Herpes Simplex Virus 1 UL34 Mutants That Affect Membrane Budding Regulation and Nuclear Lamina Disruption

Abstract

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.

Herpes simplex virus type-1 cVAC formation in neuronal cells is mediated by dynein motor function and glycoprotein retrieval from the plasma membrane

“…To determine whether CC1 expression inhibits events that preceded arrival of capsids in the cytoplasm, we quantified cytoplasmic capsids in cells infected with a recombinant virus that expresses a fluorescently tagged capsid protein as previously described ( Fig. 7A ) ( 27 ). In CAD cells that overexpressed CC1, the number of cytoplasmic capsids was no lower than in infected cells that did not express CC1 and was significantly higher than in cells infected with a UL34-null virus, which is defective in nuclear egress ( Fig.…”

“…For cytoplasmic capsid quantification, the number of cytoplasmic capsids in infected cells was quantified using a modified protocol that has been previously described ( 27 ). Hoechst 33342 staining and gE staining were used to define the nuclear and cytoplasmic boundaries of the cell, respectively.…”

HSV Tegument Protein pUL51 Interacts with Host Dynactin for Viral Spread

“…The boundary of each cell was defined by either gE signals or ubiquitous cytoplasmic fluorescent proteins. For cytoplasmic capsid quantification, the number of cytoplasmic capsids in infected cells were quantified by using a modified protocol that has been previously described (5). Hoechst 33342 staining and gE staining were used to define the nuclear and cytoplasmic boundaries of the cell, respectively.…”

“…These genes are essential for the viral life cycle in vivo, and many of them play roles in viral assembly and egress. Some of these conserved assembly proteins participate in self-assembly complexes, such as major and minor capsid proteins; while others, including the viral nuclear egress complex, the virus protein kinases pUS3 and pUL13, pUL21, pUL36, VP26, pUS9, and gE interact with host proteins to mediate other aspects of viral egress (5)(6)(7)(8)(9)(10)(11)(12)(13). Targeting these viral-host interactions for therapeutics may reduce development of drug resistance since the host partner is not subject to selective pressure (14).…”