The TATA Motif Specifies the Differential Activation of Minimal Promoters by Varicella Zoster Virus Immediate-early Regulatory Protein IE62

ABSTRACT Varicella-zoster virus (VZV) causes varicella and establishes latency in sensory nerve ganglia, but the characteristics of VZV latency are not well defined. Immunohistochemical detection of the VZV immediate-early 63 (IE63) protein in ganglion neurons has been described, but there are significant discrepancies in estimates of the frequency of IE63-positive neurons, varying from a rare event to abundant expression. We examined IE63 expression in cadaver ganglia using a high-potency rabbit anti-IE63 antibody and corresponding preimmune serum. Using standard immunohistochemical techniques, we evaluated 10 ganglia that contained VZV DNA from seven individuals. These experiments showed that neuronal pigments were a confounding variable; however, by examining sections coded to prevent investigator bias and applying statistical analysis, we determined that IE63 protein, if present, is in a very small proportion of neurons (<2.8%). To refine estimates of IE63 protein abundance, we modified our protocol by incorporating a biological stain to exclude the pigment signal and evaluated 27 ganglia from 18 individuals. We identified IE63 protein in neurons within only one ganglion, in which VZV glycoprotein E and an immune cell infiltrate were also demonstrated. Antigen preservation was shown by detection of neuronal synaptophysin. These data provide evidence that the expression of IE63 protein, which has been referred to as a latency-associated protein, is rare. Refining estimates of VZV protein expression in neurons is important for developing a hypothesis about the mechanisms by which VZV latency may be maintained.

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The Immediate-Early 63 Protein of Varicella-Zoster Virus: Analysis of Functional Domains Required for Replication In Vitro and for T-Cell and Skin Tropism in the SCIDhu Model In Vivo

Journal of Virology ◽

10.1128/jvi.78.3.1181-1194.2004 ◽

2004 ◽

Vol 78 (3) ◽

pp. 1181-1194 ◽

Cited By ~ 40

Author(s):

Armin Baiker ◽

Christoph Bagowski ◽

Hideki Ito ◽

Marvin Sommer ◽

Leigh Zerboni ◽

...

Keyword(s):

T Cell ◽

Varicella Zoster Virus ◽

Nuclear Localization ◽

Regulatory Protein ◽

Immediate Early ◽

Functional Domains ◽

Reading Frame ◽

Varicella Zoster

ABSTRACT The immediate-early 63-kDa (IE63) protein of varicella-zoster virus (VZV) is a phosphoprotein encoded by open reading frame (ORF) ORF63/ORF70. To identify functional domains, 22 ORF63 mutations were evaluated for effects on IE63 binding to the major VZV transactivator, IE62, and on IE63 phosphorylation and nuclear localization in transient transfections, and after insertion into the viral genome with VZV cosmids. The IE62 binding site was mapped to IE63 amino acids 55 to 67, with R59/L60 being critical residues. Alanine substitutions within the IE63 center region showed that S165, S173, and S185 were phosphorylated by cellular kinases. Four mutations that changed two putative nuclear localization signal (NLS) sequences altered IE63 distribution to a cytoplasmic/nuclear pattern. Only three of 22 mutations in ORF63 were compatible with recovery of infectious VZV from our cosmids, but infectivity was restored by inserting intact ORF63 into each mutated cosmid. The viable IE63 mutants had a single alanine substitution, altering T171, S181, or S185. These mutants, rOKA/ORF63rev[T171], rOKA/ORF63rev[S181], and rOKA/ORF63rev[S185], produced less infectious virus and had a decreased plaque phenotype in vitro. ORF47 kinase protein and glycoprotein E (gE) synthesis was reduced, indicating that IE63 contributed to optimal expression of early and late gene products. The three IE63 mutants replicated in skin xenografts in the SCIDhu mouse model, but virulence was markedly attenuated. In contrast, infectivity in T-cell xenografts was not altered. Comparative analysis suggested that IE63 resembled the herpes simplex virus type 1 US1.5 protein, which is expressed colinearly with ICP22 (US1). In summary, most mutations of ORF63 made with our VZV cosmid system were lethal for infectivity. The few IE63 changes that were tolerated resulted in VZV mutants with an impaired capacity to replicate in vitro. However, the IE63 mutants were attenuated in skin but not T cells in vivo, indicating that the contribution of the IE63 tegument/regulatory protein to VZV pathogenesis depends upon the differentiated human cell type which is targeted for infection within the intact tissue microenvironment.

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Functional Characterization of the Serine-Rich Tract of Varicella-Zoster Virus IE62

Journal of Virology ◽

10.1128/jvi.02096-15 ◽

2015 ◽

Vol 90 (2) ◽

pp. 959-971 ◽

Cited By ~ 4

Author(s):

Seong K. Kim ◽

Akhalesh K. Shakya ◽

Seongman Kim ◽

Dennis J. O'Callaghan

Keyword(s):

Ribosomal Protein ◽

Varicella Zoster Virus ◽

Functional Characterization ◽

Regulatory Protein ◽

Chimeric Protein ◽

Immediate Early ◽

Viral Gene ◽

Activation Domain ◽

Varicella Zoster ◽

Hsv 1

ABSTRACTThe immediate early 62 protein (IE62) of varicella-zoster virus (VZV), a major viraltrans-activator, initiates the virus life cycle and is a key component of pathogenesis. The IE62 possesses several domains essential fortrans-activation, including an acidictrans-activation domain (TAD), a serine-rich tract (SRT), and binding domains for USF, TFIIB, and TATA box binding protein (TBP). Transient-transfection assays showed that the VZV IE62 lacking the SRTtrans-activated the early VZV ORF61 promoter at only 16% of the level of the full-length IE62. When the SRT of IE62 was replaced with the SRT of equine herpesvirus 1 (EHV-1) IEP, itstrans-activation activity was completely restored. Herpes simplex virus 1 (HSV-1) ICP4 that lacks a TAD very weakly (1.5-fold)trans-activated the ORF61 promoter. An IE62 TAD-ICP4 chimeric protein exhibitedtrans-activation ability (10.2-fold), indicating that the IE62 TAD functions with the SRT of HSV-1 ICP4 totrans-activate viral promoters. When the serine and acidic residues of the SRT were replaced with Ala, Leu, and Gly,trans-activation activities of the modified IE62 proteins IE62-SRTΔSe and IE62-SRTΔAc were reduced to 46% and 29% of wild-type activity, respectively. Bimolecular complementation assays showed that the TAD of IE62, EHV-1 IEP, and HSV-1 VP16 interacted with Mediator 25 in human melanoma MeWo cells. The SRT of IE62 interacted with the nucleolar-ribosomal protein EAP, which resulted in the formation of globular structures within the nucleus. These results suggest that the SRT plays an important role in VZV viral gene expression and replication.IMPORTANCEThe immediate early 62 protein (IE62) of varicella-zoster virus (VZV) is a major viraltrans-activator and is essential for viral growth. Our data show that the serine-rich tract (SRT) of VZV IE62, which is well conserved within the alphaherpesviruses, is needed fortrans-activation mediated by the acidictrans-activation domain (TAD). The TADs of IE62, EHV-1 IEP, and HSV-1 VP16 interacted with cellular Mediator 25 in bimolecular complementation assays. The interaction of the IE62 SRT with nucleolar-ribosomal protein EAP resulted in the formation of globular structures within the nucleus. Understanding the mechanisms by which the TAD and SRT of IE62 contribute to the function of this essential regulatory protein is important in understanding the gene program of this human pathogen.

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The varicella–zoster virus–mediated delayed host shutoff: open reading frame 17 has no major function, whereas immediate–early 63 protein represses heterologous gene expression

Microbes and Infection ◽

10.1016/j.micinf.2005.05.010 ◽

2005 ◽

Vol 7 (15) ◽

pp. 1519-1529 ◽

Cited By ~ 12

Author(s):

Nathalie Desloges ◽

Markus Rahaus ◽

Manfred H. Wolff

Keyword(s):

Gene Expression ◽

Varicella Zoster Virus ◽

Heterologous Gene Expression ◽

Open Reading Frame ◽

Heterologous Gene ◽

Immediate Early ◽

Reading Frame ◽

Varicella Zoster ◽

Major Function ◽

Host Shutoff

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Erratum to: Immunological evasion of immediate-early varicella zoster virus proteins

Immunogenetics ◽

10.1007/s00251-016-0912-3 ◽

2016 ◽

Vol 68 (6-7) ◽

pp. 487-487

Author(s):

Pieter Meysman ◽

Dmitry Fedorov ◽

Viggo Van Tendeloo ◽

Benson Ogunjimi ◽

Kris Laukens

Keyword(s):

Varicella Zoster Virus ◽

Immediate Early ◽

Varicella Zoster ◽

Virus Proteins

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The cellular transcription factor USF cooperates with varicella-zoster virus immediate-early protein 62 to symmetrically activate a bidirectional viral promoter

Molecular and Cellular Biology ◽

10.1128/mcb.14.10.6896-6906.1994 ◽

1994 ◽

Vol 14 (10) ◽

pp. 6896-6906 ◽

Cited By ~ 3

Author(s):

J L Meier ◽

X Luo ◽

M Sawadogo ◽

S E Straus

Keyword(s):

Dna Binding ◽

Varicella Zoster Virus ◽

Dominant Negative ◽

Immediate Early ◽

Activation Process ◽

Viral Promoter ◽

Varicella Zoster ◽

Cellular Transcription Factor ◽

Early Protein ◽

Target Promoters

The mechanisms governing the function of cellular USF and herpesvirus immediate-early transcription factors are subjects of considerable interest. In this regard, we identified a novel form of coordinate gene regulation involving a cooperative interplay between cellular USF and the varicella-zoster virus immediate-early protein 62 (IE 62). A single USF-binding site defines the potential level of IE 62-dependent activation of a bidirectional viral early promoter of the DNA polymerase and major DNA-binding protein genes. We also report a dominant negative USF-2 mutant lacking the DNA-binding domain that permits the delineation of the biological role of both USF-1 and USF-2 in this activation process. The symmetrical stimulation of the bidirectional viral promoter by IE 62 is achieved at concentrations of USF-1 (43 kDa) or USF-2 (44 kDa) already existing in cells. Our observations support the notion that cellular USF can intervene in and possibly target promoters for activation by a herpesvirus immediate-early protein.

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