Kylene Kehn

The HTLV-I Tax oncoprotein targets the retinoblastoma protein for proteasomal degradation

Human T-cell leukemia virus type-I (HTLV-I), the etiologic agent of adult T-cell leukemia (ATL), is estimated to affect 10–20 million people worldwide. The transforming ability of HTLV-I has been largely attributed to the viral protein Tax, which modulates the activity of several well-known cell cycle regulators. An important cell cycle regulator, the retinoblastoma (Rb) protein, is often inactivated in many cancers including virally induced cancers. Upon examination of Rb status, we observed a decrease in Rb protein expression in HTLV-1-infected cell lines as well as in ex vivo ATL patient samples. Transient transfection assays indicated that decreased Rb protein levels were Tax dependent. Here, we demonstrate for the first time that Tax directly associates with Rb. This interaction was localized within the B pocket of Rb and the C-terminus of Tax (aa 245–353). Within the C-terminus of Tax, we have identified an LXCXE-like motif, that when mutated resulted in the loss of Tax/Rb interaction. Furthermore, through the use of proteasome inhibitors, such as MG-132, in vivo and proteasome degradation assays in vitro, we found that Tax destabilizes the hypo-phosphorylated (active) form of Rb via the proteasome pathway. Therefore, we propose a model whereby Tax targets Rb to the proteasome by acting as a molecular bridge bringing Rb into contact with the proteasome for degradation.

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.

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.

Acetylated Tat Regulates Human Immunodeficiency Virus Type 1 Splicing through Its Interaction with the Splicing Regulator p32

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) potent transactivator Tat protein mediates pleiotropic effects on various cell functions. Posttranslational modification of Tat affects its activity during viral transcription. Tat binds to TAR and subsequently becomes acetylated on lysine residues by histone acetyltransferases. Novel protein-protein interaction domains on acetylated Tat are then established, which are necessary for both sustained transcriptional activation of the HIV-1 promoter and viral transcription elongation. In this study, we investigated the identity of proteins that preferentially bound acetylated Tat. Using a proteomic approach, we identified a number of proteins that preferentially bound AcTat, among which p32, a cofactor of splicing factor ASF/SF-2, was identified. We found that p32 was recruited to the HIV-1 genome, suggesting a mechanism by which acetylation of Tat may inhibit HIV-1 splicing needed for the production of full-length transcripts. Using Tat from different clades, harboring a different number of acetylation sites, as well as Tat mutated at lysine residues, we demonstrated that Tat acetylation affected splicing in vivo. Finally, using confocal microscopy, we found that p32 and Tat colocalize in vivo in HIV-1-infected cells.

The role of cyclin D2 and p21/waf1 in human T-cell leukemia virus type 1 infected cells

BackgroundThe human T-cell leukemia virus type 1 (HTLV-1) Tax protein indirectly influences transcriptional activation, signal transduction, cell cycle control, and apoptosis. The function of Tax primarily relies on protein-protein interactions. We have previously shown that Tax upregulates the cell cycle checkpoint proteins p21/waf1 and cyclin D2. Here we describe the consequences of upregulating these G1/S checkpoint regulators in HTLV-1 infected cells.ResultsTo further decipher any physical and functional interactions between cyclin D2 and p21/waf1, we used a series of biochemical assays from HTLV-1 infected and uninfected cells. Immunoprecipitations from HTLV-1 infected cells showed p21/waf1 in a stable complex with cyclin D2/cdk4. This complex is active as it phosphorylates the Rb protein in kinase assays. Confocal fluorescent microscopy indicated that p21/waf1 and cyclin D2 colocalize in HTLV-1 infected, but not in uninfected cells. Furthermore, in vitro kinase assays using purified proteins demonstrated that the addition of p21/waf1 to cyclin D2/cdk4 increased the kinase activity of cdk4.ConclusionThese data suggest that the p21/cyclin D2/cdk4 complex is not an inhibitory complex and that p21/waf1 could potentially function as an assembly factor for the cyclin D2/cdk4 complex in HTLV-1 infected cells. A by-product of this assembly with cyclin D2/cdk4 is the sequestration of p21/waf1 away from the cyclin E/cdk2 complex, allowing this active cyclin-cdk complex to phosphorylate Rb pocket proteins efficiently and push cells through the G1/S checkpoint. These two distinct functional and physical activities of p21/waf1 suggest that RNA tumor viruses manipulate the G1/S checkpoint by deregulating cyclin and cdk complexes.

Identifying the Membrane Proteome of HIV-1 Latently Infected Cells *□

Profiling integral plasma membrane proteins is of particular importance for the identification of new biomarkers for diagnosis and for drug development. We report in this study the identification of surface markers by performing comparative proteomics of established human immunodeficiency virus-1 (HIV-1) latent cell models and parental cell lines. To this end we isolated integral membrane proteins using a biotin-directed affinity purification method. Isolated proteins were separated by two-dimensional gel electrophoresis and identified by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) after in gel digestion. Seventeen different proteins were found to vary on the surface of T-cells due to HIV-1 infection. Of these proteins, 47% were integral membrane proteins, and 18% were membrane-associated. Through the use of complementary techniques such as Western blotting and fluorescent staining, we confirmed the differential expression of some of the proteins identified by MALDI-TOF including Brutons tyrosine kinase and X-linked inhibitor of apoptosis. Finally, using phosphatidylinositol 3-kinase inhibitors and flavopiridol to inhibit Brutons tyrosine kinase localization at the membrane and X-linked inhibitor of apoptosis protein expression, respectively, we showed that HIV-1 latently infected cells are more sensitive to these drugs than uninfected cells. This suggests that HIV-1 latently infected cells may be targeted with drugs that alter several pathways that are essential for the establishment and maintenance of latency.

Functional consequences of cyclin D1/BRCA1 interaction in breast cancer cells.

The inheritance of one defective BRCA1 or BRCA2 allele predisposes an individual to developing breast and ovarian cancers. BRCA1 is a multifunctional tumor suppressor protein, which through interaction with a vast array of proteins has implications in processes such as cell cycle, transcription, DNA damage response and chromatin remodeling. Conversely, the oncogene, cyclin D1 is overexpressed in about 35% of all breast cancer cases. In this study, we provide detailed analyses on the phosphorylation state of BRCA1 by cyclin D1/cdk4 complexes. In particular, we have identified Ser 632 of BRCA1 as a cyclin D1/cdk4 phosphorylation site in vitro. Using chromatin immunoprecipitation assays, we observed that the inhibition of cyclin D1/cdk4 activity resulted in increased BRCA1 DNA binding at particular promoters in vivo. In addition, we identified multiple novel genes that are bound by BRCA1 in vivo. Collectively, these results indicate that cyclin D1/cdk4-mediated phosphorylation of BRCA1 inhibits the ability of BRCA1 to be recruited to particular promoters in vivo. Therefore, cyclin D1/Cdk4 phosphorylation of BRCA1 could provide a mechanism to interfere with the DNA-dependent activities of BRCA1.

Pharmacological cyclin-dependent kinase inhibitors as HIV-1 antiviral therapeutics.

Human immunodeficiency virus type 1 (HIV-1) can infect quiescent cells; however, viral production is restricted to actively proliferating cells. Recent evidence has indicated that HIV-1 viral proteins, Vpr and Tat, perturb the cell cycle to optimize HIV-1 replication. Vpr arrests the cell cycle at G2 by inactivating the cyclin B/cdk1 complex. Tat regulates the cell cycle by altering factors involved in proliferation and differentiation (i.e. the cdk inhibitor p21/waf1) and associating with cyclin/cdk complexes (i.e. cyclin E/cdk2, cyclin H/cdk7, and cyclin T/cdk9). These studies indicate the importance of host cellular factors, such as cyclin/cdk complexes, in regulating HIV-1 replication and therefore represent novel targets for antiviral therapeutics. Recently, the efficacy of pharmalogical cdk inhibitors (PCIs) in abrogating viral replication has been under development. To date there are 25-30 PCIs that have been synthesized against known cdks, several of which have been shown to inhibit HIV-1 and other AIDS-associated viruses in vitro and in vivo. Targeting these critical cyclin/cdk complexes needed for viral propagation may solve the problems inherent in current HAART therapy, including the emergence of drug-resistant viruses. Thus, PCIs have the potential to become novel therapeutic antiviral drugs that can inhibit HIV-1 transcription and opens the possibility of new avenues of treatment.

Paradoxical effects of a stress signal on pro- and anti-apoptotic machinery in HTLV-1 Tax expressing cells

Adult T-cell leukemia (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) are associated with Human T-cell lymphotropic virus type 1 (HTLV-1) infection. The viral transactivator, Tax is able to mediate the cell cycle progression by targeting key regulators of the cell cycle such as p21/waf1, p16/ink4a, p53, cyclins D1–3/cdk complexes, and the mitotic spindle checkpoint MAD apparatus, thereby deregulating cellular DNA damage and checkpoint control.Genome expression profiling of infected cells exemplified by the development of DNA microarrays represents a major advance in genome-wide functional analysis. Utilizing cDNA microarray analysis, we have observed an apparent opposing and paradoxical regulatory network of host cell gene expression upon the introduction of DNA damage stress signal. We find the apparent induction of cell cycle inhibitors, and pro- as well as anti-apoptotic gene expression is directly linked to whether cells are at either G1, S, or G2/M phases of the cell cycle. Specifically, a G1/S block is induced by p21/waf1 and p16/ink4a, while pro-apoptotic expression at S, and G2/M is associated with caspase activation, and anti-apoptotic gene expression is associated with up regulation of Bcl-2 family member, namely bfl-1 gene. Therefore, the microarray results indicating expression of both pro- and anti-apoptotic genes could easily be explained by the particular stage of the cell cycle. Mechanism and the functional outcome of induction for both pathways are discussed.

Therapeutic targets for HIV-1 infection in the host proteome

BackgroundDespite the success of HAART, patients often stop treatment due to the inception of side effects. Furthermore, viral resistance often develops, making one or more of the drugs ineffective. Identification of novel targets for therapy that may not develop resistance is sorely needed. Therefore, to identify cellular proteins that may be up-regulated in HIV infection and play a role in infection, we analyzed the effects of Tat on cellular gene expression during various phases of the cell cycle.ResultsSOM and k-means clustering analyses revealed a dramatic alteration in transcriptional activity at the G1/S checkpoint. Tat regulates the expression of a variety of gene ontologies, including DNA-binding proteins, receptors, and membrane proteins. Using siRNA to knock down expression of several gene targets, we show that an Oct1/2 binding protein, an HIV Rev binding protein, cyclin A, and PPGB, a cathepsin that binds NA, are important for viral replication following induction from latency and de novo infection of PBMCs.ConclusionBased on exhaustive and stringent data analysis, we have compiled a list of gene products that may serve as potential therapeutic targets for the inhibition of HIV-1 replication. Several genes have been established as important for HIV-1 infection and replication, including Pou2AF1 (OBF-1), complement factor H related 3, CD4 receptor, ICAM-1, NA, and cyclin A1. There were also several genes whose role in relation to HIV-1 infection have not been established and may also be novel and efficacious therapeutic targets and thus necessitate further study. Importantly, targeting certain cellular protein kinases, receptors, membrane proteins, and/or cytokines/chemokines may result in adverse effects. If there is the presence of two or more proteins with similar functions, where only one protein is critical for HIV-1 transcription, and thus, targeted, we may decrease the chance of developing treatments with negative side effects.