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1.
Virol Sin ; 25(1): 36-44, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20960282

ABSTRACT

A group of SARS-like coronaviruses (SL-CoV) have been identified in horseshoe bats. Despite SL-CoVs and SARS-CoV share identical genome structure and high-level sequence similarity, SL-CoV does not bind to the same cellular receptor as for SARS-CoV and the N-terminus of the S proteins only share 64% amino acid identity, suggesting there are fundamental differences between these two groups of coronaviruses. To gain insight into the basis of this difference, we established a recombinant adenovirus system expressing the S protein from SL-CoV (rAd-Rp3-S) to investigate its immune characterization. Our results showed that immunized mice generated strong humoral immune responses against the SL-CoV S protein. Moreover, a strong cellular immune response demonstrated by elevated IFN-γ and IL-6 levels was also observed in these mice. However, the induced antibody from these mice had weaker cross-reaction with the SARS-CoV S protein, and did not neutralize HIV pseudotyped with SARS-CoV S protein. These results demonstrated that the immunogenicity of the SL-CoV S protein is distinct from that of SARS-CoV, which may cause the immunological differences between human SARS-CoV and bat SL-CoV. Furthermore, the recombinant virus could serve as a potential vaccine candidate against bat SL-CoV infection.


Subject(s)
Chiroptera/virology , Membrane Glycoproteins/immunology , Severe acute respiratory syndrome-related coronavirus/immunology , Viral Envelope Proteins/immunology , Adenoviridae/genetics , Animals , Antibodies, Neutralizing , Antibodies, Viral/blood , Cross Reactions , Female , Gene Expression , Genetic Vectors , HIV/genetics , Humans , Interferon-gamma/metabolism , Interleukin-6/metabolism , Leukocytes, Mononuclear/immunology , Membrane Glycoproteins/genetics , Mice , Mice, Inbred BALB C , Neutralization Tests , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Severe acute respiratory syndrome-related coronavirus/genetics , Severe acute respiratory syndrome-related coronavirus/isolation & purification , Spike Glycoprotein, Coronavirus , Viral Envelope Proteins/genetics , Viral Vaccines/administration & dosage , Viral Vaccines/immunology
2.
Arch Virol ; 155(12): 1951-7, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20820822

ABSTRACT

In a study of 315 HBV specimens obtained from southern China, 240 (76.9%) were assigned to genotype B, 72 (22.9%) were genotype C, two (0.6%) were genotype A and one (0.3%) was genotype D. Statistical analysis revealed that variables such as age, gender, HBV vaccination rate, hepatitis anamnesis rate, anti-HBs and HBeAg prevalence and virus load were insignificant between genotype B (n = 240) and genotype C cases (n = 72) (P > 0.05). However, the frequency of amino acid (aa) substitutions in the major hydrophilic region (MHR; aa 99-169) and the putative HLA class I-restricted cytotoxic T lymphocyte (CTL) epitope region of the S gene, as well as the overlapping polymerase/RT region (aa 32-212), were significantly higher in genotype C group than genotype B (P < 0.001). These results suggest that the higher variability within genotype C carriers may account for the pathogenic potential.


Subject(s)
Genetic Variation , Hepatitis B Surface Antigens/genetics , Hepatitis B virus/genetics , Hepatitis B virus/isolation & purification , Hepatitis B/virology , Adolescent , Adult , Amino Acid Substitution , Child , Child, Preschool , China , DNA-Directed DNA Polymerase/genetics , Epitopes, T-Lymphocyte/genetics , Female , Genotype , Hepatitis B virus/classification , Humans , Infant , Male , Middle Aged , Phylogeny , Sequence Analysis, DNA , Viral Proteins/genetics , Young Adult
3.
PLoS One ; 5(1): e8798, 2010 Jan 20.
Article in English | MEDLINE | ID: mdl-20098704

ABSTRACT

BACKGROUND: The NMDA receptor represents a particularly important site of ethanol action in the CNS. We recently reported that NMDA receptor 2B (NR2B) gene expression was persistently up-regulated following chronic intermittent ethanol (CIE) treatment. Increasing evidence that epigenetic mechanisms are involved in dynamic and long-lasting regulation of gene expression in multiple neuroadaptive processes prompted us to investigate the role of DNA methylation in mediating CIE-induced up-regulation of NR2B gene transcription. To dissect the changes of DNA methylation in the NR2B gene, we have screened a large number of CpG sites within its 5'-regulatory area following CIE treatment. METHODS: Primary cortical cultured neurons were subjected to ethanol treatment in a CIE paradigm. Bisulfite conversion followed by pyrosequencing was used for quantitative measurement and analysis of CpG methylation status within the 5'-regulatory area of the NR2B gene; chromatin immunoprecipitation (ChIP) assay was used to examine DNA levels associated with methylation and transcription factor binding. Electrophoretic mobility shift assay (EMSA) and in vitro DNA methylation assays were performed to determine the direct impact of DNA methylation on the interaction between DNA and transcription factor and promoter activity. RESULTS: Analysis of individual CpG methylation sites within the NR2B 5'regulatory area revealed three regions with clusters of site-specific CpG demethylation following CIE treatment and withdrawal. This was confirmed by ChIP showing similar decreases of methylated DNA in the same regions. The CIE-induced demethylation is characterized by being located near certain transcription factor binding sequences, AP-1 and CRE, and occurred during treatment as well as after ethanol withdrawal. Furthermore, the increase in vitro of methylated DNA decreased transcription factor binding activity and promoter activity. An additional ChIP assay indicated that the CIE-induced DNA demethylation is accompanied by increased occupation by transcription factors. CONCLUSIONS: These results suggest an important role of DNA demethylation in mediating CIE-induced NR2B gene up-regulation, thus implicating a novel molecular site of alcohol action.


Subject(s)
DNA Methylation , Ethanol/pharmacology , Receptors, N-Methyl-D-Aspartate/genetics , Regulatory Sequences, Nucleic Acid , Transcription, Genetic/drug effects , Up-Regulation/drug effects , Animals , Base Sequence , Cells, Cultured , Chromatin Immunoprecipitation , CpG Islands , DNA Primers , Electrophoretic Mobility Shift Assay , Epigenesis, Genetic , Mice , Mice, Inbred C57BL , Polymerase Chain Reaction , Promoter Regions, Genetic
4.
Methods Mol Biol ; 567: 113-32, 2009.
Article in English | MEDLINE | ID: mdl-19588089

ABSTRACT

The ability to determine genome-wide location of transcription factor binding sites (TFBS) is crucial for elucidating gene regulatory networks in human cells during normal development and disease such as tumorigenesis. To achieve this goal, we developed a method called serial analysis of binding elements for transcription factors (SABE) for globally identifying TFBS in human or other mammalian genomes. In this method, a specific antibody targeting a DNA-binding transcription factor of interest is used to pull down the transcription factor and its bound DNA elements through chromatin immunoprecipitation (ChIP). ChIP DNA fragments are further enriched by subtractive hybridization against non-enriched DNA and analyzed through generation of sequence tags similar to serial analysis of gene expression (SAGE). The SABE method circumvents the need for microarrays and is able to identify immunoprecipitated loci in an unbiased manner. The combination of ChIP, subtractive hybridization, and SAGE-type methods is advantageous over other similar strategies to reduce the level of intrinsic noise sequences that is typically present in ChIP samples from human or other mammalian cells.


Subject(s)
Chromatin Immunoprecipitation/methods , Regulatory Elements, Transcriptional/genetics , Sequence Analysis, DNA/methods , Transcription Factors/metabolism , Animals , Binding Sites/genetics , Humans , Models, Biological , Polymerase Chain Reaction/methods , Protein Binding
5.
Virology ; 386(2): 290-302, 2009 Apr 10.
Article in English | MEDLINE | ID: mdl-19233445

ABSTRACT

Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) encoded by ORF50 is a lytic switch protein for viral reactivation from latency. The expression of RTA activates the expression of downstream viral genes, and is necessary for triggering the full viral lytic program. Using chromatin immunoprecipitation assay coupled with a KSHV whole-genome tiling microarray (ChIP-on-chip) approach, we identified a set of 19 RTA binding sites in the KSHV genome in a KSHV-infected cell line BCBL-1. These binding sites are located in the regions of promoters, introns, or exons of KSHV genes including ORF8, ORFK4.1, ORFK5, PAN, ORF16, ORF29, ORF45, ORF50, ORFK8, ORFK10.1, ORF59, ORFK12, ORF71/72, ORFK14/ORF74, and ORFK15, the two origins of lytic replication OriLyt-L and OriLyt-R, and the microRNA cluster. We confirmed these RTA binding sites by ChIP and quantitative real-time PCR. We further mapped the RTA binding site in the first intron of the ORFK15 gene, and determined that it is RTA-responsive. The ORFK15 RTA binding sequence TTCCAGGAA TTCCTGGAA consists of a palindromic structure of two tandem repeats, of which each itself is also an imperfect inverted repeat. Reporter assay and electrophoretic mobility shift assay confirmed the binding of the RTA protein to this sequence in vitro. Sequence alignment with other RTA binding sites identified the RTA consensus binding motif as TTCCAGGAT(N)(0-16)TTCCTGGGA. Interestingly, most of the identified RTA binding sites contain only half or part of this RTA binding motif. These results suggest the complexity of RTA binding in vivo, and the involvement of other cellular or viral transcription factors during RTA transactivation of target genes.


Subject(s)
Genome, Viral , Herpesvirus 8, Human/genetics , Immediate-Early Proteins/metabolism , Trans-Activators/metabolism , Viral Proteins/metabolism , Base Sequence , Binding Sites , Cell Line , DNA, Viral/metabolism , Gene Expression Regulation, Viral , Herpesvirus 8, Human/metabolism , Humans , Immediate-Early Proteins/genetics , Molecular Sequence Data , Multigene Family , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic , Protein Binding , Sequence Alignment , Trans-Activators/genetics , Viral Proteins/genetics
6.
Cancer Treat Res ; 133: 69-127, 2007.
Article in English | MEDLINE | ID: mdl-17672038

ABSTRACT

KSHV has been established as the causative agent of KS, PEL, and MCD, malignancies occurring more frequently in AIDS patients. The aggressive nature of KSHV in the context of HIV infection suggests that interactions between the two viruses enhance pathogenesis. KSHV latent infection and lytic reactivation are characterized by distinct gene expression profiles, and both latency and lytic reactivation seem to be required for malignant progression. As a sophisticated oncogenic virus, KSHV has evolved to possess a formidable repertoire of potent mechanisms that enable it to target and manipulate host cell pathways, leading to increased cell proliferation, increased cell survival, dysregulated angiogenesis, evasion of immunity, and malignant progression in the immunocompromised host. Worldwide, approximately 40.3 million people are currently living with HIV infection. Of these, a significant number are coinfected with KSHV. The complex interplay between the two viruses dramatically elevates the risk for development of KSHV-induced malignancies, KS, PEL, and MCD. Although HAART significantly reduces HIV viral load, the entire T-cell repertoire and immune function may not be completely restored. In fact, clinically significant immune deficiency is not necessary for the induction of KSHV-related malignancy. Because of variables such as lack of access to therapy noncompliance with prescribed treatment, failure to respond to treatment and the development of drug-resistant strains of HIV, KSHV-induced malignancies will continue to present as major health concerns.


Subject(s)
Acquired Immunodeficiency Syndrome/complications , Acquired Immunodeficiency Syndrome/virology , Herpesvirus 8, Human/genetics , Herpesvirus 8, Human/pathogenicity , Neoplasms/complications , Neoplasms/virology , Animals , Herpesvirus 8, Human/chemistry , Humans , Sarcoma, Kaposi/complications , Sarcoma, Kaposi/virology
7.
Virus Genes ; 35(2): 215-23, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17546494

ABSTRACT

The HIV-1 LTR is regulated by multiple signaling pathways responsive to T cell activation. In this study, we have examined the contribution of the MAPK, calcineurin-NFAT and TNFalpha-NF-kappaB pathways on induction of chromosomally integrated HIV-1 LTR reporter genes. We find that induction by T-cell receptor (CD3) cross-linking and PMA is completely dependent upon a binding site for RBF-2 (USF1/2-TFII-I), known as RBEIII at -120. The MAPK pathway is essential for induction of the wild type LTR by these treatments, as the MEK inhibitors PD98059 and U0126 block induction by both PMA treatment and CD3 cross-linking. Stimulation of cells with ionomycin on its own has no effect on the integrated LTR, indicating that calcineurin-NFAT is incapable of causing induction in the absence of additional signals, but stimulation with both PMA and ionomycin produces a synergistic response. In contrast, stimulation of NF-kappaB by treatment with TNFalpha causes induction of both the wild type and RBEIII mutant LTRs, an effect that is independent of MAPK signaling. USF1, USF2 and TFII-I from unstimulated cells are capable of binding RBEIII in vitro, and furthermore can be observed on the LTR in vivo by chromatin imunoprecipitation from untreated cells. DNA binding activity of USF1/2 is marginally stimulated by PMA/ ionomycin treatment, and all three factors appear to remain associated with the LTR throughout the course of induction. These results implicate major roles for the MAPK pathway and RBF-2 (USF1/2-TFII-I) in coordinating events necessary for transition of latent integrated HIV-1 to active transcription in response to T cell signaling.


Subject(s)
HIV Long Terminal Repeat/genetics , HIV-1/genetics , MAP Kinase Signaling System/physiology , Transcription Factors, TFII/physiology , Upstream Stimulatory Factors/physiology , Virus Integration/genetics , ras Proteins/physiology , Chromosomes, Human/enzymology , Chromosomes, Human/virology , Gene Expression Regulation, Viral/physiology , Humans , Jurkat Cells , Lymphocyte Activation/physiology , Proviruses/enzymology , Proviruses/genetics , Proviruses/metabolism , T-Lymphocytes/enzymology , Transcription, Genetic/physiology
8.
Nat Protoc ; 1(3): 1481-93, 2006.
Article in English | MEDLINE | ID: mdl-17406439

ABSTRACT

Serial analysis of binding elements (SABE) is a method that can be used to identify the genome-wide location of transcription factor binding sites in human or other mammalian cells. In this method, a specific antibody targeting a DNA-binding transcription factor of interest is used to pull down the transcription factor and its bound DNA elements through chromatin immunoprecipitation (ChIP). ChIP DNA fragments are further enriched by subtractive hybridization against non-enriched DNA using representational difference analysis (RDA) and analyzed through the generation of sequence tags similar to serial analysis of gene expression (SAGE). The SABE method circumvents the need for microarrays and is able to identify immunoprecipitated loci in an unbiased manner. The combination of ChIP, RDA and SAGE-type methods has advantages over other similar strategies in reducing the level of intrinsic noise sequences that are typically present in ChIP samples from human cells. This protocol takes about 2 weeks to complete.


Subject(s)
Chromatin Immunoprecipitation/methods , DNA/isolation & purification , Genome, Human/genetics , Transcription Factors/isolation & purification , Binding Sites , DNA/metabolism , Gene Library , Humans , Transcription Factors/metabolism
9.
Proc Natl Acad Sci U S A ; 102(13): 4813-8, 2005 Mar 29.
Article in English | MEDLINE | ID: mdl-15781865

ABSTRACT

The ability to determine the global location of transcription factor binding sites in vivo is important for a comprehensive understanding of gene regulation in human cells. We have developed a technology, called serial analysis of binding elements (SABE), involving subtractive hybridization of chromatin immunoprecipitation-enriched DNA fragments followed by the generation and analysis of concatamerized sequence tags. We applied the SABE technology to search for p53 target genes in the human genome, and have identified several previously described p53 targets in addition to numerous potentially novel targets, including the DNA mismatch repair genes MLH1 and PMS2. Both of these genes were determined to be responsive to DNA damage and p53 activation in normal human fibroblasts, and have p53-response elements within their first intron. These two genes may serve as a sensor in DNA repair mechanisms and a critical determinant for the decision between cell-cycle arrest and apoptosis. These results also demonstrate the potential for use of SABE as a broadly applicable means to globally identify regulatory elements for human transcription factors in vivo.


Subject(s)
Adenosine Triphosphatases/metabolism , Base Pair Mismatch/genetics , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Genome, Human , Genomics/methods , Neoplasm Proteins/metabolism , Nuclear Proteins/metabolism , Tumor Suppressor Protein p53/metabolism , Adaptor Proteins, Signal Transducing , Adenosine Triphosphatases/genetics , Binding Sites/genetics , Carrier Proteins , Chromatin Immunoprecipitation/methods , DNA Primers , DNA Repair Enzymes/genetics , DNA-Binding Proteins/genetics , Humans , Jurkat Cells , Luciferases , Mismatch Repair Endonuclease PMS2 , MutL Protein Homolog 1 , Neoplasm Proteins/genetics , Nuclear Proteins/genetics , Nucleic Acid Hybridization/methods , Oligonucleotide Probes , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA/methods
10.
J Virol ; 79(7): 4396-406, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15767439

ABSTRACT

Human immunodeficiency virus type 1 (HIV-1) replication is coupled to T-cell activation through its dependence on host cell transcription factors. Despite the enormous sequence variability of these factors, several cis elements for host factors are highly conserved within the 5' long terminal repeats (LTRs) of viruses from AIDS patients; among these is the RBEIII upstream element for the Ras response element binding factor 2 (RBF-2). Here we show that RBF-2 is comprised of a USF1/USF2 heterodimer and TFII-I, which bind cooperatively to RBEIII. Recombinant USF1/USF2 binds to the RBEIII core sequence 160-fold less efficiently than it binds to an E box element, but the interaction with RBEIII is stimulated by TFII-I. Chromosomally integrated HIV-1 LTRs bearing an RBEIII mutation have slightly elevated basal transcription in unstimulated Jurkat cells but are unresponsive to cross-linking of the T-cell receptor or stimulation with phorbol myristate acetate (PMA) and ionomycin. Induction is inhibited by dominant interfering USF and TFII-I but not by the dominant negative I-kappaB protein. USF1, USF2, and TFII-I bind to the integrated wild-type LTR in unstimulated cells and become phosphorylated during the induction of transcription upon stimulation with PMA. These results demonstrate that USF1/USF2 and TFII-I interact cooperatively at the upstream RBEIII element and are necessary for the induction of latent HIV-1 in response to T-cell activation signals.


Subject(s)
DNA-Binding Proteins/metabolism , Gene Expression Regulation, Viral , HIV Long Terminal Repeat/physiology , HIV-1/genetics , T-Lymphocytes/virology , Transcription Factors, TFII/metabolism , Transcription Factors/metabolism , Virus Integration , Virus Replication , Animals , COS Cells , Dimerization , Electrophoretic Mobility Shift Assay , HIV-1/physiology , Humans , Jurkat Cells , Lymphocyte Activation , Phosphorylation , Protein Binding , Receptors, Antigen, T-Cell/physiology , T-Lymphocytes/immunology , Transcription, Genetic , Upstream Stimulatory Factors , Virus Activation
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