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A functional role for TorsinA in herpes simplex virus 1 nuclear egress.

ABSTRACT: Herpes simplex virus 1 (HSV-1) capsids leave the nucleus by a process of envelopment and de-envelopment at the nuclear envelope (NE) that is accompanied by structural alterations of the NE. As capsids translocate across the NE, transient primary enveloped virions form in the perinuclear space. Here, we provide evidence that torsinA (TA), a ubiquitously expressed ATPase, has a role in HSV-1 nuclear egress. TA resides within the lumen of the endoplasmic reticulum (ER)/NE and functions in maintaining normal NE architecture. We show that perturbation of TA normal function by overexpressing torsinA wild type (TAwt) inhibits HSV-1 production. Ultrastructural analysis of infected cells overexpressing TAwt revealed reduced levels of surface virions in addition to accumulation of novel, double-membrane structures called virus-like vesicles (VLVs). Although mainly found in the cytoplasm, VLVs resemble primary virions in their size, by the appearance of the inner membrane, and by the presence of pUL34, a structural component of primary virions. Collectively, our data suggest a model in which interference of TA normal function by overexpression impairs de-envelopment of the primary virions leading to their accumulation in a cytoplasmic membrane compartment. This implies novel functions for TA at the NE.

SUBMITTER: Maric M

PROVIDER: S-EPMC3196446 | biostudies-other | 2011 Oct

REPOSITORIES: biostudies-other

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A functional role for TorsinA in herpes simplex virus 1 nuclear egress.

Maric Martina M Shao Jianqiang J Ryan Randi J RJ Wong Chun-Shu CS Gonzalez-Alegre Pedro P Roller Richard J RJ

Journal of virology 20110720 19

Herpes simplex virus 1 (HSV-1) capsids leave the nucleus by a process of envelopment and de-envelopment at the nuclear envelope (NE) that is accompanied by structural alterations of the NE. As capsids translocate across the NE, transient primary enveloped virions form in the perinuclear space. Here, we provide evidence that torsinA (TA), a ubiquitously expressed ATPase, has a role in HSV-1 nuclear egress. TA resides within the lumen of the endoplasmic reticulum (ER)/NE and functions in maintaini ...[more]

PMID: 21775450

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Project description:This study developed a system consisting of two rounds of screening cellular proteins involved in the nuclear egress of herpes simplex virus 1 (HSV-1). Using this system, we first screened cellular proteins that interacted with the HSV-1 nuclear egress complex (NEC) consisting of UL34 and UL31 in HSV-1-infected cells, which are critical for the nuclear egress of HSV-1, by tandem affinity purification coupled with mass spectrometry-based proteomics technology. Next, we performed CRISPR/Cas9-based screening of live HSV-1-infected reporter cells under fluorescence microscopy using single guide RNAs targeting the cellular proteins identified in the first proteomic screening to detect the mislocalization of the lamin-associated protein emerin, which is a phenotype for defects in HSV-1 nuclear egress. This study focused on a cellular orphan transporter SLC35E1, one of the cellular proteins identified by the screening system. Knockout of SLC35E1 reduced HSV-1 replication and induced membranous invaginations containing perinuclear enveloped virions (PEVs) adjacent to the nuclear membrane (NM), aberrant accumulation of PEVs in the perinuclear space between the inner and outer NMs and the invagination structures, and mislocalization of the NEC. These effects were similar to those of previously reported mutation(s) in HSV-1 proteins and depletion of cellular proteins that are important for HSV-1 de-envelopment, one of the steps required for HSV-1 nuclear egress. Our newly established screening system enabled us to identify a novel cellular protein required for efficient HSV-1 de-envelopment. IMPORTANCE The identification of cellular protein(s) that interact with viral effector proteins and function in important viral procedures is necessary for enhancing our understanding of the mechanics of various viral processes. In this study, we established a new system consisting of interactome screening for the herpes simplex virus 1 (HSV-1) nuclear egress complex (NEC), followed by loss-of-function screening to target the identified putative NEC-interacting cellular proteins to detect a defect in HSV-1 nuclear egress. This newly established system identified SLC35E1, an orphan transporter, as a novel cellular protein required for efficient HSV-1 de-envelopment, providing an insight into the mechanisms involved in this viral procedure.

Project description:Viral proteins pUL16 and pUL21 are required for efficient nuclear egress of herpes simplex virus 2 capsids. To better understand the role of these proteins in nuclear egress, we established whether nuclear egress complex (NEC) distribution and/or function was altered in the absence of either pUL16 or pUL21. NEC distribution in cells infected with pUL16-deficient viruses was indistinguishable from that observed in cells infected with wild-type viruses. In contrast, NEC distribution was aberrant in cells infected with pUL21-deficient virus and, instead, showed some similarity to the aberrant NEC distribution pattern observed in cells infected with pUs3-deficient virus. These results indicated that pUL16 plays a role in nuclear egress that is distinct from that of pUL21 and pUs3. Higher-resolution examination of nuclear envelope ultrastructure in cells infected with pUL21-deficient viruses by transmission electron microscopy showed different types of nuclear envelope perturbations, including some that were not observed in cells infected with pUs3 deficient virus. The formation of the nuclear envelope perturbations observed in pUL21-deficient virus infections was dependent on a functional NEC, revealing a novel role for pUL21 in regulating NEC activity. The results of comparisons of nuclear envelope ultrastructure in cells infected with viruses lacking pUs3, pUL16, or both pUs3 and pUL16 were consistent with a role for pUL16 in advance of primary capsid envelopment and shed new light on how pUs3 functions in nuclear egress.IMPORTANCE The membrane deformation activity of the herpesvirus nuclear egress complex (NEC) allows capsids to transit through both nuclear membranes into the cytoplasm. NEC activity must be precisely controlled during viral infection, and yet our knowledge of how NEC activity is controlled is incomplete. To determine how pUL16 and pUL21, two viral proteins required for nuclear egress of herpes simplex virus 2, function in nuclear egress, we examined how the lack of each protein impacted NEC distribution. These analyses revealed a function of pUL16 in nuclear egress distinct from that of pUL21, uncovered a novel role for pUL21 in regulating NEC activity, and shed new light on how a viral kinase, pUs3, regulates nuclear egress. Nuclear egress of capsids is required for all herpesviruses. A complete understanding of all aspects of nuclear egress, including how viral NEC activity is controlled, may yield strategies to disrupt this process and aid the development of herpes-specific antiviral therapies.

Project description:Nuclear egress of herpesviruses is accompanied by changes in the architecture of the nuclear membrane and nuclear lamina that are thought to facilitate capsid access to the inner nuclear membrane (INM) and curvature of patches of the INM around the capsid during budding. Here we report the properties of a point mutant of pUL34 (Q163A) that fails to induce gross changes in nuclear architecture or redistribution of lamin A/C. The UL34(Q163A) mutant shows a roughly 100-fold defect in single-step growth, and it forms small plaques. This mutant has a defect in nuclear egress, and furthermore, it fails to disrupt nuclear shape or cause observable displacement of lamin A/C despite retaining the ability to recruit the pUS3 and PKC protein kinases and to mediate phosphorylation of emerin. Extragenic suppressors of the UL34(Q163A) phenotype were isolated, and all of them carry a single mutation of arginine 229 to leucine in UL31. Surprisingly, although this UL31 mutation largely restores virus replication, it does not correct the lamina disruption defect, suggesting that, in Vero cells, changes in nuclear shape and gross displacements of lamin A/C may facilitate but are unnecessary for nuclear egress. IMPORTANCE:Herpesvirus nuclear egress is an essential and conserved process that requires close association of the viral capsid with the inner nuclear membrane and budding of the capsid into that membrane. Access to the nuclear membrane and tight curvature of that membrane are thought to require disruption of the nuclear lamina that underlies the inner nuclear membrane, and consistent with this idea, herpesvirus infection induces biochemical and architectural changes at the nuclear membrane. The significance of the nuclear membrane architectural changes is poorly characterized. The results presented here address that deficiency in our understanding and show that a combination of mutations in two of the viral nuclear egress factors results in a failure to accomplish at least two components of lamina disruption while still allowing relatively efficient viral replication, suggesting that changes in nuclear shape and displacement of lamins are not necessary for herpes simplex virus 1 (HSV-1) nuclear egress.

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A functional role for TorsinA in herpes simplex virus 1 nuclear egress.

Publications

A functional role for TorsinA in herpes simplex virus 1 nuclear egress.

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