Kuan Teh Jeang

Human T Cell Leukemia Virus Type 1 Oncoprotein Tax Targets the Human Mitotic Checkpoint Protein MAD1

In searching for cellular targets of the HTLV-I oncoprotein Tax, we identified TXBP181, which we characterized as the human homolog of yeast mitotic checkpoint MAD1 protein. Evidence supporting TXBP181 as HsMAD1 includes sequence conservation with yeast MAD1, hyperphosphorylation during S/G2/M phases and upon treatment of cells with nocodazole, and binding to HsMAD2. HsMAD1 functions as a homodimer. It localizes to the centrosome during metaphase and to the spindle midzone and the midbody during anaphase and telophase. Expression of either Tax or a transdominant-negative TXBP181 results in multinucleated cells, a phenotype consistent with a loss of HsMAD1 function. We propose a model of viral transformation in which Tax targets TXBP181, thereby abrogating a mitotic checkpoint.

Multifaceted Activities of the HIV-1 Transactivator of Transcription, Tat

Human immunodeficiency virus, type 1 (HIV-1) is the etiological agent for the acquired immunodeficiency syndrome (AIDS). HIV-1 is a retrovirus that encodes a small nuclear transcriptional activator protein, Tat (Fig. 1). In vivo, Tat is required for virus replication and is conserved in the genomes of all primate lentiviruses (1). Over the past decade, the transcriptional function(s) of Tat (reviewed in detail several years ago (2)) has been intensely investigated. It has become clear that a primary role for Tat is in regulating productive and processive transcription from the HIV-1 long terminal repeat (LTR). Tat also has other activities; some are consistent with that of a secreted growth factor (3–5) and a potentiator of reverse transcription (6). Here we review recent insights into the multifaceted activities of Tat.

HIV-1 tat transcriptional activity is regulated by acetylation.

The human immunodeficiency virus (HIV) trans‐ activator protein, Tat, stimulates transcription from the viral long‐terminal repeats (LTR) through an RNA hairpin element, trans‐activation responsive region (TAR). We and others have shown that trans‐activator protein (Tat)‐associated histone acetyltransferases (TAHs), p300 and p300/CBP‐associating factor (PCAF), assist functionally in the activation of chromosomally integrated HIV‐1 LTR. Here, we show that p300 and PCAF also directly acetylate Tat. We defined two sites of acetylation located in different functional domains of Tat. p300 acetylated Lys50 in the TAR RNA binding domain, while PCAF acetylated Lys28 in the activation domain of Tat. In support of a functional role for acetylation in vivo, histone deacetylase inhibitor (trichostatin A) synergized with Tat in transcriptional activation of the HIV‐1 LTR. Synergism was TAR‐dependent and required the intact presence of both Lys28 and Lys50. Mechanistically, acetylation at Lys28 by PCAF enhanced Tat binding to the Tat‐associated kinase, CDK9/P‐TEFb, while acetylation by p300 at Lys50 of Tat promoted the dissociation of Tat from TAR RNA that occurs during early transcription elongation. These data suggest that acetylation of Tat regulates two discrete and functionally critical steps in transcription, binding to an RNAP II CTD‐kinase and release of Tat from TAR RNA.

Activation of Integrated Provirus Requires Histone Acetyltransferase p300 AND P/CAF ARE COACTIVATORS FOR HIV-1 Tat

A unique aspect of the retrovirus life cycle is the obligatory integration of the provirus into host cell chromosomes. Unlike viruses that do not integrate, retroviruses must conserve an ability to activate transcription from a chromatin context. Human immunodeficiency virus (HIV)-1 encodes an unusual and an unusually potent transcriptional transactivator, Tat, which binds to a nascent viral leader RNA, TAR. The action of Tat has been well studied in various reductive model systems; however, the physiological mechanism through which Tat gains access to chromatin-associated proviral long terminal repeats (LTRs) is not understood. We show here that a nuclear histone acetyltransferase activity associates with Tat. Intracellularly, we found that Tat forms a ternary complex with p300 and P/CAF, two histone acetyltransferases (HATs). A murine cell defect in Tat transactivation of the HIV-1 LTR was linked to the reduced abundance of p300 and P/CAF. Thus, overexpression of p300 and P/CAF reconstituted Tat transactivation of the HIV-1 LTR in NIH3T3 cells to a level similar to that observed for human cells. By using transdominant p300 or P/CAF mutants that lack enzymatic activity, we delineated a requirement for the HAT component from the latter but not the former in Tat function. Finally, we observed that Tat-associated HAT is preferentially important for transactivation of integrated, but not unintegrated, HIV-1 LTR.

Genome-wide expression changes induced by HTLV-1 tax: evidence for MLK-3 mixed lineage kinase involvement in Tax-mediated NF-κB activation

The Tax protein of human T-lymphotropic virus type 1 (HTLV-1), an oncoprotein that transactivates viral and cellular genes, plays a key role in HTLV-1 replication and pathogenesis. We used cDNA microarrays to examine Tax-mediated transcriptional changes in the human Jurkat T-cell lines JPX-9 and JPX-M which express Tax and Tax-mutant protein, respectively, under the control of an inducible promoter. Approximately 300 of the over 2000 genes examined were differentially expressed in the presence of Tax. These genes were grouped according to their function and are discussed in the context of existing findings in the literature. There was strong agreement between our results and genes previously reported as being Tax-responsive. Genes that were differentially expressed in the presence of Tax included those related to apoptosis, the cell cycle and DNA repair, signaling factors, immune modulators, cytokines and growth factors, and adhesion molecules. Functionally, we provide evidence that one of these genes, the mixed-lineage kinase MLK-3, is involved in Tax-mediated NF-κB signaling. Our current results provide additional insights into Tax-mediated signaling.

Host restriction factors in retroviral infection: promises in virus-host interaction

Retroviruses have an intricate life cycle. There is much to be learned from studying retrovirus-host interactions. Among retroviruses, the primate lentiviruses have one of the more complex genome structures with three categories of viral genes: structural, regulatory, and accessory genes. Over time, we have gained increasing understanding of the lentivirus life cycle from studying host factors that support virus replication. Similarly, studies on host restriction factors that inhibit viral replication have also made significant contributions to our knowledge. Here, we review recent progress on the rapidly growing field of restriction factors, focusing on the antiretroviral activities of APOBEC3G, TRIM5, tetherin, SAMHD1, MOV10, and cellular microRNAs (miRNAs), and the counter-activities of Vif, Vpu, Vpr, Vpx, and Nef.

MicroRNA silencing of tumor suppressor DLC‐1 promotes efficient hepatitis C virus replication in primary human hepatocytes

MicroRNAs (miRNAs) are approximately 22‐nucleotide noncoding RNAs that constitute silencers of target gene expression. Aberrant expression of miRNA has been linked to a variety of cancers, including hepatocellular carcinoma (HCC). Hepatitis C virus (HCV) infection is considered a major cause of chronic liver disease and HCC, although the mechanism of virus infection–associated hepatocarcinogenesis remains unclear. We report a direct role of miRNAs induced in HCV‐infected primary human hepatocytes that target the tumor suppressor gene DLC‐1 (a Rho GTPase‐activating protein), which is frequently deleted in HCC, and other solid human tumors. MicroRNA miR‐141 that targets DLC‐1 was accentuated in cells infected with HCV genotypes 1a, 1b, and 2a. We present several lines of evidence that efficient HCV replication requires miR‐141–mediated suppression of DLC‐1. An increase in miR‐141 correlated with the inhibition of DLC‐1 protein in HCV‐infected cells. Depletion of miR‐141 with oligonucleotides complementary to the miRNAs inhibited virus replication, whereas artificially increased levels of intracellular miR‐141 enhanced HCV replication. HCV‐infected hepatocytes showed enhanced cell proliferation that can be countered by overexpression of DLC‐1. Conclusion: The collective results of this study suggest a novel mechanism of HCV infection–associated miRNA‐mediated regulation of a tumor suppressor protein that has the ability to influence cell proliferation and HCV infection–mediated liver cancer. (HEPATOLOGY 2011)

Ubiquitin-Specific Peptidase 20 Targets TRAF6 and Human T Cell Leukemia Virus Type 1 Tax To Negatively Regulate NF-κB Signaling

ABSTRACT NF-κB plays a key role in innate and acquired immunity. Its activity is regulated through intricate signaling networks. Persistent or excessive activation of NF-κB induces diseases, such as autoimmune disorders and malignant neoplasms. Infection by human T cell leukemia virus type 1 (HTLV-1) causes a fatal hematopoietic malignancy termed adult T cell leukemia (ATL). The HTLV-1 viral oncoprotein Tax functions pivotally in leukemogenesis through its potent activation of NF-κB. Recent findings suggest that protein ubiquitination is crucial for proper regulation of NF-κB signaling and for Tax activity. Here, we report that ubiquitin-specific peptidase USP20 deubiquitinates TRAF6 and Tax and suppresses interleukin 1β (IL-1β)- and Tax-induced NF-κB activation. Our results point to USP20 as a key negative regulator of Tax-induced NF-κB signaling.

p53 negatively regulates the expression of FAT10, a gene upregulated in various cancers.

FAT10 is a member of the ubiquitin-like modifier family of proteins and has been implicated to play important roles in antigen presentation, cytokine response, apoptosis and mitosis. We have recently demonstrated the upregulation of FAT10 gene expression in 90% of hepatocellular carcinoma patients. Here, we identified and characterized the promoter of the FAT10 gene to elucidate the mechanism of FAT10 gene expression. Notably, we found that the 5′ untranslated region (5′UTR), from the transcription start site to 15 bases before the translational start site, displays significant promoter activity. Regions upstream of the 5′UTR (from +26 to −1997) do not confer any promoter activity. Curiously, FAT10 promoter activity and expression is significantly repressed in KB3-1 and HepG2 cells, which have wild-type p53, than in p53-negative Hep3B cells. The role of p53 in regulating FAT10 expression was evident by the significant downregulation (P<0.05) of FAT10 mRNA expression and promoter activity when wild-type p53 was transfected into p53-null Hep3B cells. Conversely, inhibiting p53 expression through siRNA against p53 significantly enhanced FAT10 expression and promoter activity. p53 was found to bind in vivo to the 5′ half consensus sequence of p53-binding site located at the FAT10 promoter. Hence, we propose that FAT10 is a downstream target of p53 and dysregulation of FAT10 expression in p53-defective cells could contribute to carcinogenesis.

Promoter activity of Tat at steps subsequent to TATA-binding protein recruitment.

Artificial recruitment of TATA-binding protein (TBP) to many eukaryotic promoters bypasses DNA-bound activator function. The human immunodeficiency virus type 1 (HIV-1) Tat is an unconventional activator that up-regulates transcription from the HIV-1 long terminal repeat (LTR) through binding to a nascent RNA sequence, TAR. Because this LTR and its cognate activator have atypical features compared to a standard RNA polymerase II (RNAP II) transcriptional unit, the precise limiting steps for HIV-1 transcription and how Tat resolves these limitations remain incompletely understood. We thus constructed human TBP fused to the DNA-binding domain of GAL4 to determine whether recruitment of TBP is one rate-limiting step in HIV-1 LTR transcription and whether Tat functions to recruit TBP. As a control, we compared the activity of the adenovirus E1b promoter. Our findings indicate that TBP tethering to the E1b promoter fully effected transcription to the same degree achievable with the potent GAL4-VP16 activator. By contrast, TBP recruitment to the HIV-1 LTR, although necessary for conferring Tat responsiveness, did not bypass a physical need for Tat in achieving activated transcription. These results document that the HIV-1 and the E1b promoters are transcriptionally limited at different steps; the major rate-limiting step for E1b is recruitment of TBP, while activation of the HIV-1 LTR requires steps in addition to TBP recruitment. We suggest that Tat acts to accelerate rate-limiting steps after TBP recruitment.