scholarly journals Herpes Simplex Virus Type 1 Infection Induces Activation and Recruitment of Protein Kinase C to the Nuclear Membrane and Increased Phosphorylation of Lamin B

2006 ◽  
Vol 80 (1) ◽  
pp. 494-504 ◽  
Author(s):  
Richard Park ◽  
Joel D. Baines
Keyword(s):  
Protein Kinase C ◽  
Herpes Simplex ◽  
Protein Kinase ◽  
Nuclear Membrane ◽  
Nuclear Lamina ◽  
Lamin B ◽  
Kinase C ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT We report that herpes simplex virus type 1 (HSV-1) infection leads to the recruitment of protein kinase C (PKC) to the nuclear rim. In HEp-2 cells, PKC recruitment to the nuclear rim was initiated between 8 h and 12 h postinfection. PKCδ, a proapoptotic kinase, was completely recruited to the nuclear rim upon infection with HSV-1. PKCα was less dramatically relocalized mostly at the nuclear rim upon infection, although some PKCα remained in the cytoplasm. PKCζ-specific immunofluorescence was not significantly relocated to the nuclear rim. The UL34 and UL31 proteins, as well as their association, were each required for PKC recruitment to the nuclear rim. The HSV-1 US3 protein product, a kinase which regulates the phosphorylation state and localization of UL34, was not required for PKC recruitment to the nuclear rim; however, it was required for proper localization along the nuclear rim, as PKC appeared unevenly distributed along the nuclear rim of cells infected with US3 null and kinase-dead mutants. HSV-1 infection induced the phosphorylation of both lamin B and PKC. Elevated lamin B phosphorylation in HSV-1-infected cells was partially reduced by inhibitors of PKC. The data suggest a model in which kinases that normally disassemble the nuclear lamina during apoptosis are recruited to the nuclear membrane through functions requiring UL31 and UL34. We hypothesize that the recruitment of PKC functions to phosphorylate lamin B to help modify the nuclear lamina and promote budding of nucleocapsids at the inner nuclear membrane.

scholarly journals Fate of the Inner Nuclear Membrane Protein Lamin B Receptor and Nuclear Lamins in Herpes Simplex Virus Type 1 Infection

2001 ◽  
Vol 75 (18) ◽  
pp. 8818-8830 ◽  
Author(s):  
Emily S. Scott ◽  
Peter O'Hare
Keyword(s):  
Herpes Simplex ◽  
Nuclear Membrane ◽  
Nuclear Lamina ◽  
Lamin B ◽  
Inner Nuclear Membrane ◽  
Lamin B Receptor ◽  
Infected Cells ◽  
Simplex Virus ◽  
Inner Nuclear Membrane Protein

ABSTRACT During herpesvirus egress, capsids bud through the inner nuclear membrane. Underlying this membrane is the nuclear lamina, a meshwork of intermediate filaments with which it is tightly associated. Details of alterations to the lamina and the inner nuclear membrane during infection and the mechanisms involved in capsid transport across these structures remain unclear. Here we describe the fate of key protein components of the nuclear envelope and lamina during herpes simplex virus type 1 (HSV-1) infection. We followed the distribution of the inner nuclear membrane protein lamin B receptor (LBR) and lamins A and B2 tagged with green fluorescent protein (GFP) in live infected cells. Together with additional results from indirect immunofluorescence, our studies reveal major morphologic distortion of nuclear-rim LBR and lamins A/C, B1, and B2. By 8 h p.i., we also observed a significant redistribution of LBR-GFP to the endoplasmic reticulum, where it colocalized with a subpopulation of cytoplasmic glycoprotein B by immunofluorescence. In addition, analysis by fluorescence recovery after photobleaching reveals that LBR-GFP exhibited increased diffusional mobility within the nuclear membrane of infected cells. This is consistent with the disruption of interactions between LBR and the underlying lamina. In addition to studying stably expressed GFP-lamins by fluorescence microscopy, we studied endogenous A- and B-type lamins in infected cells by Western blotting. Both approaches reveal a loss of lamins associated with virus infection. These data indicate major disruption of the nuclear envelope and lamina of HSV-1-infected cells and are consistent with a virus-induced dismantling of the nuclear lamina, possibly in order to gain access to the inner nuclear membrane.

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

2009 ◽  
Vol 84 (4) ◽  
pp. 2110-2121 ◽  
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.

scholarly journals Cytomegaloviral proteins pUL50 and pUL53 are associated with the nuclear lamina and interact with cellular protein kinase C

2007 ◽  
Vol 88 (10) ◽  
pp. 2642-2650 ◽  
Author(s):  
Jens Milbradt ◽  
Sabrina Auerochs ◽  
Manfred Marschall
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Direct Interaction ◽  
Nuclear Lamina ◽  
Cellular Protein ◽  
Immunofluorescent Staining ◽  
Lamin B ◽  
Kinase C ◽  
Lamin B Receptor

Human cytomegalovirus-encoded pUL50 and pUL53 belong to a group of conserved herpesviral nuclear proteins. This study describes: (i) the co-localization of pUL50 with components of the nuclear lamina such as lamins A/C and lamin B receptor by double immunofluorescent staining, (ii) a strong pUL50-mediated relocalization of pUL53 from a diffuse nuclear pattern towards a nuclear rim localization, (iii) a direct interaction between pUL50 and pUL53, as well as between pUL50 and protein kinase C (PKC), shown by yeast two-hybrid and co-immunoprecipitation analyses, (iv) in vitro phosphorylation of pUL50, which is highly suggestive of PKC activity, and finally (v) partial relocalization of PKC by pUL50/pUL53 from its main cytoplasmic localization to a marked nuclear lamina accumulation. These data suggest a role for pUL50 and pUL53 in the recruitment of PKC, an event that is considered to be important for cytomegalovirus-induced distortion of the nuclear lamina.

scholarly journals Herpes Simplex Virus Type 1 Immediate-Early Protein Vmw110 Induces the Proteasome-Dependent Degradation of the Catalytic Subunit of DNA-Dependent Protein Kinase

1999 ◽  
Vol 73 (1) ◽  
pp. 650-657 ◽  
Author(s):  
Jane Parkinson ◽  
Susan P. Lees-Miller ◽  
Roger D. Everett
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Protein Kinase ◽  
Herpes Simplex Virus Type ◽  
Dependent Protein Kinase ◽  
Early Protein ◽  
Dependent Protein ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpes simplex virus type 1 (HSV-1) infection causes the active degradation of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), and this process is reliant on the expression of the HSV-1 immediate-early protein Vmw110. In this study we investigated in more detail the mechanism by which the degradation occurs, the domains of Vmw110 which are required, and whether Vmw110 is by itself sufficient for the effect. We found that proteasome inhibitors prevented the degradation of DNA-PKcs, indicating the involvement of a proteasome pathway. Furthermore, the continued activity of DNA-PK during infection in the presence of these inhibitors indicated that Vmw110 does not directly alter the enzyme activity of DNA-PKcs prior to its degradation in a normal infection. Indeed, Vmw110 was found to bind to neither the catalytic nor Ku subunits of DNA-PK. Using mutant Vmw110 viruses we show that the RING finger domain of Vmw110 is essential for the induced degradation of DNA-PKcs but that the ability of Vmw110 to bind to a cellular ubiquitin-specific protease (HAUSP) is not required. When expressed in the absence of other viral proteins, Vmw110 was sufficient to cause the degradation of DNA-PKcs, indicating that the effect on the stability of DNA-PKcs was a direct consequence of Vmw110 activity and not an indirect Vmw110-dependent effect of virus infection. Finally, the Vmw110-induced degradation of DNA-PKcs and loss in DNA-PK activity appears to be beneficial to HSV-1 infection, as virus replication was more efficient in cells lacking DNA-PKcs, especially at low multiplicities of infection.

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

Cells ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 120 ◽  
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.

scholarly journals 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

2001 ◽  
Vol 75 (18) ◽  
pp. 8803-8817 ◽  
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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