scholarly journals Identification of Phosphorylation Sites within the Herpes Simplex Virus Tegument Protein VP22

1999 ◽  
Vol 73 (7) ◽  
pp. 6203-6206 ◽  
Author(s):  
Gillian Elliott ◽  
Dawn O’Reilly ◽  
Peter O’Hare
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Serine Phosphorylation ◽  
Virus Protein ◽  
Phosphorylation Sites ◽  
Infected Cells ◽  
Simplex Virus ◽  
Terminal Site ◽  
Protein Vp22

ABSTRACT The herpes simplex virus protein VP22 is a major phosphoprotein of infected cells. In this study, we identify two serine phosphorylation sites within VP22 and show that the N-terminal site is a substrate for casein kinase II, while the extreme C-terminal site is a substrate for another, as yet unidentified, cellular kinase. Furthermore, we show that a mutant of VP22 which has both sites altered is unable to incorporate phosphate in vivo, confirming that there are no other phosphorylation sites within VP22.

scholarly journals Deletion of the Herpes Simplex Virus VP22-Encoding Gene (UL49) Alters the Expression, Localization, and Virion Incorporation of ICP0

2005 ◽  
Vol 79 (15) ◽  
pp. 9735-9745 ◽  
Author(s):  
Gillian Elliott ◽  
Wali Hafezi ◽  
Alison Whiteley ◽  
Emmanuelle Bernard
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Assembly ◽  
Tegument Protein ◽  
Virus Protein ◽  
Obvious Effect ◽  
Virus Growth ◽  
Infected Cells ◽  
Simplex Virus ◽  
Protein Vp22

ABSTRACT The role of the herpes simplex virus tegument protein VP22 is not yet known. Here we describe the characterization of a virus in which the entire VP22 open reading frame has been deleted. We show that VP22 is not essential for virus growth but that virus lacking VP22 (Δ22) displays a cell-specific replication defect in epithelial MDBK cells. Virus particles assembled in the absence of VP22 show few obvious differences to wild-type (WT) particles, except for a moderate reduction in glycoproteins gD and gB. In addition, the Δ22 virus exhibits a general delay in the initiation of virus protein synthesis, but this is not due to a glycoprotein-related defect in virus entry. Intriguingly, however, the absence of VP22 has an obvious effect on the intracellular level of the immediate-early (IE) protein ICP0. Moreover, following translocation from the nucleus to the cytoplasm, ICP0 is unable to localize to the characteristic cytoplasmic sites observed in a WT infection. We demonstrate that, in WT-infected cells, VP22 and ICP0 are concentrated in the same cytoplasmic sites. Furthermore, we show that, while ICP0 and ICP4 are components of WT extracellular virions, the altered localization of ICP0 in the cytoplasm of Δ22-infected cells correlates with an absence of both ICP0 and ICP4 from Δ22 virions. Hence, while a role has not yet been defined for virion IE proteins in virus infection, our results suggest that their incorporation is a specific event requiring the tegument protein VP22. This report provides the first direct evidence that VP22 influences virus assembly.

scholarly journals Phosphorylation of the Herpes Simplex Virus Tegument Protein VP22 Has No Effect on Incorporation of VP22 into the Virus but Is Involved in Optimal Expression and Virion Packaging of ICP0

2005 ◽  
Vol 79 (22) ◽  
pp. 14057-14068 ◽  
Author(s):  
Corinne Potel ◽  
Gillian Elliott
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Replication ◽  
Serine Phosphorylation ◽  
Tegument Protein ◽  
Microtubule Network ◽  
Mdbk Cells ◽  
Infected Cells ◽  
Simplex Virus ◽  
Protein Vp22

ABSTRACT Herpes simplex virus VP22 is a major tegument protein of unknown function. Very recently, we reported that the predominant effect of deleting the VP22 gene was on the expression, localization, and virion incorporation of ICP0. In addition, the Δ22 virus replicated poorly in epithelial MDBK cells. We have also previously shown that VP22 interacts with the tegument protein VP16 and the cellular microtubule network. While the majority of VP22 in infected cells is highly phosphorylated, the nonphosphorylated form of VP22 is the predominant species in the virion, suggesting a differential requirement for phosphorylation through virus replication. Hence, to study the significance of VP22 phosphorylation, we have now constructed two recombinant viruses expressing green fluorescent protein-VP22 (G22) in which the previously identified serine phosphorylation sites have been mutated either to alanine to abolish the phosphorylation status of VP22 (G22P−) or to glutamic acid to mimic permanent phosphorylation (G22P+). Localization studies indicated that the G22P− protein associated tightly with microtubules in some infected cells, suggesting that VP22 phosphorylation may control its interaction with the microtubule network. By contrast, VP22 phosphorylation had no effect on its ability to interact with VP16 and, importantly, had no effect on the relative packaging of VP22. Intriguingly, virion packaging of ICP0 was reduced in the G22P+ virus while ICP0 expression was reduced in the G22P− virus, suggesting that these two ICP0 defects, previously observed in the Δ22 virus, were attributable to different forms of VP22. Furthermore, the Δ22 virus replication defect in MDBK cells correlated with the expression of constitutively charged VP22 in the G22P+ virus. Taken together, these results suggest an important role for VP22 phosphorylation in its relationship with ICP0.

scholarly journals Herpes simplex virus type 2 membrane protein UL56 associates with the kinesin motor protein KIF1A

2005 ◽  
Vol 86 (3) ◽  
pp. 527-533 ◽  
Author(s):  
Tetsuo Koshizuka ◽  
Yasushi Kawaguchi ◽  
Yukihiro Nishiyama
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Membrane Protein ◽  
Transmembrane Domain ◽  
Yeast Two Hybrid ◽  
Infected Cells ◽  
Simplex Virus ◽  
Two Hybrid

The herpes simplex virus UL56 gene product is a C-terminal-anchored, type II membrane protein of unknown function. UL56 was found to interact with KIF1A, a member of the kinesin-3 family, in a yeast two-hybrid screen and a GST pull-down assay. KIF1A mediates the transport of synaptic vesicle precursors and is essential for the function and viability of neurons. When overexpressed, KIF1A co-localized with full-sized UL56, but no clear co-localization was observed when co-expressed with the UL56 mutant protein lacking its C-terminal transmembrane domain (TMD). Although the C-terminal TMD was not essential for the interaction with KIF1A in the yeast two-hybrid screen and GST pull-down assays, these results indicate that the C-terminal TMD, as well as aa 69–217, of UL56 are important for the interaction with KIF1A in vivo. The hypothesis that the UL56 protein affects vesicular trafficking in infected cells, potentially by acting as a receptor for motor proteins in neurons, is discussed.

scholarly journals Role of Phosphatidylethanolamine Biosynthesis in Herpes Simplex Virus 1-Infected Cells in Progeny Virus Morphogenesis in the Cytoplasm and in Viral Pathogenicity In Vivo

2020 ◽  
Vol 94 (24) ◽  
Author(s):  
Jun Arii ◽  
Ayano Fukui ◽  
Yuta Shimanaka ◽  
Nozomu Kono ◽  
Hiroyuki Arai ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Viral Replication ◽  
Biosynthetic Pathway ◽  
Herpes Simplex Virus 1 ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Glycerophospholipids are major components of cell membranes. Phosphatidylethanolamine (PE) is a glycerophospholipid that is involved in multiple cellular processes, such as membrane fusion, the cell cycle, autophagy, and apoptosis. In this study, we investigated the role of PE biosynthesis in herpes simplex virus 1 (HSV-1) infection by knocking out the host cell gene encoding phosphate cytidylyltransferase 2, ethanolamine (Pcyt2), which is a key rate-limiting enzyme in one of the two major pathways for PE biosynthesis. Pcyt2 knockout reduced HSV-1 replication and caused an accumulation of unenveloped and partially enveloped nucleocapsids in the cytoplasm of an HSV-1-infected cell culture. A similar phenotype was observed when infected cells were treated with meclizine, which is an inhibitor of Pcyt2. In addition, treatment of HSV-1-infected mice with meclizine significantly reduced HSV-1 replication in the mouse brains and improved their survival rates. These results indicated that PE biosynthesis mediated by Pcyt2 was required for efficient HSV-1 envelopment in the cytoplasm of infected cells and for viral replication and pathogenicity in vivo. The results also identified the PE biosynthetic pathway as a possible novel target for antiviral therapy of HSV-associated diseases and raised an interesting possibility for meclizine repositioning for treatment of these diseases, since it is an over-the-counter drug that has been used for decades against nausea and vertigo in motion sickness. IMPORTANCE Glycerophospholipids in cell membranes and virus envelopes often affect viral entry and budding. However, the role of glycerophospholipids in membrane-associated events in viral replication in herpesvirus-infected cells has not been reported to date. In this study, we have presented data showing that cellular PE biosynthesis mediated by Pcyt2 is important for HSV-1 envelopment in the cytoplasm, as well as for viral replication and pathogenicity in vivo. This is the first report showing the importance of PE biosynthesis in herpesvirus infections. Our results showed that inhibition of Pcyt2, a key cell enzyme for PE synthesis, significantly inhibited HSV-1 replication and pathogenicity in mice. This suggested that the PE biosynthetic pathway, as well as the HSV-1 virion maturation pathway, can be a target for the development of novel anti-HSV drugs.

scholarly journals Analysis of the Phosphorylation Sites of Herpes Simplex Virus Type 1 Regulatory Protein ICP27

1999 ◽  
Vol 73 (4) ◽  
pp. 3246-3257 ◽  
Author(s):  
Yan Zhi ◽  
Rozanne M. Sandri-Goldin
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Viral Infection ◽  
Herpes Simplex Virus Type ◽  
Regulatory Protein ◽  
Phosphorylation Sites ◽  
Simplex Virus ◽  
Phosphate Groups

ABSTRACT The herpes simplex virus type 1 (HSV-1) regulatory protein ICP27 is a 63-kDa phosphoprotein required for viral replication. ICP27 has been shown to contain both stable phosphate groups and phosphate groups that cycle on and off during infection (K. W. Wilcox, A. Kohn, E. Sklyanskaya, and B. Roizman, J. Virol. 33:167–182, 1980). Despite extensive genetic analysis of the ICP27 gene, there is no information available about the sites of the ICP27 molecule that are phosphorylated during viral infection. In this study, we mapped several of the phosphorylation sites of ICP27 following in vivo radiolabeling. Phosphoamino acid analysis showed that serine is the only amino acid that is phosphorylated during infection. Two-dimensional phosphopeptide mapping showed a complex tryptic phosphopeptide pattern with at least four major peptides and several minor peptides. In addition, ICP27 purified from transfected cells yielded a similar phosphopeptide pattern, suggesting that cellular kinases phosphorylate ICP27 during viral infection. In vitro labeling showed that protein kinase A (PKA), PKC, and casein kinase II (CKII) were able to differentially phosphorylate ICP27, resulting in distinct phosphopeptide patterns. The major phosphorylation sites of ICP27 appeared to cluster in the N-terminal portion of the protein, such that a frameshift mutant that encodes amino acids 1 to 163 yielded a phosphopeptide pattern very similar to that seen with the wild-type protein. Further, using small deletion and point mutations in kinase consensus sites, we have elucidated individual serine residues that are phosphorylated in vivo. Specifically, the serine at residue 114 was highly phosphorylated by PKA and the serine residues at positions 16 and 18 serve as targets for CKII phosphorylation in vivo. These kinase consensus site mutants were still capable of complementing the growth of an ICP27-null mutant virus. Interestingly, phosphorylation of the serine at residue 114, which lies within the major nuclear localization signal, appeared to modulate the efficiency of nuclear import of ICP27.

scholarly journals Anti-viral activity of Staphylococcus aureus lysates against herpes simplex virus type-I infection: an in vitro and in vivo study

2021 ◽  
Vol 14 (10) ◽  
pp. 1463-1472
Author(s):  
Tian-Lan Lin ◽  
◽  
Wei-Ting Zeng ◽  
Fang Duan ◽  
Yin-Hui Pei ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Herpes Simplex Virus Type ◽  
Plaque Formation ◽  
Type I ◽  
Gfp Expression ◽  
Infected Cells ◽  
Simplex Virus

AIM: To investigate the effect of Staphylococcus aureus (S. aures) lysates (SALs) on herpes simplex virus type-I (HSV1) infection in human corneal epithelial (HCE) cells and in a mouse model of HSV1 keratitis. METHODS: HCE, Vero, HeLa, and BV2 cells were infected with HSV1 [HSV1 f strain, HSV1f; HSV-1-H129 with green fluorescent protein (GFP) knock-in, HSV1g]. Pre- or post-infection, SAL at various concentrations was added to the culture medium for 24h. GFP fluorescence in HSV1g or plaque formation by HSV1f were examined. The effects of heat-treated SAL, precooled acetone-precipitated SAL, and SAL subjected to ultrafiltration (100 kDa) were evaluated. The effects of other bacterial components and lysates on HSV1 infection were also tested, including lipoteichoic acid (LTA), peptidoglycan (PGN), staphylococcal protein A (SPA), and α-hemolysin from S. aureus (α-toxin) as well as lysates from a wild-type S. aureus strain, S. epidermidis, and Escherichia coli (W-SAL, SEL, and ECL, respectively). In addition, SAL eye drops were applied topically to BALB/c mice with HSV1 keratitis, followed by in vivo observations. RESULTS: The cytopathic effect, plaque formation (HSV1f), and GFP expression (HSV1g) in infected cells were inhibited by SAL in a dose-dependent manner. The active component of SAL (≥100 kDa) was heat-sensitive and retained activity after acetone precipitation. In HSV1g-infected cells, treatment with LTA-sa, α-toxin, PGN-sa, or SPA did not inhibit GFP expression. SAL, W-SAL, and SEL (but not ECL) decreased GFP expression. In mice with HSV1 keratitis, SAL reduced corneal lesions by 71%. CONCLUSION: The results of this study demonstrate that SAL can be used to inhibit HSV1 infection, particularly keratitis. Further studies are needed to determine the active components and mechanism underlying the effects of SAL.

scholarly journals No evidence for aberrant expression of amyloid β and phosphorylated tau proteins in herpes simplex virus-infected human neurons in vivo

2021 ◽  
Author(s):  
Diana N Tran ◽  
Amy TCM Bakx ◽  
Vera van Dis ◽  
Eleonora Aronica ◽  
Robert M Verdijk ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Amyloid Β ◽  
Tau Proteins ◽  
Aberrant Expression ◽  
Human Neurons ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv 1

Increasing evidence implicates the neurotropic herpes simplex virus 1 (HSV-1) in the pathogenesis of Alzheimer's Disease (AD). However, it is unclear whether previously reported findings in HSV-1 cell culture and animal models can be translated to humans. Here, we analyzed clinical specimens from latently HSV-1 infected individuals and individuals with lytic HSV infection of the brain (herpes simplex encephalitis; HSE). Latent HSV-1 DNA load in trigeminal ganglia was identical between AD patients and controls, and latently HSV-infected neurons did not express amyloid-β (β) or hyperphosphorylated tau (pTau). Some HSE patient brains presented with ageing-related intraneuronal Aβ accumulations, neurofibrillary tangles (NFT) or extracellular Aβ plaques, but these were neither restricted to HSV-infected neurons nor brain regions containing virus-infected cells. Analysis of unique brain material from an AD patient with concurrent HSE showed that HSV-infected cells frequently localized close to Aβ plaques and NFT, but did not exacerbate AD-related pathology in relation to HSV infection. HSE-associated neuroinflammation was not associated with specific Aβ or pTau phenotypes. Collectively, the data indicate that neither latent nor lytic HSV infection of human neurons in vivo is directly associated with aberrant Aβ or pTau protein expression.

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