Herpes simplex virus (HSV) is a neuroinvasive virus that has been used as a model organism for studying common properties of all herpesviruses. HSV induces host organelle rearrangement and forms multiple, dispersed assembly compartments in epithelial cells, which complicates the study of HSV assembly. In this study, we show that HSV forms a visually distinct unitary cytoplasmic viral assembly center (cVAC) in both cancerous and primary neuronal cells that concentrates viral structural proteins and is a major site of capsid envelopment. The HSV cVAC also concentrates host membranes that are important for viral assembly, such as Golgi- and recycling endosome-derived membranes. Lastly, we show that HSV cVAC formation and/or maintenance depends on an intact microtubule network and a viral tegument protein, pUL51. Our observations suggest that the neuronal cVAC is a uniquely useful model to study common herpesvirus assembly pathways, and cell-specific pathways for membrane reorganization. Importance Herpesvirus particles are complex and contain many different proteins that must come together in an organized and coordinated fashion. Many viruses solve this coordination problem by creating a specialized assembly factory in the host cell, and the formation of such factories provides a promising target for interfering with virus production. Herpes simplex virus 1 (HSV-1) infects several types of cells, including neurons, but has not previously been shown to form such an organized factory in the non-neuronal cells in which its assembly has been best studied. Here we show that HSV-1 forms an organized assembly factory in neuronal cells, and we identify some of the viral and host cell factors that are important for its formation.
Nuclear envelope budding in herpesvirus nuclear egress may be negatively regulated, since the pUL31/pUL34 nuclear egress complex heterodimer can induce membrane budding without capsids when expressed ectopically or on artificial membranes in vitro, but not in the infected cell. We have previously described a pUL34 mutant that contained alanine substitutions at R158 and R161, and that showed impaired growth, impaired pUL31/pUL34 interaction, and unregulated budding. Here we determine the phenotypic contributions of the individual substitutions to these phenotypes. Neither substitution alone was able to reproduce the impaired growth or NEC interaction phenotypes. Either substitution, however, could fully reproduce the unregulated budding phenotype, suggesting that mis-regulated budding may not substantially impair virus replication. Additionally, the R158A substitution caused re-localization of the NEC to intranuclear punctate structures and recruited lamin A/C to those structures, suggesting that this residue might be important for recruitment of kinases for dispersal of nuclear lamins. Importance Herpesvirus nuclear egress is a complex, regulated process coordinated by two virus proteins that are conserved among the herpesviruses that form a heterodimeric nuclear egress complex (NEC). The NEC drives budding of capsids at the inner nuclear membrane, and recruits other viral and host cell proteins for disruption of the nuclear lamina, membrane scission and fusion. The structural basis of individual activities of the NEC, apart from membrane budding, are not clear, nor is the basis of the regulation of membrane budding. Here we explore the properties of NEC mutants that have an unregulated budding phenotype, determine the significance of that regulation for virus replication, and also characterize a structural requirement for nuclear lamina disruption.
Dynein motors are microtubule associated protein complexes that mediate multiple essential cellular processes, such as long-distance cargo trafficking and stabilization of the microtubule organization center. Most of these functions and their regulations depend on the dynein motor subunit dynactin. By using an infection-inducible system, we disrupted dynein motor function after HSV entry by overexpressing a dominant-negative inhibitor of dynein, resulting in a 5-fold growth defect in Vero cells and 1000-fold growth defect in CAD neuronal cells. Also, we found that in infected CAD cells, the dynein complex was recruited to viral assembly sites regardless of microtubule polymerization. Based on these observations, we then identified a novel interaction between conserved HSV-1 tegument protein pUL51 and p150Glued. pUL51 is a palmitoylated Golgi membrane-associated protein that is required for efficient virus assembly and spread. Overexpression of pUL51 alone was sufficient to recruit p150Glued to Golgi membranes. Sequences that are important and sufficient for pUL51-p150Glued interaction were mapped to residues 90 to 124 in pUL51 and residues 548 to 911 in p150Glued. Deletion of a.a 90-124 in pUL51 resulted in a moderate viral growth defect, a profound spread defect, and failure to accumulate both dynactin and the viral spread factor glycoprotein E (gE) at cell-cell junctions. A synthetic peptide that contains pUL51 a.a 90-125 could also inhibit viral growth and spread in pUL51-dependent manner. Taken together, our results suggest that the proper function of pUL51 in efficient viral assembly and spread depends on its interaction with p150Glued.
22Herpes simplex virus (HSV) is a neuroinvasive virus that has been used as a model organism 23for studying common properties of all herpesviruses. HSV induces host organelle 24 rearrangement and forms dispersed assembly compartments in epithelial cells, which 25 complicates the study of HSV assembly. In this study, we show that HSV forms a visually 26 distinct unitary cytoplasmic viral assembly center (cVAC) in both cancerous and primary 27 neuronal cells that concentrates viral structural proteins and is the site of capsid envelopment. 28The HSV cVAC also concentrates host membranes that are important for viral assembly, such 29as Golgi-and recycling endosome-derived membranes. Lastly, we show that HSV cVAC 30 formation and/or maintenance depends on an intact microtubule network and a viral tegument 31 protein, pUL51. Our observations suggest that the neuronal cVAC is a uniquely useful model to 32 study common herpesvirus assembly pathways, and cell-specific pathways for membrane 33 reorganization. 34 35
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