HIV-1 Tat binds to SH3 domains: Cellular and viral outcome of Tat/Grb2 interaction

The Src-homology 3 (SH3) domain is one of the most frequent protein recognition modules (PRMs), being represented in signal transduction pathways and in several pathologies such as cancer and AIDS. Grb2 (growth factor receptor-bound protein 2) is an adaptor protein that contains two SH3 domains and is involved in receptor tyrosine kinase (RTK) signal transduction pathways. The HIV-1 transactivator factor Tat is required for viral replication and it has been shown to bind directly or indirectly to several host proteins, deregulating their functions. In this study, we show interaction between the cellular factor Grb2 and the HIV-1 trans-activating protein Tat. The binding is mediated by the proline-rich sequence of Tat and the SH3 domain of Grb2. As the adaptor protein Grb2 participates in a wide variety of signaling pathways, we characterized at least one of the possible downstream effects of the Tat/Grb2 interaction on the well-known IGF-1R/Raf/MAPK cascade. We show that the binding of Tat to Grb2 impairs activation of the Raf/MAPK pathway, while potentiating the PKA/Raf inhibitory pathway. The Tat/Grb2 interaction affects also viral function by inhibiting the Tat-mediated transactivation of HIV-1 LTR and viral replication in infected primary microglia.     

Induction of OGG1 gene expression by HIV-1 Tat

To identify the cellular gene target for Tat, we performed gene expression profile analysis and found that Tat up-regulates the expression of the OGG1 (8-oxoguanine-DNA glycosylase-1) gene, which encodes an enzyme responsible for repairing the oxidatively damaged guanosine, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG). We observed that Tat induced OGG1 gene expression by enhancing its promoter activity without changing its mRNA stability. We found that the upstream AP-4 site within the OGG1 promoter is responsible and that Tat interacted with AP-4 and removed AP-4 from the OGG1 promoter by in vivo chromatin immunoprecipitation assay. Thus, Tat appears to activate OGG1 expression by sequestrating AP-4. Interestingly, although Tat induces oxidative stress known to generate 8-oxo-dG, which causes the G:C to T:A transversion, we observed that the amount of 8-oxo-dG was reduced by Tat. When OGG1 was knocked down by small interfering RNA, Tat increased the amount of 8-oxo-dG, thus confirming the role of OGG1 in preventing the formation of 8-oxo-dG. These findings collectively indicate the possibility that Tat may play a role in maintenance of the genetic integrity of the proviral and host cellular genomes by up-regulating OGG1 as a feed-forward mechanism.     

光敏色素与转录因子结合直接调控植物基因表达和发育

植物的光控发育一直是植物学中一个非常活跃的研究领域,是近该领域又取得重大突破性进展,人们通过酵母双杂交技术克隆到在体内与光敏色素相互作用的转录因子,并证实被光活化的光敏色素直接进入细胞核与转录因子结构合启动基因表达,本文就此研究进展作简要介绍。     

A cellular protein binds to a conserved sequence in the adenovirus type 2 enhancer.

A sensitive gel retention assay has been utilized to detect proteins from uninfected Hela nuclei which interact with the adenovirus type 2 enhancer. This assay has been employed to monitor fractionation of nuclear extracts. Three enhancer binding factors were resolved by chromatography on DEAE-Sepharose and one of the factors was further purified by chromatography on heparin-Sepharose. DNase protection experiments have shown that the heparin-Sepharose fraction contains a factor which binds predominantly to the conserved sequence GTGGAAATTT present at position 160 in the adenovirus type 2 genome and found in many viral and cellular enhancers. Protection of this sequence from DNase I digestion was abolished by competition with a synthetic duplex oligonucleotide spanning bases 144-181. This region corresponds to the sequence defined by Hen et al. as possessing enhancer function. Competition experiments indicated that the enhancer binding factor also bound, albeit with reduced affinity, to multiple sites in the Ela upstream region located between positions 192 and 353. Within the sequences which compete are regions with homology to the high affinity site at position 160. The enhancer binding factor also binds with high affinity to sequences within the SV40 enhancer demonstrating that this factor interacts with sequences common to both the adenovirus and SV40 enhancers.     

Ficolin-2 binds to HIV-1 gp120 and blocks viral infection

Ficolin-2 is a lectin complement pathway activator present in normal human plasma and usually associated with infectious diseases, but little is known about the role of ficolin-2 in human immunodeficiency virus (HIV) infection. Here, we describe our novel findings that serum ficolin-2 concentrations of 103 HIV-1 patients were much higher compared to those of 57 healthy donors. In vitro analysis showed that HIV-1 infection could enhance ficolin-2 expression. We further demonstrated that recombinant ficolin-2 protein could bind with HIV-1 envelope glycoprotein gp120, and subsequently induce complement dependent cytotoxicity. Moreover, ficolin-2 could block the entry of HIV-1 into target cells (TZM-b1 and MT-2 cells) and infection in a ficolin-2 dosedependent manner. To our knowledge, this is the first report about the protective role of ficolin-2 against HIV-1 infection and our study suggests that ficolin-2 is an important human innate immune molecule against HIV.

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Stochastic gene expression in a lentiviral positive-feedback loop: HIV-1 Tat fluctuations drive phenotypic diversity

HIV-1 Tat transactivation is vital for completion of the viral life cycle and has been implicated in determining proviral latency. We present an extensive experimental/computational study of an HIV-1 model vector (LTR-GFP-IRES-Tat) and show that stochastic fluctuations in Tat influence the viral latency decision. Low GFP/Tat expression was found to generate bifurcating phenotypes with clonal populations derived from single proviral integrations simultaneously exhibiting very high and near zero GFP expression. Although phenotypic bifurcation (PheB) was correlated with distinct genomic integration patterns, neither these patterns nor other extrinsic cellular factors (cell cycle/size, aneuploidy, chromatin silencing, etc.) explained PheB. Stochastic computational modeling successfully accounted for PheB and correctly predicted the dynamics of a Tat mutant that were subsequently confirmed by experiment. Thus, Tat stochastics appear sufficient to generate PheB (and potentially proviral latency), illustrating the importance of stochastic fluctuations in gene expression in a mammalian system.     

Trans-dominant Tat mutants with alterations in the basic domain inhibit HIV-1 gene expression

The Tat protein of the human immunodeficiency virus type 1 (HIV-1) is required for efficient viral gene expression. By means of mutational analyses, several domains of the Tat protein that are required for complete activation of HIV-1 gene expression have been defined. These include an amino-terminal activating domain, a cysteine-rich dimerization domain, and a basic domain important in the binding of Tat to the trans-activation response element (TAR) and in Tat nuclear localization. Recently, we described a mutation, known as delta tat, which resulted in a protein with a truncated basic domain. This protein had a "trans-dominant" phenotype in that it inhibited wild-type Tat activation of the HIV-1 LTR. To further characterize the requirements for generating a Tat trans-dominant phenotype, we constructed a variety of Tat proteins with truncations or substitutions in the basic domain. A number of these proteins showed a trans-dominant phenotype. These Tat mutants also inhibited activation of the HIV-1 LTR by a protein composed of Tat fused to the prokaryotic R17 (phage MS2) RNA-binding protein in which the R17 recognition element was inserted in the HIV-1 LTR in place of TAR. Thus, an intact TAR element was not required for this inhibition. We also studied the cellular localization of Tat and a trans-dominant Tat mutant by means of immunofluorescence staining with the use of antibodies reactive to different domains of the Tat protein. The results indicated that Tat becomes localized predominantly in the nucleus both in the presence and absence of the trans-dominant Tat construct, suggesting that the trans-dominant mutant does not inhibit Tat nuclear localization. These studies further define the requirements for the creation of trans-dominant Tat mutants, and suggest that the mechanism of trans-dominant Tat inhibition may be either the formation of an inactive complex between wild-type and mutant Tat or sequestration of cellular factors involved in regulating HIV-1 gene expression.     

c-Myc binds to 5' flanking sequence motifs of the dihydrofolate reductase gene in cellular extracts: role in proliferation.

The dihydrofolate reductase is a key enzyme of the folate metabolism which supplies the cell with dTTPs for DNA synthesis. Using cellular extracts, we demonstrate the formation of c-Myc/Max heterodimers at the dihydrofolate reductase (DHFR) 5' flanking CANNTG (E-box) motifs. The presence of these complexes correlates with c-Myc levels and active cellular proliferation.     

HnRNP-A1 binds directly to double-stranded DNA in vitro within a 36 bp sequence

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) is known as an RNA- and single-stranded DNA-binding protein involved in alternative splicing of mRNA, RNA transport and maintenance of chromosome telomere length. In this study we tested whether this protein could bind directly to double-stranded DNA (dsDNA). Using PCR amplification of target DNA-sequences from human chromosome 11q13 followed by their incubation with hnRNP-A1 and atomic force microscopy (AFM) of the DNA/protein complexes, we found that this protein bound to DNA within a 36 bp sequence. These results were confirmed by electrophoretic mobility shift assay (EMSA). This sequence was found widely dispersed throughout the genome. There is no overlap between the 36 bp sequence and known target sequences in RNA for binding hnRNP-A1.     

HIV-1 structural gene expression requires the binding of multiple Rev monomers to the viral RRE: implications for HIV-1 latency

Expression of the structural proteins of HIV-1 requires the direct interaction of the viral Rev trans-activator with its cis-acting RNA target sequence, the Rev response element or RRE. Here, we demonstrate that this specific RNA-binding event is, as expected, mediated by the conserved arginine-rich motif of Rev. However, we also show that amino acid residues located proximal to this basic domain that are critical for in vivo Rev function are dispensable for sequence-specific binding to the RRE. Instead, these sequences are required for the multimerization of Rev on the viral RRE target sequence. The observation that Rev function requires the sequential binding of multiple Rev molecules to the RRE provides a biochemical explanation for the observed threshold effect for Rev function in vivo and suggests a molecular model for the high incidence of latent infection by HIV-1.     

The HIV-1 Vpu protein: a multifunctional enhancer of viral particle release

HIV accessory genes are expressed throughout the viral life cycle and regulate wide-ranging aspects of virus replication including viral infectivity (Vif and Nef), viral gene expression (Vpr) and progeny virion production (Vpu). While in many cases the molecular basis of accessory protein function is not fully understood, a consensus is emerging that these viral products are generally devoid of enzymatic activity and instead act as multifunctional adapters, subverting normal cellular processes to serve the needs of the virus. This review focuses on presenting our current knowledge of the HIV-1-specific Vpu protein and its essential role in regulating viral particle release, viral load and expression of the CD4 receptor.     

Immunogenicity of HIV-1 IIIB and SHIV 89.6P Tat and Tat toxoids in rhesus macaques: induction of humoral and cellular immune responses

This study compared immune responses in rhesus macaques immunized with unmodified HIV-1 IIIB Tat, SHIV89.6P Tat, and carboxymethylated IIIB and 89.6P Tat toxoids. Immunization with either IIIB or 89.6P preparation induced high titer and broadly crossreactive serum anti-Tat IgG that recognized HIV-1 subtype-E and SIVmac251 Tat. However, the response was delayed, and titers were lower in 89.6P vaccination groups. Serum anti-Tat IgG recognized peptides corresponding to the amino-terminus, basic domain, and carboxy-terminal region. Cellular proliferative responses to Tat toxoids corresponding to the immunogen were evident in vitro in both IIIB and 89.6P groups. Crossreactive proliferative responses were observed in IIIB groups in response to stimulation with 89.6P or SIVmac251 Tat toxoids, but were much less prevalent in 89.6P groups. The truncated 86 amino acid IIIB Tat appears to be more immunogenic than the 102 amino acid 89.6P Tat with respect to both humoral and cellular immune responses, and may be a better vaccine component. Despite induction of robust humoral and cellular immune responses (including both CD4+ and CD8+ T-cell responses) to Tat, all animals were infected upon intravenous challenge with 30 MID(50) of SHIV89.6P and outcome of vaccine groups was not different from controls. Sequencing both Tat exons from serum viral RNA revealed no evidence of escape mutants. These results suggest that with intravenous SHIV89.6P challenge in rhesus macaques, precipitous CD4+ T-cell decline overwhelms potentially protective immune responses. Alternatively, Tat specific CD8+ T-cell responses may not appropriately recognize infected cells in vivo in this model. In view of evidence demonstrating Tat specific CTLs in the SIV model and in humans infected with HIV-1, results in this pathogenic SHIV model may not apparently predict the efficacy of this approach in human studies. The potency and cross-reactivity of these immune responses confirm Tat toxoid as an excellent candidate vaccine component.     

MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks

Histone variant H2AX phosphorylation in response to DNA damage is the major signal for recruitment of DNA-damage-response proteins to regions of damaged chromatin. Loss of H2AX causes radiosensitivity, genome instability, and DNA double-strand-break repair defects, yet the mechanisms underlying these phenotypes remain obscure. Here, we demonstrate that mammalian MDC1/NFBD1 directly binds to phospho-H2AX (gammaH2AX) by specifically interacting with the phosphoepitope at the gammaH2AX carboxyl terminus. Moreover, through a combination of biochemical, cell-biological, and X-ray crystallographic approaches, we reveal the molecular details of the MDC1/NFBD1-gammaH2AX complex. These data provide compelling evidence that the MDC1/NFBD1 BRCT repeat domain is the major mediator of gammaH2AX recognition following DNA damage. We further show that MDC1/NFBD1-gammaH2AX complex formation regulates H2AX phosphorylation and is required for normal radioresistance and efficient accumulation of DNA-damage-response proteins on damaged chromatin. Thus, binding of MDC1/NFBD1 to gammaH2AX plays a central role in the mammalian response to DNA damage.     

HIV-1 nucleocapsid protein binds to the viral DNA initiation sequences and chaperones their kissing interactions

The chaperone properties of the human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) are required for the two obligatory strand transfer reactions occurring during viral DNA synthesis. The second strand transfer relies on the destabilization and the subsequent annealing of the primer binding site sequences (PBS) at the 3' end of the (-) and (+) DNA strands. To characterize the binding and chaperone properties of NC on the (-)PBS and (+)PBS sequences, we monitored by steady-state and time-resolved fluorescence spectroscopy as well as by fluorescence correlation spectroscopy the interaction of NC with wild type and mutant oligonucleotides corresponding to the (-)PBS and (+)PBS hairpins. NC was found to bind with high affinity to the loop, the stem and the single-stranded protruding sequence of both PBS sequences. NC induces only a limited destabilization of the secondary structure of both sequences, activating the transient melting of the stem only during its "breathing" period. This probably results from the high stability of the PBS due to the four G-C pairs in the stem. In contrast, NC directs the formation of "kissing" homodimers efficiently for both (-)PBS and (+)PBS sequences. Salt-induced dimerization and mutations in the (-)PBS sequence suggest that these homodimers may be stabilized by two intermolecular G-C Watson-Crick base-pairs between the partly self-complementary loops. The propensity of NC to promote the dimerization of partly complementary sequences may favor secondary contacts between viral sequences and thus, recombination and viral diversity.