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Biomedical subjects

A Sergeant

Publications and source records attributed to A Sergeant.

At least 19 recordsLinked to original sources
PubMed2002

MEF2-mediated recruitment of class II HDAC at the EBV immediate early gene BZLF1 links latency and chromatin remodeling.

In B lymphocytes induced to proliferate in vitro by the Epstein-Barr virus (EBV), extra-chromosomal viral episomes packaged in chromatin persist in the nucleus, and there is no productive cycle. A switch from this latency to the productive cycle is observed after induced expression of the EBV BZLF1 gene product, the transcription factor EB1. We present evidence that, during latency, proteins of the myocyte enhancer binding factor 2 (MEF2) family are bound to the BZLF1 promoter and recruit class II histone deacetylases. Furthermore, we propose that latency is determined primarily by a specific and local recruitment of class II histone deacetylase (HDAC) by MEF2D to the BZLF1 gene promoter. The switch from latency to the productive cycle could be due in part to post-translational modification of MEF2 proteins and changes in the local acetylation state of the chromatin.

B-Lymphocytes↗
PubMed2000

The herpes simplex virus 1 Us11 protein cooperates with suboptimal amounts of human immunodeficiency virus type 1 (HIV-1) Rev protein to rescue HIV-1 production.

The human immunodeficiency virus type 1 (HIV-1) RNA-binding Rev protein governs the expression of structural and enzymatic viral proteins at a posttranscriptional level. Binding of Rev to the stem-loop IIB (SLIIB) sequence of the Rev-response element (RRE) within unspliced and singly spliced viral mRNAs and to the nuclear export signal-binding receptor, hCRM1 (or exportin 1), is required for the export of these transcripts to the cytoplasm. We have previously shown that herpes simplex virus type 1 (HSV-1) RNA-binding Us11 protein is able to bind the RRE and substitute for Rev in inducing the expression of HIV-1 envelope glycoproteins. We show here that Us11 cannot substitute for Rev in rescuing a rev-deleted HIV-1 provirus. However, HIV-1 production is observed when Us11 is expressed with suboptimal amounts of Rev. An in vivo RNA-protein binding assay indicates that Us11 is unable to directly interact with the SLIIB RNA but can bind Rev assembled on that stem-loop structure. This association of US11 with Rev, which was confirmed by in vivo coimmunoprecipitation and GST-pulldown assays, therefore underlies a biological Us11-Rev cooperation. Furthermore this cooperation was shown to remain susceptible to the effect of leptomycin B, which blocks the binding of hCRM1 to the nuclear export signal of Rev. These observations performed with intron-containing constructs provide evidence that HSV-1 Us11 protein is not directly involved in the cytoplasmic accumulation of viral mRNAs but may be rather acting as an auxiliary protein, thus allowing this retroviral protein to fulfill the nuclear export of these transcripts and to rescue HIV-1 production.

Biological Transport↗
PubMed2000

Ubinuclein, a novel nuclear protein interacting with cellular and viral transcription factors.

The major target tissues for Epstein-Barr virus (EBV) infection are B lymphocytes and epithelial cells of the oropharyngeal zone. The product of the EBV BZLF1 early gene, EB1, a member of the basic leucine-zipper family of transcription factors, interacts with both viral and cellular promoters and transcription factors, modulating the reactivation of latent EBV infection. Here, we characterize a novel cellular protein interacting with the basic domains of EB1 and c-Jun, and competing of their binding to the AP1 consensus site. The transcript is present in a wide variety of human adult, fetal, and tumor tissues, and the protein is detected in the nuclei throughout the human epidermis and as either grainy or punctuate nuclear staining in the cultured keratinocytes. The overexpression of tagged cDNA constructs in keratinocytes revealed that the NH(2) terminus is essential for the nuclear localization, while the central domain is responsible for the interaction with EB1 and for the phenotype of transfected keratinocytes similar to terminal differentiation. The gene was identified in tail-to-tail orientation with the periplakin gene (PPL) in human chromosome 16p13.3 and in a syntenic region in mouse chromosome 16. We designated this novel ubiquitously expressed nuclear protein as ubinuclein and the corresponding gene as UBN1.

Adult↗
PubMed2000

Kaposi's sarcoma-associated herpesvirus and Kaposi's sarcoma.

Kaposi's sarcoma-associated herpesvirus (KSHV) is present in all epidemiologic forms of Kaposi's sarcoma (KS). The KSHV genome contains several open reading frames which are potentially implicated in the development of KS. Some are unique to KSHV; others are homologous to cellular genes. The putative role of these genes in the genesis of KS is discussed.

Cell Transformation, Viral↗
PubMed2000

Characterization of the epstein-barr virus BRRF1 gene, located between early genes BZLF1 and BRLF1.

The switch from latency to a productive cycle in Epstein-Barr virus (EBV)-infected B cells proliferating in vitro is thought to be due to the transcriptional activation of two viral genes, BZLF1 and BRLF1, encoding two transcription factors called EB1 and R respectively. However, a third gene, BRRF1 is contained in the BZLF1/BRLF1 locus, overlapping with BRLF1 but in inverse orientation. We have characterized the 5' end of the BRRF1 mRNA and the promoter, PNa, at which BRRF1 pre-mRNA is initiated. We show that although a single BRRF1 mRNA species is induced by 12-O-tetradecanoylphorbol 13-acetate/sodium butyrate in several EBV-infected B cell lines, in Akata cells treated with anti-IgG two BRRF1 mRNAs can be detected. Transcription initiated at the BRRF1 promoter was activated by EB1 but not by R, and EB1-binding sites which contribute to the EB1-activated transcription have been mapped to between positions -469 and +1. A 34 kDa protein could be translated from the BRRF1 mRNA both in vitro and in vivo, and was found predominantly in the nucleus of HeLa cells transfected with a BRRF1 expression vector. Thus there are three promoters in the region of the EBV chromatin containing the BZLF1/BRLF1 genes, two of which, PZ and PNa, potentially share regulatory elements.

Animals↗
PubMed2000

Epstein-Barr virus EB2 protein exports unspliced RNA via a Crm-1-independent pathway.

Human herpesviruses encode posttranscriptional activators that are believed to up-regulate viral replication by facilitating early and late gene expression. We have reported previously that the Epstein-Barr virus protein EB2 (also called M or SM) promotes nuclear export of RNAs that are poor substrates for spliceosome assembly, an effect that closely resembles the human immunodeficiency virus type 1 Rev-dependent nuclear export of unspliced viral RNA. Here we present experimental data showing that EB2 efficiently promotes the nuclear export of unspliced RNA expressed from a Rev reporter construct. Site-directed mutagenesis as well as domain swapping experiments indicate that a leucine-rich region found in the EB2 protein, which matches the consensus sequence for the leucine-rich nuclear export signal, is not a nuclear export signal per se. Accordingly, leptomycin B (LMB), a specific Crm-1 inhibitor, impairs Rev- but not EB2-dependent nuclear export of unspliced RNA. Moreover, EB2 nucleocytoplasmic shuttling visualized by a heterokaryon assay is, unlike Rev shuttling, not affected by LMB. We also show that overexpression of an N-terminal deletion mutant of Nup214/can, a major nucleoporin of the nuclear pore complex involved in several aspects of nuclear transport, blocks both Rev- and EB2-dependent nuclear export of RNA. These results strongly suggest that EB2 nuclear export of unspliced RNA is mediated by a Crm-1-independent pathway.

3T3 Cells↗
PubMed1999

A new nucleoporin-like protein interacts with both HIV-1 Rev nuclear export signal and CRM-1.

The HIV-1 Rev is a shuttling protein required for the nuclear export of unspliced and partially spliced viral mRNA. In this study, we have identified a new Rev-interacting protein, that specifically interacts with the Rev nuclear export signal both in yeast and mammalian cells. This protein has features found in nucleoporins including many phenylalanine-glycine repeats, a very high serine content, a putative zinc finger, and a coiled-coil domain; we thus called it NLP-1 (nucleoporin-like protein 1). In addition, gene expression analysis and wheat germ agglutinin chromatography experiments suggested that NLP-1 is an ubiquitous O-glycosylated nuclear protein. Recently, a cellular factor called CRM-1 has been shown to be an essential nuclear export factor interacting directly with nuclear export signals including the Rev nuclear export signal in a RanGTP-dependent manner. We show here that NLP-1, like the previously described Rev-interacting protein hRIP/Rab and several nucleoporins, also interacts with CRM-1 both in yeast and mammalian cells.

Binding Sites↗
PubMed1999

Characterization of the cDNA coding for rat brain cysteine sulfinate decarboxylase: brain and liver enzymes are identical proteins encoded by two distinct mRNAs.

Cysteine sulfinate decarboxylase (CSD) is considered as the rate-limiting enzyme in the biosynthesis of taurine, a possible osmoregulator in brain. Through cloning and sequencing of RT-PCR and RACE-PCR products of rat brain mRNAs, a 2,396-bp cDNA sequence was obtained encoding a protein of 493 amino acids (calculated molecular mass, 55.2 kDa). The corresponding fusion protein showed a substrate specificity similar to that of the endogenous enzyme. The sequence of the encoded protein is identical to that encoded by liver CSD cDNA. Among other characterized amino acid decarboxylases, CSD shows the highest homology (54%) with either isoform of glutamic acid decarboxylase (GAD65 and GAD67). A single mRNA band, approximately 2.5 kb, was detected by northern blot in RNA extracts of brain, liver, and kidney. However, brain and liver CSD cDNA sequences differed in the 5' untranslated region. This indicates two forms of CSD mRNA. Analysis of PCR-amplified products of genomic DNA suggests that the brain form results from the use of a 3' alternative internal splicing site within an exon specifically found in liver CSD mRNA. Through selective RT-PCR the brain form was detected in brain only, whereas the liver form was found in liver and kidney. These results indicate a tissue-specific regulation of CSD genomic expression.

3' Untranslated Regions↗
PubMed1999

Kaposi's sarcoma-associated herpesvirus (human herpesvirus-8) encodes a homologue of the Epstein-Barr virus bZip protein EB1.

Analysis of the recently completed genomic sequence of Kaposi's sarcoma-associated herpesvirus (human herpesvirus-8) revealed that ORF 50 encodes a protein with homology to the Epstein-Barr virus (EBV) transcription factor R. In this report, we show that ORF K8, contiguous to ORF 50, is interrupted by two introns and that the spliced RNA is translated into a bZip protein that has homology to the EBV transcription factor EB1. The newly characterized K8 protein forms homodimers but does not heterodimerize with other members of the bZip protein family.

Amino Acid Sequence↗
PubMed1999

The C-terminal region but not the Arg-X-Pro repeat of Epstein-Barr virus protein EB2 is required for its effect on RNA splicing and transport.

The Epstein-Barr virus BMLF1 gene product EB2 has been shown to efficiently transform immortalized Rat1 and NIH 3T3 cells, to bind RNA, and to shuttle from the nucleus to the cytoplasm. In transient-expression assays EB2 seems to affect mRNA nuclear export of intronless RNAs and pre-mRNA 3' processing, but no direct proof of EB2 being involved in RNA processing and transport has been provided, and no specific functional domain of EB2 has been mapped. Here we significantly extend these findings and directly demonstrate that (i) EB2 inhibits the cytoplasmic accumulation of mRNAs, but only if they are generated from precursors containing weak (cryptic) 5' splice sites, (ii) EB2 has no effect on the cytoplasmic accumulation of mRNA generated from precursors containing constitutive splice sites, and (iii) EB2 has no effect on the 3' processing of precursor RNAs containing canonical and noncanonical cleavage-polyadenylation signals. We also show that in the presence of EB2, intron-containing and intronless RNAs accumulate in the cytoplasm. EB2 contains an Arg-X-Pro tripeptide repeated eight times, similar to that described as an RNA-binding domain in the herpes simplex virus type 1 protein US11. As glutathione S-transferase fusion proteins, both EB2 and the Arg-X-Pro repeat bound RNA in vitro. However, by using EB2 deletion mutants, we demonstrated that the effect of EB2 on splicing and RNA transport requires the C-terminal half of the protein but not the Arg-X-Pro repeat.

Alternative Splicing↗
PubMed1998

Transcriptional repression by the Epstein-Barr virus EBNA3A protein tethered to DNA does not require RBP-Jkappa.

The Epstein-Barr virus (EBV) proteins EBNA1, EBNA2, EBNA3A, EBNA3C, LMP1 and EBNA-LP are essential for the in vitro immortalization of primary B lymphocytes by EBV. EBNA2 is a transcriptional activator of viral and cellular genes. Both EBNA3A and EBNA3C have been shown to specifically inhibit EBNA2-activated transcription by direct interaction with RBP-Jkappa, a cellular DNA-binding factor known to recruit EBNA2 to EBNA2-responsive genes. This interaction interferes with the binding of RBP-Jkappa to DNA in vitro, and this is probably the mechanism by which EBNA3A and EBNA3C repress EBNA2-activated transcription in vivo. EBNA3A and EBNA3C also directly repress transcription when tethered to a promoter via the DNA-binding domain of the yeast Gal4 protein. As RBP-Jkappa has been previously shown to be a repressor in mammalian cells, this repression could be due to the recruitment of RBP-Jkappa by Gal4-EBNA3A and 3C. In this study, we have precisely mapped the domain of EBNA3A involved in the interaction with RBP-Jkappa and we have shown that interaction with RBP-Jkappa is not required for the Gal4-EBNA3A-mediated repression. Furthermore, we have characterized in EBNA3A a domain of 143 amino acids which is necessary and sufficient for EBNA3A-dependent repression.

DNA-Binding Proteins↗
PubMed1997

A particular DNA structure is required for the function of a cis-acting component of the Epstein-Barr virus OriLyt origin of replication.

OriLyt, thecis-acting element of Epstein-Barr virus lytic origin of replication, consists of upstream and downstream components. The upstream component plays a dual role in transcription and replication. The downstream component contains a homopurine-homopyrimidine sequence which forms an H palindrome. We show that the downstream component can adopt a triple helix structure in vitro, that the 5' border of the homopyrimidine sequence is sensitive to P1 nuclease when carried by a supercoiled plasmid and that an oligonucleotide complementary to the homopyrimidine strand is taken up by a plasmid carrying the OriLyt H palindrome. We also show that all mutations which alter the H palindrome impair both oligonucleotide uptake and OriLyt-dependent replication. Interestingly, compensatory mutations which restore an H palindrome also restore oligonucleotide uptake by the mutated plasmids and their OriLyt-dependent replication. Thus, there is a strong correlation between the inability of the OriLyt H palindrome to form a non-B-DNA structure in vitro and impairment of OriLyt-dependent replication. This suggests that the presence of a non-B-DNA structure in the OriLyt downstream component is required for OriLyt-dependent replication.

Base Sequence↗
PubMed1997

Human estrogen receptor (ER) gene promoter-P1: estradiol-independent activity and estradiol inducibility in ER+ and ER- cells.

Estrogen receptor (ER) is expressed at a low level in normal tissues such as breast and uterus but at a high level in breast and endometrial carcinomas. A proximal element (ERF-1) located between positions +133 and +204 relative to the promoter P1 major initiation site has been recently identified in ER+ cells and contributes to the differential promoter activity between ER+ and ER- cells. In this study, MCF7 and HeLa cells were transfected with chloramphenicol acetyltransferase constructs containing ER gene promoter P1 sequences. We show here that the sequences lying between nucleotides +13 to +212 are also essential for transcription at the ER gene promoter P1 in ER- cells, which do not express ERF-1. Interestingly, on gel shift experiments, a complex specific to ER- cells forms in the region spanning nucleotides +123 to +210. We also show that promoter P1 is responsive to estradiol in cells expressing endogenous (MCF7) or exogenous ER. We further demonstrate, using mutational analysis and gel retardation assays, that the three half-estrogen response elements located between nucleotides -420 and -892 are responsible for the estradiol inducibility of promoter P1. Because estradiol has a mitogenic effect on both breast and endometrial epithelial cells, our data would give an insight into the role of estrogens in the occurrence of breast and endometrial carcinomas.

Baculoviridae↗
PubMed1996

Molecular cloning and sequence analysis of the cDNA encoding rat liver cysteine sulfinate decarboxylase (CSD).

The taurine biosynthesis enzyme, cysteine sulfinate decarboxylase (CSD), was purified to homogeneity from rat liver. Three CSD peptides generated by tryptic cleavage were isolated and partially sequenced. Two of them showed a marked homology with glutamate decarboxylase and their respective position on the CSD amino acid sequence was postulated accordingly. Using appropriate degenerated primers derived from these two peptides, a PCR amplified DNA fragment was generated from liver poly(A)+ mRNA, cloned and used as a probe to screen a rat liver cDNA library. Three cDNAs, length around 1800 bp, were isolated which all contained an open reading frame (ORF) encoding a 493 amino acid protein with a calculated molecular mass of 55.2 kDa close to the experimental values for CSD. The encoded protein contained the sequence of the three peptides isolated from homogenous liver CSD. Our data confirm and significantly extend those recently published (Kaisaki et al. (1995) Biochim. Biophys. Acta 1262, 79-82). Indeed, an additional base pair found 1371 bp downstream from the initiation codon led to a shift in the open reading frame which extended the carboxy-terminal end by 15 amino acid residues and altogether modified 36 amino acids. The validity of this correction is supported by the finding that the corrected reading frame encoded a peptide issued from CSD tryptic cleavage that was not encoded anywhere in the CSD sequence previously reported.

Amino Acid Sequence↗
PubMed1996

Epstein-Barr virus (EBV) EB1/Zta protein provided in trans and competent for the activation of productive cycle genes does not activate the BZLF1 gene in the EBV genome.

The Epstein-Barr Virus (EBV) gene BZLF1 encodes the transcription fact or EB1 (also known as Zta) which is essential for the switch from latency to the lytic cycle: EB1 expressed from a plasmid transfected into B cell lines carrying latent EBV episomes, induces a productive viral cycle. Furthermore, EB1-specific DNA-binding sequences (ZREs) have been found in the promoters of many EBV early genes, including the BZLF1 promoter PZ and the PR promoter. At promoter PR, bicistronic mRNAs are initiated which contain, from 5' to 3', the BRLF1 and the BZLF1 open reading frames (ORFs) encoding respectively the R and EB1 proteins. The current model for the activation of the EBV lytic cycle implies that downregulation of the PZ promoter activity is a key element for latency and that a limiting step in the activation of the productive cycle is the translation of EB1. Once made, EB1 autoactivates promoter PZ, activates the PR promoter at which an mRNA coding for the EBV transcription factor R is initiated and activates the EBV early genes and the ORIlyt, due to unrestricted accessibility of the EB1-responsive elements in the viral genome. We show here that EB1 expressed from a plasmid activated most if not all of the EBV early genes in the viral genome but not its own gene, BZLF1. Moreover, transfected EB1 induced the transcription of the bicistronic mRNAs from which R is efficiently translated but not EB1. Our results demonstrate that EB1 provided in trans, although competent to activate the productive cycle genes, was not sufficient to overcome the downregulation of the PZ promoter.

Base Sequence↗
PubMed1996

Repression by RAZ of Epstein-Barr virus bZIP transcription factor EB1 is dimerization independent.

The hallmark of Epstein-Barr virus (EBV) infection is the establishment of a viral genome transcription pattern called latency. The EBV BZLF1 gene product EB1 (also known as ZEBRA or Zta) is a transcription factor which is essential for the switch from latency to the lytic cycle. It has been proposed that latency is maintained (i) by the inhibition of EB1 translation via antisense hybridization of EBNA1 and EB1 hnRNAs, or (ii) by the inactivation of the EB1 activating function via the direct interaction of EB1 with RelA, the retinoic acid receptor and p53, or via the titration of EB1 in RAZ:EB1 inactive heterodimers that are unable to bind to DNA. RAZ, a fusion protein which contains the EB1 C-terminal dimerization and DNA-binding domains fused to the N-terminal 86 amino acids of the EBV BRLF1 gene product R, has been described as a trans-dominant negative regulator of EB1-activated transcription. We demonstrate here that although RAZ efficiently represses EB1-mediated transcriptional activation, the amount of RAZ protein expressed is incompatible with repression through the titration of EB1 in inactive EB1:RAZ heterodimers. Furthermore, we also demonstrate that RAZ efficiently represses transcription activated by an EB1 mutant carrying the GCN4 homodimerization domain (EB1 gcn4), despite the inability of RAZ and EB1 gcn4 to form stable heterodimers.

Animals↗