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See detailPhosphorylation of varicella-zoster virus IE63 protein by casein kinases influences its cellular localization and gene regulation activity
Bontems, Sébastien ULg; Di Valentin, Emmanuel ULg; Baudoux, Laurence et al

in Journal of Biological Chemistry (2002), 277(23), 21050-21060

During the early phase of varicella-zoster virus (VZV) infection, Immediate Early protein 63 (IE63) is expressed rapidly and abundantly in the nucleus, while during latency, this protein is confined ... [more ▼]

During the early phase of varicella-zoster virus (VZV) infection, Immediate Early protein 63 (IE63) is expressed rapidly and abundantly in the nucleus, while during latency, this protein is confined mostly to the cytoplasm. Because phosphorylation is known to regulate many cellular events, we investigated the importance of this modification on the cellular localization of IE63 and on its regulatory properties. We demonstrate here that cellular casein kinases I and II are implicated in the in vitro and in vivo phosphorylation of IE63. A mutational approach also indicated that phosphorylation of the protein is important for its correct cellular localization in a cell type-dependent fashion. Using an activity test, we demonstrated that IE63 was able to repress the gene expression driven by two VZV promoters and that phosphorylation of the protein was required for its full repressive properties. Finally, we showed that IE63 was capable of exerting its repressive activity in the cytoplasm, as well as in the nucleus, suggesting a regulation at the transcriptional and/or post-transcriptional level. [less ▲]

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See detailGene activation by varicella-zoster virus IE4 protein requires its dimerization and involves both the arginine-rich sequence, the central part, and the carboxyl-terminal cysteine-rich region
Baudoux, Laurence; Defechereux, Patricia; Rentier, Bernard ULg et al

in Journal of Biological Chemistry (2000), 275(42), 32822-32831

Varicella-zoster virus (VZV) open reading frame 4-encoded protein (IE4) possesses transactivating properties for VZV genes as well as for those of heterologous viruses. Since most transcription factors ... [more ▼]

Varicella-zoster virus (VZV) open reading frame 4-encoded protein (IE4) possesses transactivating properties for VZV genes as well as for those of heterologous viruses. Since most transcription factors act as dimers, IE4 dimerization was studied using the mammalian two-hybrid system. Introduction of mutations in the IE4 open reading frame demonstrated that both the central region and the carboxyl-terminal cysteine-rich domain were important for efficient dimerization. Within the carboxyl-terminal domain, substitution of amino acids encompassing residues 443-447 totally abolished dimerization. Gene activation by IE4 was studied by transient transfection with an IE4 expression plasmid and a reporter gene under the control of either the human immunodeficiency virus, type 1, long terminal repeat or the VZV thymidine kinase promoter. Regions of IE4 important for dimerization were also shown to be crucial for transactivation. In addition, the arginine-rich domains Rb and Re of the amino-terminal region were also demonstrated to be important for transactivation, whereas the Ra domain as well as an acidic and bZIP-containing regions were shown to be dispensable for gene transactivation. A nucleocytoplasmic shuttling of IE4 has also been characterized, involving a nuclear localization signal identified within the Rb domain and a nuclear export mechanism partially depending on Crm-1. [less ▲]

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See detailOverview of the replication cycle of varicella-zoster virus
Sadzot-Delvaux, Catherine ULg; Baudoux, Laurence; Defechereux, Patricia et al

in Schmidt, A.; Wolff, M. H.; Schünemann, S. (Eds.) Varicella-Zoster Virus : Molecular Biology, Pathogenesis and Clinical Aspects (1999)

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See detailVaricella-zoster virus open reading frame 4 encodes an immediate-early protein with posttranscriptional regulatory properties
Defechereux, Patricia; Debrus, Serge; Baudoux, Laurence et al

in Journal of Virology (1997), 71(9), 7073-7079

Varicella-zoster virus (VZV) encodes four putative immediate-early proteins based on sequence homology with herpes simplex virus type I: the products of ORF4, -61, -62, and -63. Until now, only two VZV ... [more ▼]

Varicella-zoster virus (VZV) encodes four putative immediate-early proteins based on sequence homology with herpes simplex virus type I: the products of ORF4, -61, -62, and -63. Until now, only two VZV proteins have been described as being truly expressed with immediate-early kinetics (IE62 and IE63). The ORF4-encoded protein can stimulate gene expression either alone or in synergy with the major regulatory protein IE62. Making use of a sequential combination of transcription and protein synthesis inhibitors (actinomycin D and cycloheximide, respectively), we demonstrated the immediate-early nature of the ORF4 gene product, which can thus be named IE4. The fact that IE4 is expressed with kinetics similar to that of IE62 further underlines the possible cooperation between these two VZV proteins in gene expression. Analysis of the IE4-mediated autologous or heterologous viral gene expression at the mRNA levels clearly indicated that IE4 may have several mechanisms of action. Activation of the two VZV genes tested could occur partly by a posttranscriptional mechanism, since increases in chloramphenicol acetyltransferase (CAT) mRNA levels do not account for the stimulation of CAT activity. On the other hand, stimulation of the human immunodeficiency virus type 1 long terminal repeat-or the cytomegalovirus promoter-associated CAT activity is correlated with an increase in the corresponding CAT mRNA. [less ▲]

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See detailVaricella-zoster virus immediate early proteins 4 and 62 interact with cellular transcription factors
Defechereux, Patricia; Baudoux, Laurence; Rentier, Bernard ULg et al

Poster (1997)

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See detailBinding sites analysis of the major varicella-zoster virus regulatory IE62 protein
Baudoux, Laurence; Remacle, V.; Defechereux, Patricia et al

Poster (1997)

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See detailLessons to be learned from varicella-zoster virus
Rentier, Bernard ULg; Piette, Jacques ULg; Baudoux, Laurence et al

in Veterinary Microbiology (1996), 53(1-2), 55-66

Varicella-zoster virus (VZV) is an alphaherpesvirus responsible for two human diseases: chicken pox and shingles. The virus has a respiratory port of entry. After two successive viremias, it reaches the ... [more ▼]

Varicella-zoster virus (VZV) is an alphaherpesvirus responsible for two human diseases: chicken pox and shingles. The virus has a respiratory port of entry. After two successive viremias, it reaches the skin where it causes typical lesions. There, it penetrates the peripheral nervous system and it remains latent in dorsal root ganglia. It is still debatable whether VZV persists in neurons or in satellite cells. During latency, VZV expresses a limited set of transcripts of its immediate early (IE) and early (E) genes but no protein has been detected. Mechanisms of reactivation from ganglia have not been identified. However, dysfunction of the cellular immune system appears to be involved in this process. The cell-associated nature of VZV has made it difficult to identify a temporal order of gene expression, but there appears to be a cascade mechanism as for HSV-1. The lack of high titre cell-free virions or recombination mutants has hindered so far the understanding of VZV gene functions. Five genes, ORFs 4, 10, 61, 62, and 63 that encode regulatory proteins could be involved in VZV latency. ORF4p activates gene promoters with basal activities. ORF10p seems to activate the ORF 62 promoter. ORF61p has trans-activating and trans-repressing activities. The major IE protein ORF62p, a virion component, has DNA-binding and regulatory functions, transactivates many VZV promoters and even regulates its own expression. ORF63p is a nuclear IE protein of yet unclear regulatory functions, abundantly expressed very early in infection. We have established an animal model of VZV latency in the rat nervous system, enabling us to study the expression of viral mRNA and protein expression during latency, and yielding results similar to those found in humans. This model is beginning to shed light on the molecular events in VZV persistent infection and on the regulatory mechanisms that maintain the virus in a latent stage in nerve cells. [less ▲]

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See detailEnhancement of Varicella-Zoster virus infection in cell lines expressing ORF4- or ORF62-encoded proteins
Schoonbroodt, Sonia; Piette, Jacques ULg; Baudoux, Laurence et al

in Journal of Medical Virology (1996), 49(4), 264-273

Varicella-Zoster virus (VZV) open reading frames 4 (ORF4) and 62 (ORF62) encode putative immediate-early proteins (ORF4p and ORF62p, respectively) which are strong transactivators of other VZV genes and ... [more ▼]

Varicella-Zoster virus (VZV) open reading frames 4 (ORF4) and 62 (ORF62) encode putative immediate-early proteins (ORF4p and ORF62p, respectively) which are strong transactivators of other VZV genes and are involved in the very early stages of viral infection. ORF4p and ORF62p transactivate immediate-early and early gene promoters but have little or no effect on late gene promoters. To investigate the effect of ORF4p or ORF62p overexpression on the viral replication cycle, we constructed Vero cell lines expressing those genes under the control of the human cytomegalovirus major immediate-early promoter. VZV OKA infection of these stably transformed cell lines was followed-up using VZV glycoprotein E (gE) antigen quantification and virus titration. Upon serial passaging of infection in these cell lines expressing functionally active ORF4p or ORF62p, a 5- to 10-fold increase in viral gE antigen production was observed. Viral titers also demonstrated a 2- to 5-fold increase in viral production in these transformed cell lines. These results emphasize the role that both ORF4p and ORF62p play in enhancing the VZV replicative cycle. (C) 1996 Wiley-Liss, Inc. [less ▲]

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See detailIntracellular distribution of the ORF4 gene product of varicella-zoster virus is influenced by the IE62 protein
Defechereux, Patricia; Debrus, Serge; Baudoux, Laurence et al

in Journal of General Virology (1996), 77(Part 7), 1505-1513

Varicella-zoster virus (VZV) open reading frame 4-encoded protein (IE4) possesses transactivating properties for VZV genes as well as for genes of heterologous viruses, The major regulatory immediate ... [more ▼]

Varicella-zoster virus (VZV) open reading frame 4-encoded protein (IE4) possesses transactivating properties for VZV genes as well as for genes of heterologous viruses, The major regulatory immediate-early protein of VZV (IE62) is a transactivator of VZV gene expression, In transfection assays, IE4 has been shown to enhance activation induced by IE62, To investigate the functional interactions underlying this observation, indirect immunofluorescence studies were undertaken to determine whether IE62 could influence IE4 intracellular localization in transfected cells, In single transfections, IE4 was predominantly found in cytoplasm, In cotransfection with IE62, the IE4 localization pattern was altered, with nuclear staining predominating over cytoplasmic staining, This effect was specific to the IE62 protein since the gene products of ORF63 and ORF61, which are also regulatory proteins, did not influence IE4 distribution, The use of IE62 mutants indicated that IE62 influence is independent of its transactivation function and that the integrity of regions 3 and 4 is required, IE62 remained nuclear whether IE4 was present or not, These observations underline differences in the regulation of gene expression between VZV proteins and their herpes simplex virus type 1 homologues, In infected cells, IE4 was only sometimes found to colocalize with IE62 in nuclei, This observation suggests that when all VZV proteins are present, complex interactions probably occur which could diminish the influence of IE62. [less ▲]

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See detailAnalysis of VZV IE62 protein DNA binding sites on VZV gene promoters
Baudoux, Laurence; Remacle, V.; Defechereux, Patricia et al

Conference (1996)

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See detailMutational analysis of varicella-zoster virus major immediate-early protein IE62
Baudoux, Laurence; Defechereux, Patricia; Schoonbroodt, Sonia et al

in Nucleic Acids Research (1995), 23(8), 1341-1349

The varicella-zoster virus (VZV) open reading frame 62 encodes an immediate-early protein (IE62) that transactivates expression of various VZV promoters and autoregulates its own expression in transient ... [more ▼]

The varicella-zoster virus (VZV) open reading frame 62 encodes an immediate-early protein (IE62) that transactivates expression of various VZV promoters and autoregulates its own expression in transient expression assays. In Vero cells, IE62 was shown to transactivate the expression of all putative immediate-early (IE) and early (E) genes of VZV with an up-regulating effect at low intracellular concentrations. To define the functional domains involved in the regulatory properties of IE62, a large number of in-frame insertions and deletions were introduced into a plasmid-borne copy of the gene encoding IE62. Studies of the regulatory activities of the resultant mutant polypeptides in transient expression assays allowed to delineate protein regions important for repression of its own promoter and for transactivation of a VZV putative immediate-early gene (ORF61) promoter and an early gene (ORF29) promoter. This mutational analysis resulted in the identification of a new functional domain situated at the border between regions 4 and 5 which plays a crucial role in the IE62 regulatory functions. This domain turned out to be very well conserved amongst homologous alphaherpesvirus regulatory proteins and appeared to be rich in bulky hydrophobic and proline residues, similar to the proline-rich region of the CAAT box binding protein CTF-1. By immunofluorescence, a nuclear localization signal has been mapped in region 3. [less ▲]

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See detailVaricella-zoster virus gene regulation
Piette, Jacques ULg; Defechereux, Patricia; Baudoux, Laurence et al

in Neurology (1995), 45(12, suppl. 8), 23-27

The varicella-zoster virus genome contains 71 open reading frames (ORFs), five of which (ORF62, ORF4, ORF63, ORF61, and ORF10) encode regulatory proteins. ORF62 codes for the major immediate early protein ... [more ▼]

The varicella-zoster virus genome contains 71 open reading frames (ORFs), five of which (ORF62, ORF4, ORF63, ORF61, and ORF10) encode regulatory proteins. ORF62 codes for the major immediate early protein of the virus exhibiting DNA-binding and regulatory functions. This protein, localized in the cell nucleus, is a functional homologue to ICP4 of herpes simplex virus type 1 (HSV-1). It trans-activates several varicella-zoster virus promoters of the various gene classes and autoregulates its own expression. ORF4 protein activates gene promoters provided they have basal activities, but it is not a functional homologue of HSV-1 ICP27. Gene regulation activity appears to be linked to its cysteine-rich C-terminal region. ORF63 codes for an immediate early protein mainly located in the cell nucleus. The regulatory functions it performs are still unclear. ORF61 protein is the functional homologue of HSV-1 ICPO. Its N-terminal region exhibits a RING domain responsible for trans-activating and trans-repressing activities. ORF10 protein exhibits similarities with HSV-1 VP16 and activates the ORF62 promoter. [less ▲]

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