of the Alphaherpesvirinae in keratinocytes, epithelial cells, or fibroblasts and latency in sensory neurons. Infections with the herpes simplex viruses (HSV-1 or HSV-2) or varicella-zoster virus occur...
Herpesviruses are an ancient family of highly-prevalent human and animal pathogens that acquire their membrane envelopes in the cytoplasm of infected cells. While multiple conserved viral proteins are known to be required for efficient herpesvirus production, many of these proteins lack identifiable structural homologues and the molecular details of herpesvirus assembly remain unclear. We have characterized the complex of assembly proteins pUL7 and pUL51 from herpes simplex virus (HSV)-1, an α-herpesvirus, using multi-angle light scattering and small-angle X-ray scattering with chemical crosslinking. HSV-1 pUL7 and pUL51 form a stable 1:2 complex that is capable of higher-order oligomerization in solution. We solved the crystal structure of this complex, revealing a core heterodimer comprising pUL7 bound to residues 41-125 of pUL51. While pUL7 adopts a previously-unseen compact fold, the extended helix-turn-helix conformation of pUL51 resembles the cellular endosomal complex required for transport (ESCRT)-III component CHMP4B, suggesting a direct role for pUL51 in promoting membrane scission during virus assembly. We demonstrate that the interaction between pUL7 and pUL51 homologues is conserved across human α-, β-and γ-herpesviruses, as is their association with trans-Golgi membranes in cultured cells. However, pUL7 and pUL51 homologues do not form complexes with their respective partners from different virus families, suggesting that the molecular details of the interaction interface have diverged. Our results demonstrate that the pUL7:pUL51 complex is conserved across the herpesviruses and provide a structural framework for understanding its role in herpesvirus assembly. Significance StatementHerpesviruses are extremely common human pathogens that cause diseases ranging from cold sores to cancer. Herpesvirus acquire their membrane envelope in the cytoplasm via a conserved pathway, the molecular details of which remain unclear. We have solved the structure of a complex between herpes simplex virus (HSV)-1 proteins pUL7 and pUL51, two proteins that are required for efficient HSV-1 assembly. We show that formation of this complex is conserved across distantly-related human herpesviruses, as is the association of these homologues with cellular membranes that are used for virion assembly. While pUL7 adopts a previously-unseen fold, pUL51 resembles key cellular membrane-remodeling complex components, suggesting that the pUL7:pUL51 complex may play a direct role in deforming membranes to promote virion assembly.
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