Analysis of a Charge Cluster Mutation of Herpes Simplex Virus Type 1 UL34 and Its Extragenic Suppressor Suggests a Novel Interaction between pUL34 and pUL31 That Is Necessary for Membrane Curvature around Capsids

ABSTRACT Interaction between pUL34 and pUL31 is essential for targeting both proteins to the inner nuclear membrane (INM). Sequences mediating the targeting interaction have been mapped by others with both proteins. We have previously reported identification of charge cluster mutants of herpes simplex virus type 1 UL34 that localize properly to the inner nuclear membrane, indicating interaction with UL31, but fail to complement a UL34 deletion. We have characterized one mutation (CL04) that alters a charge cluster near the N terminus of pUL34 and observed the following. (i) The CL04 mutant has a dominant-negative effect on pUL34 function, indicating disruption of some critical interaction. (ii) In infections with CL04 pUL34, capsids accumulate in close association with the INM, but no perinuclear enveloped viruses, cytoplasmic capsids, or virions or cell surface virions were observed, suggesting that CL04 UL34 does not support INM curvature around the capsid. (iii) Passage of UL34-null virus on a stable cell line that expresses CL04 resulted in selection of extragenic suppressor mutants that grew efficiently using the mutant pUL34. (iv) All extragenic suppressors contained an R229→L mutation in pUL31 that was sufficient to suppress the CL04 phenotype. (v) Immunolocalization and coimmunoprecipitation experiments with truncated forms of pUL34 and pUL31 confirm that N-terminal sequences of pUL34 and a C-terminal domain of pUL31 mediate interaction but not nuclear membrane targeting. pUL34 and pUL31 may make two essential interactions—one for the targeting of the complex to the nuclear envelope and another for nuclear membrane curvature around capsids.

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Nucleolin Is Required for Efficient Nuclear Egress of Herpes Simplex Virus Type 1 Nucleocapsids

Journal of Virology ◽

10.1128/jvi.02007-09 ◽

2009 ◽

Vol 84 (4) ◽

pp. 2110-2121 ◽

Cited By ~ 29

Author(s):

Ken Sagou ◽

Masashi Uema ◽

Yasushi Kawaguchi

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Nuclear Membrane ◽

Herpes Simplex Virus Type ◽

Viral Dna ◽

Nuclear Egress ◽

Infected Cells ◽

Simplex Virus ◽

Hsv 1

ABSTRACT Herpesvirus nucleocapsids assemble in the nucleus and must cross the nuclear membrane for final assembly and maturation to form infectious progeny virions in the cytoplasm. It has been proposed that nucleocapsids enter the perinuclear space by budding through the inner nuclear membrane, and these enveloped nucleocapsids then fuse with the outer nuclear membrane to enter the cytoplasm. Little is known about the mechanism(s) for nuclear egress of herpesvirus nucleocapsids and, in particular, which, if any, cellular proteins are involved in the nuclear egress pathway. UL12 is an alkaline nuclease encoded by herpes simplex virus type 1 (HSV-1) and has been suggested to be involved in viral DNA maturation and nuclear egress of nucleocapsids. Using a live-cell imaging system to study cells infected by a recombinant HSV-1 expressing UL12 fused to a fluorescent protein, we observed the previously unreported nucleolar localization of UL12 in live infected cells and, using coimmunoprecipitation analyses, showed that UL12 formed a complex with nucleolin, a nucleolus marker, in infected cells. Knockdown of nucleolin in HSV-1-infected cells reduced capsid accumulation, as well as the amount of viral DNA resistant to staphylococcal nuclease in the cytoplasm, which represented encapsidated viral DNA, but had little effect on these viral components in the nucleus. These results indicated that nucleolin is a cellular factor required for efficient nuclear egress of HSV-1 nucleocapsids in infected cells.

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Host Vesicle Fusion Protein VAPB Contributes to the Nuclear Egress Stage of Herpes Simplex Virus Type-1 (HSV-1) Replication

Cells ◽

10.3390/cells8020120 ◽

2019 ◽

Vol 8 (2) ◽

pp. 120 ◽

Cited By ~ 1

Author(s):

Natalia Saiz-Ros ◽

Rafal Czapiewski ◽

Ilaria Epifano ◽

Andrew Stevenson ◽

Selene Swanson ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Nuclear Membrane ◽

Herpes Simplex Virus Type ◽

Vesicle Fusion ◽

Nuclear Egress ◽

Simplex Virus ◽

Hsv 1

The primary envelopment/de-envelopment of Herpes viruses during nuclear exit is poorly understood. In Herpes simplex virus type-1 (HSV-1), proteins pUL31 and pUL34 are critical, while pUS3 and some others contribute; however, efficient membrane fusion may require additional host proteins. We postulated that vesicle fusion proteins present in the nuclear envelope might facilitate primary envelopment and/or de-envelopment fusion with the outer nuclear membrane. Indeed, a subpopulation of vesicle-associated membrane protein-associated protein B (VAPB), a known vesicle trafficking protein, was present in the nuclear membrane co-locating with pUL34. VAPB knockdown significantly reduced both cell-associated and supernatant virus titers. Moreover, VAPB depletion reduced cytoplasmic accumulation of virus particles and increased levels of nuclear encapsidated viral DNA. These results suggest that VAPB is an important player in the exit of primary enveloped HSV-1 virions from the nucleus. Importantly, VAPB knockdown did not alter pUL34, calnexin or GM-130 localization during infection, arguing against an indirect effect of VAPB on cellular vesicles and trafficking. Immunogold-labelling electron microscopy confirmed VAPB presence in nuclear membranes and moreover associated with primary enveloped HSV-1 particles. These data suggest that VAPB could be a cellular component of a complex that facilitates UL31/UL34/US3-mediated HSV-1 nuclear egress.

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UL31 and UL34 Proteins of Herpes Simplex Virus Type 1 Form a Complex That Accumulates at the Nuclear Rim and Is Required for Envelopment of Nucleocapsids

Journal of Virology ◽

10.1128/jvi.75.18.8803-8817.2001 ◽

2001 ◽

Vol 75 (18) ◽

pp. 8803-8817 ◽

Cited By ~ 211

Author(s):

Ashley E. Reynolds ◽

Brent J. Ryckman ◽

Joel D. Baines ◽

Yuping Zhou ◽

Li Liang ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Nuclear Envelope ◽

Gene Product ◽

Virus Type ◽

Nuclear Membrane ◽

Herpes Simplex Virus Type ◽

Simplex Virus ◽

Hsv 1

ABSTRACT The herpes simplex virus type 1 (HSV-1) UL34 protein is likely a type II membrane protein that localizes within the nuclear membrane and is required for efficient envelopment of progeny virions at the nuclear envelope, whereas the UL31 gene product of HSV-1 is a nuclear matrix-associated phosphoprotein previously shown to interact with UL34 protein in HSV-1-infected cell lysates. For these studies, polyclonal antisera directed against purified fusion proteins containing UL31 protein fused to glutathione-S-transferase (UL31-GST) and UL34 protein fused to GST (UL34-GST) were demonstrated to specifically recognize the UL31 and UL34 proteins of approximately 34,000 and 30,000 Da, respectively. The UL31 and UL34 gene products colocalized in a smooth pattern throughout the nuclear rim of infected cells by 10 h postinfection. UL34 protein also accumulated in pleiomorphic cytoplasmic structures at early times and associated with an altered nuclear envelope late in infection. Localization of UL31 protein at the nuclear rim required the presence of UL34 protein, inasmuch as cells infected with a UL34 null mutant virus contained UL31 protein primarily in central intranuclear domains separate from the nuclear rim, and to a lesser extent in the cytoplasm. Conversely, localization of UL34 protein exclusively at the nuclear rim required the presence of the UL31 gene product, inasmuch as UL34 protein was detectable at the nuclear rim, in replication compartments, and in the cytoplasm of cells infected with a UL31 null virus. When transiently expressed in the absence of other viral factors, UL31 protein localized diffusely in the nucleoplasm, whereas UL34 protein localized primarily in the cytoplasm and at the nuclear rim. In contrast, coexpression of the UL31 and UL34 proteins was sufficient to target both proteins exclusively to the nuclear rim. The proteins were also shown to directly interact in vitro in the absence of other viral proteins. In cells infected with a virus lacking the US3-encoded protein kinase, previously shown to phosphorylate the UL34 gene product, UL31 and UL34 proteins colocalized in small punctate areas that accumulated on the nuclear rim. Thus, US3 kinase is required for even distribution of UL31 and UL34 proteins throughout the nuclear rim. Taken together with the similar phenotypes of the UL31 and UL34 deletion mutants, these data strongly suggest that the UL31 and UL34 proteins form a complex that accumulates at the nuclear membrane and plays an important role in nucleocapsid envelopment at the inner nuclear membrane.

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Faculty Opinions recommendation of Herpes simplex virus type 1 infection induces activation and recruitment of protein kinase C to the nuclear membrane and increased phosphorylation of lamin B.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1029901.346642 ◽

2005 ◽

Author(s):

Rozanne Sandri-Goldin

Keyword(s):

Protein Kinase C ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Protein Kinase ◽

Nuclear Membrane ◽

Herpes Simplex Virus Type ◽

Lamin B ◽

Kinase C ◽

Simplex Virus

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Herpes Simplex Virus Type 1 Capsids Transit by the trans-Golgi Network, Where Viral Glycoproteins Accumulate Independently of Capsid Egress

Journal of Virology ◽

10.1128/jvi.79.14.8847-8860.2005 ◽

2005 ◽

Vol 79 (14) ◽

pp. 8847-8860 ◽

Cited By ~ 112

Author(s):

Sophie Turcotte ◽

Josée Letellier ◽

Roger Lippé

Keyword(s):

Plasma Membrane ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Nuclear Membrane ◽

Herpes Simplex Virus Type ◽

Viral Glycoproteins ◽

Simplex Virus ◽

Golgi Network

ABSTRACT Egress of herpes capsids from the nucleus to the plasma membrane is a complex multistep transport event that is poorly understood. The current model proposes an initial envelopment at the inner nuclear membrane of capsids newly assembled in the nucleus. The capsids are then released in cytosol by fusion with the outer nuclear membrane. They are finally reenveloped at a downstream organelle before traveling to the plasma membrane for their extracellular release. Although the trans-Golgi network (TGN) is often cited as a potential site of reenvelopment, other organelles have also been proposed, including the Golgi, endoplasmic reticulum-Golgi intermediate compartment, aggresomes, tegusomes, and early or late endosomes. To clarify this important issue, we followed herpes simplex virus type 1 egress by immunofluorescence under conditions that slowed intracellular transport and promoted the accumulation of the otherwise transient reenvelopment intermediate. The data show that the capsids transit by the TGN and point to this compartment as the main reenvelopment site, although a contribution by endosomes cannot formally be excluded. Given that viral glycoproteins are expected to accumulate where capsids acquire their envelope, we examined this prediction and found that all tested could indeed be detected at the TGN. Moreover, this accumulation occurred independently of capsid egress. Surprisingly, capsids were often found immediately adjacent to the viral glycoproteins at the TGN.

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