scholarly journals Downregulation of Class I Major Histocompatibility Complex Surface Expression by Varicella-Zoster Virus Involves Open Reading Frame 66 Protein Kinase-Dependent and -Independent Mechanisms

2007 ◽  
Vol 81 (17) ◽  
pp. 9034-9049 ◽  
Author(s):  
Amie J. Eisfeld ◽  
Michael B. Yee ◽  
Angela Erazo ◽  
Allison Abendroth ◽  
Paul R. Kinchington
Keyword(s):  
Major Histocompatibility Complex ◽  
Protein Kinase ◽  
Varicella Zoster Virus ◽  
Surface Expression ◽  
Open Reading Frame ◽  
Mhc I ◽  
Reading Frame ◽  
Varicella Zoster ◽  
Histocompatibility Complex ◽  
Infected Cells

ABSTRACT We show here that the varicella-zoster virus (VZV) open reading frame 66 (ORF66) protein kinase is one mechanism employed to reduce class I major histocompatibility complex (MHC-I) surface expression in VZV-infected cells. Cells expressing enhanced green fluorescent protein-tagged functional and inactivated ORF66 (GFP-66 and GFP-66kd) from replication-defective adenovirus vectors revealed that ORF66 reduced MHC-I surface levels in a manner dependent on kinase activity. Cells infected with recombinant VZV expressing GFP-66 exhibited a significantly greater reduction in MHC-I surface expression than that observed in cells infected with VZV disrupted in GFP-66 expression. MHC-I maturation was delayed in its transport from the endoplasmic reticulum through the Golgi in both adenovirus-transduced cells expressing only GFP-66 and in VZV-infected cells expressing high levels of GFP-66, and this was predominantly kinase dependent. MHC-I levels were reduced in VZV-infected cells, and analyses of intracellular MHC-I revealed accumulation of folded MHC-I in the Golgi region, irrespective of ORF66 expression. Thus, the ORF66 kinase is important for VZV-mediated MHC-I downregulation, but additional mechanisms also may be involved. Analyses of the VZV ORF9a protein, the ortholog of the bovine herpesvirus 1 transporter associated with antigen processing inhibitor UL49.5 revealed no effects on MHC-I. These results establish a new role for viral protein kinases in immune evasion and suggest that VZV utilizes unique mechanisms to inhibit antigen presentation.

scholarly journals Virion Association of IE62, the Varicella-Zoster Virus (VZV) Major Transcriptional Regulatory Protein, Requires Expression of the VZV Open Reading Frame 66 Protein Kinase

2001 ◽  
Vol 75 (19) ◽  
pp. 9106-9113 ◽  
Author(s):  
Paul R. Kinchington ◽  
Karen Fite ◽  
Amy Seman ◽  
Stephanie E. Turse
Keyword(s):  
Protein Kinase ◽  
Varicella Zoster Virus ◽  
Regulatory Protein ◽  
Open Reading Frame ◽  
Protein Kinase Activity ◽  
Virion Protein ◽  
Reading Frame ◽  
Varicella Zoster ◽  
Transcriptional Regulatory ◽  
Infected Cells

ABSTRACT IE62, the major transcriptional regulatory protein encoded by varicella-zoster virus (VZV), is associated with the tegument of gradient-purified virions. Here, we show that most, if not all, of the association requires the expression of open reading frame 66 (ORF66), a protein kinase. The association of IE62 with wild-type VZV virions was confirmed using immunoelectron microscopy with IE62-specific antibodies, which reacted with virions in ultrathin sections of VZV-infected cells. Fractionated purified virions from cells infected with recombinant VZV ROka contained substantial levels of the 175-kDa virion IE62 protein and also contained the ORF66 protein. However, virions from cells infected with recombinant VZV ROka66S, in which ORF66 is disrupted, lacked not only the ORF66 protein but also most of the virion 175-kDa IE62 polypeptide. The virion-associated protein kinase activity was still present in ROka66S virions, although the 175-kDa protein substrate for the virion kinase was absent, implying that the virion protein kinase is encoded by genes other than ORF66. The very low levels of IE62 in ROka66S virions indicate that ORF66 protein mediates the redistribution of IE62 to sites of tegument assembly. IE62 was resolved into several species from VZV-infected cells which showed mobility differences between ROka and ROka66S, and a specific form of IE62 was detected in ROka virions. These results are consistent with a role for the ORF66-mediated phosphorylation of IE62 that results in cytoplasmic distribution of the regulatory protein for tegument inclusion. They support a model in which VZV tegument acquisition occurs in the cytoplasm. As such, two unusual features of VZV IE62, namely, its virion inclusion and its phosphorylation and nuclear exclusion by the ORF66 protein kinase, are functionally linked.

scholarly journals Nuclear Accumulation of IE62, the Varicella-Zoster Virus (VZV) Major Transcriptional Regulatory Protein, Is Inhibited by Phosphorylation Mediated by the VZV Open Reading Frame 66 Protein Kinase

2000 ◽  
Vol 74 (5) ◽  
pp. 2265-2277 ◽  
Author(s):  
Paul R. Kinchington ◽  
Karen Fite ◽  
Stephanie E. Turse
Keyword(s):  
Protein Kinase ◽  
Varicella Zoster Virus ◽  
Nuclear Localization ◽  
Kinase Activity ◽  
Open Reading Frame ◽  
Nuclear Accumulation ◽  
Protein Kinase Activity ◽  
Reading Frame ◽  
Varicella Zoster ◽  
In Cells

ABSTRACT IE62, the major transcriptional activator protein encoded by varicella-zoster virus (VZV), locates to the nucleus when expressed in transfected cells. We show here that cytoplasmic forms of IE62 accumulate in transfected and VZV-infected cells as the result of the protein kinase activity associated with VZV open reading frame 66 (ORF66). Expression of the ORF66 protein kinase but not the VZV ORF47 protein kinase impaired the ability of coexpressed IE62 to transactivate promoter-reporter constructs. IE62 that was coexpressed with the ORF66 protein accumulated predominantly in the cytoplasm, whereas the normal nuclear localization of other proteins was not affected by the ORF66 protein. In cells infected with VZV, IE62 accumulated in the cytoplasm at late times of infection, whereas in cells infected with a VZV recombinant unable to express ORF66 protein (ROka66S), IE62 was completely nuclear. Point mutations introduced into the predicted serine/threonine catalytic domain and ATP binding domain of ORF66 abrogated its ability to influence IE62 nuclear localization, indicating that the protein kinase activity was required. The region of IE62 that was targeted by ORF66 was mapped to amino acids 602 to 733. IE62 peptides containing this region were specifically phosphorylated in cells coexpressing the ORF66 protein kinase and in cells infected with wild-type VZV but were not phosphorylated in cells infected with ROka66S. We conclude that the ORF66 protein kinase phosphorylates IE62 to induce its cytoplasmic accumulation, most likely by inhibiting IE62 nuclear import.

scholarly journals Varicella-Zoster Virus Retains Major Histocompatibility Complex Class I Proteins in the Golgi Compartment of Infected Cells

2001 ◽  
Vol 75 (10) ◽  
pp. 4878-4888 ◽  
Author(s):  
Allison Abendroth ◽  
Ines Lin ◽  
Barry Slobedman ◽  
Hidde Ploegh ◽  
Ann M. Arvin
Keyword(s):  
Major Histocompatibility Complex ◽  
T Lymphocytes ◽  
Cell Surface ◽  
Major Histocompatibility Complex Class ◽  
Complex Class ◽  
Mhc I ◽  
Varicella Zoster ◽  
Histocompatibility Complex ◽  
Golgi Compartment ◽  
Infected Cells

ABSTRACT We sought to examine the effects of varicella-zoster virus (VZV) infection on the expression of major histocompatibility complex class I (MHC I) molecules by human fibroblasts and T lymphocytes. By flow cytometry, VZV infection reduced the cell surface expression of MHC I molecules on fibroblasts significantly, yet the expression of transferrin receptor was not affected. Importantly, when human fetal thymus/liver implants in SCID-hu mice were inoculated with VZV, cell surface MHC I expression was downregulated specifically on VZV-infected human CD3+ T lymphocytes, a prominent target that sustains VZV viremia. The stage in the MHC I assembly process that was disrupted by VZV in fibroblasts was examined in pulse-chase and immunoprecipitation experiments in the presence of endoglycosidase H. MHC I complexes continued to be assembled in VZV-infected cells and were not retained in the endoplasmic reticulum. In contrast, immunofluorescence and confocal microscopy showed that VZV infection resulted in an accumulation of MHC I molecules which colocalized to the Golgi compartment. Inhibition of late viral gene expression by treatment of infected fibroblasts with phosphonoacetic acid did not influence the modulation of MHC I expression, nor did transfection of cells with plasmids expressing immediate early viral proteins. However, cells transfected with a plasmid carrying the early geneORF66 did result in a significant downregulation of MHC I expression, suggesting that this gene encodes a protein with an immunomodulatory function. Thus, VZV downregulates MHC I expression by impairing the transport of MHC I molecules from the Golgi compartment to the cell surface; this effect may enable the virus to evade CD8+ T-cell immune recognition during VZV pathogenesis, including the critical phase of T-lymphocyte-associated viremia.

scholarly journals Varicella-Zoster Virus (VZV) ORF32 Encodes a Phosphoprotein That Is Posttranslationally Modified by the VZV ORF47 Protein Kinase

1998 ◽  
Vol 72 (10) ◽  
pp. 8083-8088 ◽  
Author(s):  
Sanjay M. Reddy ◽  
Edward Cox ◽  
Ilya Iofin ◽  
Weily Soong ◽  
Jeffrey I. Cohen
Keyword(s):  
Protein Kinase ◽  
Varicella Zoster Virus ◽  
Protein Kinases ◽  
Gene Products ◽  
Osteosarcoma Cells ◽  
Reading Frame ◽  
Varicella Zoster ◽  
Intestine Alkaline Phosphatase ◽  
Infected Cells ◽  
Simplex Virus

ABSTRACT Varicella-zoster virus (VZV) encodes five gene products that do not have homologs in herpes simplex virus. One of these genes, VZV open reading frame 32 (ORF32), is predicted to encode a protein of 16 kDa. VZV ORF32 protein was shown to be phosphorylated and located in the cytosol of virus-infected cells. Antibody to ORF32 protein immunoprecipitated 16- and 18-kDa phosphoproteins from VZV-infected cells. Since VZV encodes two protein kinases that might phosphorylate ORF32 protein, immunoprecipitations were performed with cells infected with VZV mutants unable to express either of the viral protein kinases. Cells infected with VZV unable to express the ORF66 protein kinase contained both the 16- and 18-kDa ORF32 phosphoproteins; however, cells infected with the VZV ORF47 protein kinase mutant showed only the 16-kDa ORF32 phosphoprotein. Treatment of [35S]methionine-labeled proteins with calf intestine alkaline phosphatase resulted in a decrease in size of the ORF32 proteins from 16 and 18 kDa to 15 and 17 kDa, respectively. VZV unable to express ORF32 protein replicated in human melanoma cells to titers similar to those seen with parental virus; however, VZV unable to express ORF32 was impaired for replication in U20S osteosarcoma cells. Thus, VZV ORF32 protein is posttranslationally modified by the ORF47 protein kinase. Since the VZV ORF47 protein kinase has recently been shown to be critical for replication in human fetal skin and lymphocytes, its ability to modify the ORF32 protein suggests that the latter protein may have a role for VZV replication in human tissues.

scholarly journals Phosphorylation of the Varicella-Zoster Virus (VZV) Major Transcriptional Regulatory Protein IE62 by the VZV Open Reading Frame 66 Protein Kinase

2006 ◽  
Vol 80 (4) ◽  
pp. 1710-1723 ◽  
Author(s):  
Amie J. Eisfeld ◽  
Stephanie E. Turse ◽  
Sara A. Jackson ◽  
Edwina C. Lerner ◽  
Paul R. Kinchington
Keyword(s):  
Protein Kinase ◽  
Varicella Zoster Virus ◽  
Nuclear Import ◽  
Regulatory Protein ◽  
Recombinant Baculovirus ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Varicella Zoster ◽  
Transcriptional Regulatory

ABSTRACT IE62, the major transcriptional regulatory protein encoded by varicella-zoster virus (VZV), is nuclear at early times of VZV infection but then becomes predominantly cytoplasmic as a result of expression of the protein kinase encoded by open reading frame 66 (ORF66). Cytoplasmic forms of IE62 are required for its inclusion as an abundant VZV virion tegument protein. Here we show that ORF66 directly phosphorylates IE62 at two residues, with phosphorylation at S686 being sufficient to regulate IE62 nuclear import. Phosphotryptic peptide analyses established an ORF66 kinase-mediated phosphorylation of the complete IE62 protein in transfected and VZV-infected cells. Using truncated and point-mutated IE62 peptides, ORF66-directed phosphorylation was mapped to residues S686 and S722, immediately downstream of the IE62 nuclear localization signal. An IE62 protein with an S686A mutation retained efficient nuclear import activity, even in the presence of functional ORF66 protein kinase, but an IE62 protein containing an S686D alteration was imported into the nucleus inefficiently. In contrast, the nuclear import of IE62 carrying an S722A mutation was still modulated by ORF66 expression, and IE62 with an S722D mutation was imported efficiently into the nucleus. An in vitro phosphorylation assay was developed using bacterially expressed IE62-maltose binding protein fusions as substrates for immunopurified ORF66 protein kinase from recombinant baculovirus-infected insect cells. ORF66 kinase phosphorylated the IE62 peptides, with similar specificities for residues S686 and S722. These results indicate that IE62 nuclear import is modulated as a result of direct phosphorylation of IE62 by ORF66 kinase. This represents an interaction that is, so far, unique among the alphaherpesviruses.

scholarly journals Varicella-Zoster Virus Open Reading Frame 66 Protein Kinase Is Required for Efficient Viral Growth in Primary Human Corneal Stromal Fibroblast Cells

2008 ◽  
Vol 82 (15) ◽  
pp. 7653-7665 ◽  
Author(s):  
Angela Erazo ◽  
Michael B. Yee ◽  
Nikolaus Osterrieder ◽  
Paul R. Kinchington
Keyword(s):  
T Cells ◽  
Protein Kinase ◽  
Varicella Zoster Virus ◽  
Nuclear Import ◽  
Fluorescent Protein ◽  
Regulatory Protein ◽  
Open Reading Frame ◽  
Stromal Fibroblast ◽  
Reading Frame ◽  
Varicella Zoster

ABSTRACT Varicella-zoster virus (VZV) open reading frame 66 (ORF66) encodes a serine/threonine protein kinase that is not required for VZV growth in most cell types but is needed for efficient growth in T cells. The ORF66 kinase affects nuclear import and virion packaging of IE62, the major regulatory protein, and is known to regulate apoptosis in T cells. Here, we further examined the importance of ORF66 using VZV recombinants expressing green fluorescent protein (GFP)-tagged functional and kinase-negative ORF66 proteins. VZV virions with truncated or kinase-inactivated ORF66 protein were marginally reduced for growth and progeny yields in MRC-5 fibroblasts but were severely growth and replication impaired in low-passage primary human corneal stromal fibroblasts (PCF). To determine if the growth impairment was due to ORF66 kinase regulation of IE62 nuclear import, recombinant VZVs that expressed IE62 with alanine residues at S686, the suspected target by which ORF66 kinase blocks IE62 nuclear import, were made. IE62 S686A expressed by the VZV recombinant remained nuclear throughout infection and was not packaged into virions. However, the mutant virus still replicated efficiently in PCF cells. We also show that inactivation of the ORF66 kinase resulted in only marginally increased levels of apoptosis in PCF cells, which could not fully account for the cell-specific growth requirement of ORF66 kinase. Thus, the unique short region VZV kinase has important cell-type-specific functions that are separate from those affecting IE62 and apoptosis.

scholarly journals Open Reading Frame S/L of Varicella-Zoster Virus Encodes a Cytoplasmic Protein Expressed in Infected Cells

2000 ◽  
Vol 74 (23) ◽  
pp. 11311-11321 ◽  
Author(s):  
George W. Kemble ◽  
Paula Annunziato ◽  
Octavian Lungu ◽  
Ruth E. Winter ◽  
Tai-An Cha ◽  
...  
Keyword(s):  
Varicella Zoster Virus ◽  
Skin Lesions ◽  
Open Reading Frame ◽  
Plaque Morphology ◽  
Virus Reactivation ◽  
Wild Type ◽  
Reading Frame ◽  
Varicella Zoster ◽  
L Protein ◽  
Infected Cells

ABSTRACT We report the discovery of a novel gene in the varicella-zoster virus (VZV) genome, designated open reading frame (ORF) S/L. This gene, located at the left end of the prototype VZV genome isomer, expresses a polyadenylated mRNA containing a splice within the 3′ untranslated region in virus-infected cells. Sequence analysis reveals significant differences between the ORF S/Ls of wild-type and attenuated strains of VZV. Antisera raised to a bacterially expressed portion of ORF S/L reacted specifically with a 21-kDa protein synthesized in cells infected with a VZV clinical isolate and with the original vaccine strain of VZV (Oka-ATCC). Cells infected with other VZV strains, including a wild-type strain that has been extensively passaged in tissue culture and commercially produced vaccine strains of Oka, synthesize a family of proteins ranging in size from 21 to 30 kDa that react with the anti-ORF S/L antiserum. MeWO cells infected with recombinant VZV harboring mutations in the C-terminal region of the ORF S/L gene lost adherence to the stratum and adjacent cells, resulting in an altered plaque morphology. Immunohistochemical analysis of VZV-infected cells demonstrated that ORF S/L protein localizes to the cytoplasm. ORF S/L protein was present in skin lesions of individuals with primary or reactivated infection and in the neurons of a dorsal root ganglion during virus reactivation.

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