Animals ; HIV Long Terminal Repeat ; genetics ; Humans ; Promoter Regions, Genetic ; genetics ; Spumavirus ; genetics ; Viral Proteins ; genetics ; metabolism

BACKGROUND: Although Tat plays a role as a potent transactivator in the viral gene expression from the Human Immunodeficiency Virus type 1 long terminal repeat (HIV-1 LTR), it does not function efficiently in rodent cells implying the absence of a human specific factor essential for Tat-medicated transactivation in rodent cells. In previous experiments, we demonstrated that one of chimeric forms of TAR (transacting responsive element) of HIV-1 LTR compensated the restriction in rodent cells. METHODS: To characterize the nature of the compensation, we tested the effects of several upstream binding factors of HIV-1 LTR by simple substitution, and also examined the role of the configuration of the upstream binding factor(s) indirectly by constructing spacing mutants that contained insertions between Sp1 and TATA box on Tat-mediated transactivation. RESULTS: Human Sp1 had no effect whereas its associated factors displayed differential effects in human and rodent cells. In addition, none of the spacing mutants tested overcame the restriction in rodent cells. Rather, when the secondary structure of the chimeric HIV-1 TAR construct was destroyed, the compensation in rodent cells was disappeared. Interestingly, the proper interaction between Sp1 and TATA box binding proteins, which is essential for Tat-dependent transcription, was dispensable in rodent cells. CONCLUSION: This result suggests that the human-specific Tat cofactor acts to allow Tat to interact effectively in a ribonucleoprotein complex that includes Tat, cellular factors, and TAR RNA, rather than be associated with the HIV-1 LTR upstream DNA binding factors.
Compensation and Redress ; DNA ; Genes, Viral ; HIV Long Terminal Repeat ; HIV* ; HIV-1* ; Humans* ; Ribonucleoproteins ; RNA ; Rodentia* ; TATA Box ; TATA-Box Binding Protein ; Terminal Repeat Sequences ; Trans-Activators ; Transcriptional Activation

BACKGROUND: Although Tat plays a role as a potent transactivator in the viral gene expression from the Human Immunodeficiency Virus type 1 long terminal repeat (HIV-1 LTR), it does not function efficiently in rodent cells implying the absence of a human specific factor essential for Tat-medicated transactivation in rodent cells. In previous experiments, we demonstrated that one of chimeric forms of TAR (transacting responsive element) of HIV-1 LTR compensated the restriction in rodent cells. METHODS: To characterize the nature of the compensation, we tested the effects of several upstream binding factors of HIV-1 LTR by simple substitution, and also examined the role of the configuration of the upstream binding factor(s) indirectly by constructing spacing mutants that contained insertions between Sp1 and TATA box on Tat-mediated transactivation. RESULTS: Human Sp1 had no effect whereas its associated factors displayed differential effects in human and rodent cells. In addition, none of the spacing mutants tested overcame the restriction in rodent cells. Rather, when the secondary structure of the chimeric HIV-1 TAR construct was destroyed, the compensation in rodent cells was disappeared. Interestingly, the proper interaction between Sp1 and TATA box binding proteins, which is essential for Tat-dependent transcription, was dispensable in rodent cells. CONCLUSION: This result suggests that the human-specific Tat cofactor acts to allow Tat to interact effectively in a ribonucleoprotein complex that includes Tat, cellular factors, and TAR RNA, rather than be associated with the HIV-1 LTR upstream DNA binding factors.
Compensation and Redress ; DNA ; Genes, Viral ; HIV Long Terminal Repeat ; HIV* ; HIV-1* ; Humans* ; Ribonucleoproteins ; RNA ; Rodentia* ; TATA Box ; TATA-Box Binding Protein ; Terminal Repeat Sequences ; Trans-Activators ; Transcriptional Activation

Base Sequence ; Drug Resistance, Viral ; HIV Long Terminal Repeat ; HIV-1 ; classification ; drug effects ; genetics ; Humans ; Molecular Sequence Data ; Phylogeny ; Receptors, HIV ; physiology

BACKGROUNDOne of the major characteristics of the human immunodeficiency virus type 1 (HIV-1) is its unusually high degree of genetic variability, which involves in genetic diagnosis, subtyping, vaccine design, and epidemiology. HIV-1 CRF01_AE is a main prevalent HIV-1 recombinant strain in China. In this study, three full-length CRF01_AE genomes from Fujian Province, China were cloned, sequenced, and analyzed; and the further genetic diversity defining and epidemiologic analysis were carried out.

METHODSProviral DNA was extracted from non-cultured peripheral blood mononuclear cells, the near full-length HIV-1 genome was amplified and the PCR products were cloned into pCR-XL-TOPO vector and sequenced. 5'-long terminal repeat (LTR) and 3'-LTRs were amplified by additional independent PCR and cloned into pMD18T vector. Gene-based phylogenic tree was constructed and genetic distances were calculated by MEGA 3.1. Simplot was used for Bootscan analysis.

RESULTSThe phylogeny and genetic distance analysis of the three near full-length sequences confirmed that these three samples clustered with CRF01_AE isolates, more close to Thailand CRF01_AE strain CM240, and were distantly related to African CRF01_AE strain 90CF402. Analysis of their genomic organization revealed the presence of nine potential open reading frames. There were no major deletions, rearrangements, or insertions in the three sequences, but an in-frame stop codon was found in tat gene of Fj051. LTRs of the three sequences contained a few nucleotides mutation. We did not find new mosaic recombinant in the three sequences. The V3 motif was GPGQ in all the three sequences, and there were only few amino acids differences in all three V3 loop sequences.

CONCLUSIONThis report reveals the background of the three full-length CRF01_AE genomes, the most dominantly circulating HIV-1 strain in Fujian Province, China. The work is essential for the design and development of an effective AIDS vaccine for the region.

Adult ; Amino Acid Sequence ; Base Sequence ; DNA, Viral ; chemistry ; Female ; Genome, Viral ; HIV Long Terminal Repeat ; HIV-1 ; classification ; genetics ; Humans ; Male ; Molecular Sequence Data ; Phylogeny ; Recombination, Genetic

HIV-1 trans-activator of transcription (Tat) plays a critical role in HIV-1 transcription. Based on the beta-turn motif present in HIV-1 Tat, a series of novel benzodiazepine analogs were designed as beta-turn mimetics and prepared from p-chloro-nitrobenzene/2-phenylacetonitrile, p-toluidine/benzoyl chloride, or (Z)-7-nitro-5-phenyl-1H-benzo[e][1, 4]diazepin-2(3H)-one (nitrazepam) through different synthetic routes. Preliminary biological evaluation indicated that compound 30 exhibited inhibitory activity on HIV-1 tat-mediated LTR transcription with EC50 of 25.0 micromol x L(-1) and showed no obvious cytotoxic effects on TZM-BI cells under the concentration of 100 micromol x L(-1).
Benzodiazepinones ; chemical synthesis ; chemistry ; pharmacology ; Cell Line, Tumor ; HIV Long Terminal Repeat ; genetics ; HIV-1 ; genetics ; Humans ; Transcription, Genetic ; drug effects ; tat Gene Products, Human Immunodeficiency Virus ; antagonists & inhibitors

BACKGROUNDThe CKLF-like MARVEL transmembrane domain-containing family (CMTM) is a novel family of proteins linking chemokines and TM4SF. Different members exhibit diverse biological functions. In this study, the effect of intracellular CMTM2 on regulating human immunodeficiency virus type-1 (HIV-1) transcription was evaluated.

METHODSThe effects of CMTM2 on regulating full-length HIV-1 provirus and the HIV-1 long terminal repeat (LTR)-directed transcription were assessed by luciferase assay. Transcription factor assays, using the luciferase reporter plasmids of AP-1, CRE, and NF-κB were conducted to explore the signaling pathway(s) that may be regulated by CMTM2. The potential relationship between CMTM2 and the transcription factor AP-1 was further analyzed by Western blotting analyses to investigate the effect of CMTM2 on PMA-induced ERK1/2 phosphorylation.

RESULTSThe results from the current study revealed that CMTM2 acts as a negative regulator of HIV-1 transcription. CMTM2 exerted a suppressive action on both full-length HIV-1 provirus and HIV-1 LTR-directed transcription. Transcription factor assays showed that CMTM2 selectively inhibited basal AP-1 and CREB activity. Co-expression of HIV-1 Tat, a potent AP-1 and CREB activator, can not reverse CMTM2-mediated AP-1 and CREB inhibition, suggesting a potent and specific effect of CMTM2 on negatively regulating these two signaling pathways.

CONCLUSIONIntracellular CMTM2 can negatively regulate HIV-1 transcription, at least in part, by targeting the AP-1 and CREB pathways. Exploring the mechanisms further may lead to new ways to control HIV-1 replication.

Chemokines ; physiology ; Cyclic AMP Response Element-Binding Protein ; antagonists & inhibitors ; HIV Long Terminal Repeat ; HIV-1 ; genetics ; Humans ; Intracellular Space ; metabolism ; Jurkat Cells ; MARVEL Domain-Containing Proteins ; Tetradecanoylphorbol Acetate ; pharmacology ; Transcription Factor AP-1 ; antagonists & inhibitors ; Transcription, Genetic ; U937 Cells

Vpr, an auxiliary protein of HIV-1(Human immunodeficiency virus type 1), exerts important functions to promote viral replication and AIDS progression. In this study, we performed a yeast two-hybrid screening assay using human cDNA library to further investigate the molecular mechanism of various functions of Vpr RelB, a key protein in NF-kappaB signaling pathway, was identified as a Vpr interaction protein by co-immunoprecipitation. Further investigations indicated that RelB not only promoted the Vpr-mediated activation of NF-kappaB reporter gene, but also enhanced the transactivation of HIV LTR. Moreover, the results showed that RelB promoted Vpr-induced cell cycle G2/M arrest. Collectively, these results indicated that RelB might interact with Vpr and regulate its transcriptional activation and cell cycle arrest.
Cell Cycle Checkpoints ; Cell Division ; G2 Phase ; HIV Long Terminal Repeat ; HeLa Cells ; Humans ; NF-kappa B ; genetics ; Transcription Factor RelB ; physiology ; Transcriptional Activation ; vpr Gene Products, Human Immunodeficiency Virus ; physiology

Human immunodeficiency virus type 1 (HIV-1) transcription is a crucial step in the viral replication cycle, which is considered to be a potential target for inhibition of HIV-1 replication. Among the factors involved in this step, the cellular protein nuclear factor NF-kappaB is the most powerful inducer of HIV-1 transcription. HIV-1 transcription is initiated by the binding of NF-kappaB to the enhancer region in the long terminal repeat (LTR) of HIV-1. Several compounds suppress HIV-1 transcription through the inhibition of NF-kappaB activation. The mechanisms of NF-kappaB in the transcription of HIV-1 and progress of the current inhibitors of NF-kappaB are reviewed.
Anti-HIV Agents ; pharmacology ; HIV Long Terminal Repeat ; HIV-1 ; genetics ; Humans ; I-kappa B Kinase ; metabolism ; I-kappa B Proteins ; metabolism ; NF-KappaB Inhibitor alpha ; NF-kappa B ; antagonists & inhibitors ; metabolism ; Nicotinic Acids ; pharmacology ; Nitriles ; pharmacology ; Transcription, Genetic ; drug effects ; Virus Replication

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths in the world, especially in Korea where 6-12% of the general population show positive for HbsAg. The accumulating studies suggest that the HBV-X protein found in HBV DNA may be the causative factor in the development of a HCC. METHOD: We studied the role of retinoblastoma (Rb) protein in the suppression of the tumorigenicity of a HCC by using cytomegalovirus (CMV) co-transfected HBV-X protein with retinoblastoma protein (pRb) or N-terminal truncated retinoblastoma protein (pRb94) in HepG2 cell lines. RESULTS: First, culturing HepG2 cells with CMV-Rb/liposome or CMV-Rb94/liposome, we observed the suppression of cell growth by using hemocytometric counting of the cells stained by trypan blue and by using a [3H]thymidine incorporation assay. Then, by using a plasmid co-transfected with the chloramphenicol acetyl transferase(CAT) gene, we investigated the role of HBV-X gene in regulating the transcriptional activity in the HepG2 cells under the control of a kB-like sequence of HIV-1 enhancer and the suppression of its activity by pRb and pRb94. CONCLUSIONS: We concluded that both pRb and pRb94 were capable of suppressing cell growth of a HepG2 cell line containing recombinant plasmids coding HBV-X protein. Furthermore, it was demonstrated that the suppression activity of pRb94 was more potent and sustaining than that of pRb. These results suggest that if additional research is performed on the method of gene delivery, gene therapy using pRb94 might be used as a new modality for the treatment of a HCC.
Carcinoma, Hepatocellular* ; Chloramphenicol ; Clinical Coding ; Cytomegalovirus ; DNA ; Genetic Therapy ; Hep G2 Cells ; Hepatitis B Surface Antigens ; Hepatitis B virus* ; Hepatitis B* ; Hepatitis* ; HIV Enhancer ; Humans* ; Korea ; Plasmids ; Retinoblastoma Protein* ; Retinoblastoma* ; Trypan Blue