Longwen Deng

Inhibition of Human Immunodeficiency Virus Type 1 Transcription by Chemical Cyclin-Dependent Kinase Inhibitors

ABSTRACT Cyclin-dependent kinases (cdks) have recently been suggested to regulate human immunodeficiency virus type 1 (HIV-1) transcription. Previously, we have shown that expression of one cdk inhibitor, p21/Waf1, is abrogated in HIV-1 latently infected cells. Based on this result, we investigated the transcription of HIV-1 in the presence of chemical drugs that specifically inhibited cdk activity and functionally mimicked p21/Waf1 activity. HIV-1 production in virally integrated lymphocytic and monocytic cell lines, such as ACH2, 8E5, and U1, as well as activated peripheral blood mononuclear cells infected with syncytium-inducing (SI) or non-syncytium-inducing (NSI) HIV-1 strains, were all inhibited by Roscovitine, a purine derivative that reversibly competes for the ATP binding site present in cdks. The decrease in viral progeny in the HIV-1-infected cells was correlated with a decrease in the transcription of HIV-1 RNAs in cells treated with Roscovitine and not with the non-cdk general cell cycle inhibitors, such as hydroxyurea (G1/S blocker) or nocodazole (M-phase blocker). Cyclin A- and E-associated histone H1 kinases, as well as cdk 7 and 9 activities, were all inhibited in the presence of Roscovitine. The 50% inhibitory concentration of Roscovitine on cdks 9 and 7 was determined to be ∼0.6 μM. Roscovitine could selectively sensitize HIV-1-infected cells to apoptosis at concentrations that did not impede the growth and proliferation of uninfected cells. Apoptosis induced by Roscovitine was found in both latent and activated infected cells, as evident by Annexin V staining and the cleavage of the PARP protein by caspase-3. More importantly, contrary to many apoptosis-inducing agents, where the apoptosis of HIV-1-infected cells accompanies production and release of infectious HIV-1 viral particles, Roscovitine treatment selectively killed HIV-1-infected cells without virion release. Collectively, our data suggest that cdks are required for efficient HIV-1 transcription and, therefore, we propose specific cdk inhibitors as potential antiviral agents in the treatment of AIDS.

Gene expression profile of HIV-1 Tat expressing cells: a close interplay between proliferative and differentiation signals.

BackgroundExpression profiling holds great promise for rapid host genome functional analysis. It is plausible that host expression profiling in an infection could serve as a universal phenotype in virally infected cells. Here, we describe the effect of one of the most critical viral activators, Tat, in HIV-1 infected and Tat expressing cells. We utilized microarray analysis from uninfected, latently HIV-1 infected cells, as well as cells that express Tat, to decipher some of the cellular changes associated with this viral activator.ResultsUtilizing uninfected, HIV-1 latently infected cells, and Tat expressing cells, we observed that most of the cellular host genes in Tat expressing cells were down-regulated. The down-regulation in Tat expressing cells is most apparent on cellular receptors that have intrinsic receptor tyrosine kinase (RTK) activity and signal transduction members that mediate RTK function, including Ras-Raf-MEK pathway. Co-activators of transcription, such as p300/CBP and SRC-1, which mediate gene expression related to hormone receptor genes, were also found to be down-regulated. Down-regulation of receptors may allow latent HIV-1 infected cells to either hide from the immune system or avoid extracellular differentiation signals. Some of the genes that were up-regulated included co-receptors for HIV-1 entry, translation machinery, and cell cycle regulatory proteins.ConclusionsWe have demonstrated, through a microarray approach, that HIV-1 Tat is able to regulate many cellular genes that are involved in cell signaling, translation and ultimately control the host proliferative and differentiation signals.

Effect of SWI/SNF chromatin remodeling complex on HIV-1 Tat activated transcription

BackgroundHuman immunodeficiency virus type 1 (HIV-1) is the etiologic agent of acquired immunodeficiency virus (AIDS). Following entry into the host cell, the viral RNA is reverse transcribed into DNA and subsequently integrated into the host genome as a chromatin template. The integrated proviral DNA, along with the specific chromatinized environment in which integration takes place allows for the coordinated regulation of viral transcription and replication. While the specific roles of and interplay between viral and host proteins have not been fully elucidated, numerous reports indicate that HIV-1 retains the ability for self-regulation via the pleiotropic effects of its viral proteins. Though viral transcription is fully dependent upon host cellular factors and the state of host activation, recent findings indicate a complex interplay between viral proteins and host transcription regulatory machineries including histone deacetylases (HDACs), histone acetyltransferases (HATs), cyclin dependent kinases (CDKs), and histone methyltransferases (HMTs).ResultsHere, we describe the effect of Tat activated transcription at the G1/S border of the cell cycle and analyze the interaction of modified Tat with the chromatin remodeling complex, SWI/SNF. HIV-1 LTR DNA reconstituted into nucleosomes can be activated in vitro using various Tat expressing extracts. Optimally activated transcription was observed at the G1/S border of the cell cycle both in vitro and in vivo, where chromatin remodeling complex, SWI/SNF, was present on the immobilized LTR DNA. Using a number of in vitro binding as well as in vivo chromatin immunoprecipitation (ChIP) assays, we detected the presence of both BRG1 and acetylated Tat in the same complex. Finally, we demonstrate that activated transcription resulted in partial or complete removal of the nucleosome from the start site of the LTR as evidenced by a restriction enzyme accessibility assay.ConclusionWe propose a model where unmodified Tat is involved in binding to the CBP/p300 and cdk9/cyclin T1 complexes facilitating transcription initiation. Acetylated Tat dissociates from the TAR RNA structure and recruits bromodomain-binding chromatin modifying complexes such as p/CAF and SWI/SNF to possibly facilitate transcription elongation.

ROLE OF VIRAL REGULATORY AND ACCESSORY PROTEINS IN HIV-1 REPLICATION

Human immunodeficiency virus-1 (HIV-1) is the causative agent of acquired immune deficiency syndrome (AIDS), a disease characterized by CD4+ T lymphocyte depletion. HIV-1 replicates actively in a variety of cells by encoding several regulatory (Tat and Rev) and accessory (Vpr, Vif, Vpu, and Nef) proteins. Accessory proteins, thought initially to be dispensable for infection, have now been shown to be important for efficient infection in vivo. Recent evidence suggests that certain viral proteins, like Vif, have evolved to overcome the antiviral mechanisms of the host, while proteins like Nef, which are markers for disease pathogenesis in vivo, help to increase pathogenesis by targeting bystander cells. Thus, these proteins control many aspects of the virus life cycle as well as host cell function, namely gene regulation and apoptosis. Understanding the mechanisms by which the virus is able to successfully replicate in host cells and subsequently cause gradual destruction of the immune system may yield new approaches for therapeutic strategies. In this review, we attempt to integrate information on the role of these regulatory and accessory proteins, emphasizing their interactions with other viral and cellular components, and the subsequent effect on viral replication.

Chromatin Remodeling and Modification during HIV-1 Tat-activated Transcription

Human immunodeficiency virus type 1 (HIV-1) is the etiologic agent of AIDS. Following entry into the host cell, the viral RNA is reverse transcribed into DNA and subsequently integrated into the host genome as a chromatin template. Chromatin structure may be responsible for silencing retroviral gene expression. Transcriptional activation occurs after ATP-dependent chromatin remodeling complexes alter chromatin structure and positioning of nucleosomes. Histone acetyltransferases (HATs), histone deacetylases (HDACs), kinases, and methyltransferases (HMTs), covalently modify nucleosomes by adding or removing chemical moieties in the N-terminal tails of histones. Recent advances have indicated that HIV-1 encoded proteins interact with chromatin remodeling complexes and histone modifying enzymes, implying that chromatin remodeling plays an important role in the HIV-1 life cycle. Nucleosomes are positioned on the HIV-1 LTR and are barriers to transcription. Following cellular activation, these nucleosomes are modified and repositioned allowing for activation of viral gene expression. Tat recruits various HATs to the HIV-1 promoter region and can also be acetylated by some of these enzymes. Unmodified Tat is involved in binding to the CBP/p300 and cdk9/cyclin T complexes and facilitates transcription initiation. Acetylated Tat dissociates from the TAR RNA structure and recruits bromodomain-containing chromatin modifying complexes such as p/CAF and SWI/SNF to facilitate transcription elongation. This review summarizes our current knowledge and understanding of chromatin remodeling complexes and their regulation of HIV-1 replication, and highlights the important contributions HIV-1 research has made to further our understanding of the transcription process.

Overexpression of p21(waf1) in human T-cell lymphotropic virus type 1-infected cells and its association with cyclin A/cdk2.

ABSTRACT Human T-cell lymphotropic virus type 1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). T-cell transformation is mainly due to the actions of the viral phosphoprotein Tax. Tax interacts with multiple transcriptional factors, aiding the transcription of many cellular genes. Here, we report that the cyclin-dependent kinase inhibitor p21/waf1 is overexpressed in all HTLV-1-infected cell lines tested as well as in ATL and HAM/TSP patient samples. Tax was found to be able to transactivate the endogenous p21/waf1 promoter, as detected by RNase protection, as well as activate a series of wild-type and 5′-deletion constructs linked to a luciferase reporter cassette. Wild-type but not a mutant form of Tax (M47) transactivated the p21/waf1 promoter in a p53-independent manner and utilized a minimal promoter that contained E2A and TATA box sequences. The p21/waf1 protein was reproducibly observed to be complexed with cyclin A/cdk2 and not with any other known G1, S, or G2/M cyclins. Functionally, the association of p21/cyclin A/cdk2 decreased histone H1 phosphorylation in vitro, as observed in immunoprecipitations followed by kinase assays, and affected other substrates, such as the C terminus of Rb protein involved in c-Abl and histone deacetylase-1 (HDAC1) regulation. Interestingly, upon the use of a stress signal, such as gamma-irradiation, we found that the p21/cyclin A/cdk2 complex was able to block all known phosphorylation sites on the Rb molecule. Finally, using elutriated cell cycle fractions and a stress signal, we observed that the HTLV-1-infected T cells containing wild-type Tax, which had been in early or mid-G1 phase prior to gamma-irradiation, arrested in G1 and did not undergo apoptosis. This may be an important mechanism for an oncogenic virus such as HTLV-1 to stop the host at the G1/S boundary and to repair the damaged DNA upon injury, prior to S-phase entry.

Protein Profile of Tax-associated Complexes

Infection with human T-cell leukemia virus type 1 (HTLV-1) results in adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. Tax, a 40-kDa protein, regulates viral and cellular transcription, host signal transduction, the cell cycle, and apoptosis. Tax has been shown to modulate cellular CREB and NFκB pathways; however, to date, its role in binding to various host cellular proteins involved in tumorigenesis has not been fully described. In this study, we describe the Tax-associated proteins and their functions in cells using several approaches. Tax eluted from a sizing column mostly at an apparent molecular mass of 1800 kDa. Following Tax immunoprecipitation, washes with high salt buffer, two-dimensional gel separation, and mass spectrometric analysis, a total of 32 proteins was identified. Many of these proteins belong to the signal transduction and cytoskeleton pathways and transcription/chromatin remodeling. A few of these proteins, including TXBP151, have been shown previously to bind to Tax. The interaction of Tax with small GTPase-cytoskeleton proteins, such as ras GAP1m, Rac1, Cdc42, RhoA, and gelsolin, indicates how Tax may regulate migration, invasion, and adhesion in T-cell cancers. Finally, the physical and functional association of Tax with the chromatin remodeling SWI/SNF complex was assessed using in vitro chromatin remodeling assays, chromatin remodeling factor BRG1 mutant cells, and RNA interference experiments. Collectively, Tax is able to bind and regulate many cellular proteins that regulate transcription and cytoskeletal related pathways, which might explain the pleiotropic effects of Tax leading to T-cell transformation and leukemia in HTLV-1-infected patients.

Gene expression array of HTLV type 1-infected T cells : Up-regulation of transcription factors and cell cycle genes

By utilizing a human cDNA expression array blot (588 genes), we have observed overexpression of various transcription factors, cell cycle regulated kinases, and DNA repair genes in HTLV-1-infected T cells. One of the genes of interest, and focus in this study, is the cyclin-dependent kinase inhibitor, p21/waf1. The p21/waf1 transcription and protein is overexpressed in all HTLV-1-infected cell lines tested as well as ATL and HAM/TSP patient samples. While p21/waf1 has been shown to display a selectivity for G(1)/S cyclin/cdk complexes, we have observed p21/waf1 to be complexed with cyclin A/cdk2. Functionally, the association of p21/cyclin A/cdk2 decreased the histone H1 phosphorylation in vitro, as observed in immunoprecipitations followed by kinase assays, as well as affecting other substrates such as the C-terminus of Rb protein involved in c-Abl and HDAC1 regulation. Wild-type, but not a mutant form (M47) of Tax, was found to be able to transactivate the p21/waf1 promoter in a p53-independent manner. We found that the minimal p21/waf1 promoter (-49 to +49 sequence) was activated by Tax and the minimal promoter contained two E2A transcription factor binding sites located between the TATA box and the initiation site. E2A proteins, E12 and E47, as well as a related helix-loop-helix protein, HEB, are all up-regulated in HTLV-1-infected T cells. When using band shift analysis, we found that only the E1 site (overlapping the transcription start site) was a functional DNA binding site. By using a chromatin immunoprecipitation (ChIP) assay, we observed that histone H4, and not histone H3, was acetylated from the endogenous p21/waf1 promoter in vivo, implying that CBP/p300, and not the SAGA complex, was critical in complexing with E2A in up-regulation of p21/waf1 in HTLV-1-infected cells.

Loss of G1/S Checkpoint in Human Immunodeficiency Virus Type 1-Infected Cells Is Associated with a Lack of Cyclin-Dependent Kinase Inhibitor p21/Waf1

ABSTRACT Productive high-titer infection by human immunodeficiency virus type 1 (HIV-1) requires the activation of target cells. Infection of quiescent peripheral CD4 lymphocytes by HIV-1 results in incomplete, labile reverse transcripts and lack of viral progeny formation. An interplay between Tat and p53 has previously been reported, where Tat inhibited the transcription of the p53 gene, which may aid in the development of AIDS-related malignancies, and p53 expression inhibited HIV-1 long terminal repeat transcription. Here, by using a well-defined and -characterized stress signal, gamma irradiation, we find that upon gamma irradiation, HIV-1-infected cells lose their G1/S checkpoints, enter the S phase inappropriately, and eventually apoptose. The loss of the G1/S checkpoint is associated with a loss of p21/Waf1 protein and increased activity of a major G1/S kinase, namely, cyclin E/cdk2. The p21/Waf1 protein, a known cyclin-dependent kinase inhibitor, interacts with the cdk2/cyclin E complex and inhibits progression of cells into S phase. We find that loss of the G1/S checkpoint in HIV-1-infected cells may in part be due to Tats ability to bind p53 (a known activator of the p21/Waf1 promoter) and sequester its transactivation activity, as seen in both in vivo and in vitro transcription assays. The loss of p21/Waf1 in HIV-1-infected cells was specific to p21/Waf1 and did not occur with other KIP family members, such as p27 (KIP1) and p57 (KIP2). Finally, the advantage of a loss of the G1/S checkpoint for HIV-1 per se may be that it pushes the host cell into the S phase, which may then allow subsequent virus-associated processes, such as RNA splicing, transport, translation, and packaging of virion-specific genes, to occur.

Coordination of Transcription Factor Phosphorylation and Histone Methylation by the P-TEFb Kinase during Human Immunodeficiency Virus Type 1 Transcription

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Tat protein recruits positive transcription elongation factor b (P-TEFb) to the transactivation response (TAR) RNA structure to facilitate formation of processive transcription elongation complexes (TECs). Here we examine the role of the Tat/TAR-specified cyclin-dependent kinase 9 (CDK9) kinase activity in regulation of HIV-1 transcription elongation and histone methylation. In HIV-1 TECs, P-TEFb phosphorylates the RNA polymerase II (RNAP II) carboxyl-terminal domain (CTD) and the transcription elongation factors SPT5 and Tat-SF1 in a Tat/TAR-dependent manner. Using in vivo chromatin immunoprecipitation analysis, we demonstrate the following distinct properties of the HIV-1 transcription complexes. First, the RNAP II CTD is phosphorylated at Ser 2 and Ser 5 near the promoter and at downstream coding regions. Second, the stable association of SPT5 with the TECs is dependent upon P-TEFb kinase activity. Third, P-TEFb kinase activity is critical for the induction of methylation of histone H3 at lysine 4 and lysine 36 on HIV-1 genes. Flavopiridol, a potent P-TEFb kinase inhibitor, inhibits CTD phosphorylation, stable SPT5 binding, and histone methylation, suggesting that its potent antiviral activity is due to its ability to inhibit several critical and unique steps in HIV-1 transcription elongation.