USP7 deubiquitinase controls HIV-1 production by stabilizing Tat protein

Deubiquitinases (DUBs) are key regulators of complex cellular processes. HIV-1 Tat is synthesized early after infection and is mainly responsible for enhancing viral production. Here, we report that one of the DUBs, USP7, stabilized the HIV-1 Tat protein through its deubiquitination. Treatment with either a general DUB inhibitor (PR-619) or USP7-specific inhibitor (P5091) resulted in Tat protein degradation. The USP7-specific inhibitor reduced virus production in a latently infected T-lymphocytic cell line J1.1, which produces large amounts of HIV-1 upon stimulation. A potent increase in Tat-mediated HIV-1 production was observed with USP7 in a dose-dependent manner. As expected, deletion of the USP7 gene using the CRISPR-Cas9 method reduced the Tat protein and supported less virus production. Interestingly, the levels of endogenous USP7 increased after HIV-1 infection in human T-cells (MOLT-3) and in mammalian cells transfected with HIV-1 proviral DNA. Thus, HIV-1 Tat is stabilized by the host cell deubiquitinase USP7, leading to enhanced viral production, and HIV-1 in turn up-regulates the USP7 protein level.

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Shutdown of HIV-1 Transcription in T Cells by Nullbasic, a Mutant Tat Protein

mBio ◽

10.1128/mbio.00518-16 ◽

2016 ◽

Vol 7 (4) ◽

Cited By ~ 11

Author(s):

Hongping Jin ◽

Dongsheng Li ◽

Haran Sivakumaran ◽

Mary Lor ◽

Lina Rustanti ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Virus Replication ◽

Virus Production ◽

Jurkat Cells ◽

Mutant Form ◽

Tat Protein ◽

Functional Cure ◽

Latently Infected ◽

Hiv 1

ABSTRACTNullbasic is a derivative of the HIV-1 transactivator of transcription (Tat) protein that strongly inhibits HIV-1 replication in lymphocytes. Here we show that lentiviral vectors that constitutively express a Nullbasic-ZsGreen1 (NB-ZSG1) fusion protein by the eEF1α promoter led to robust long-term inhibition of HIV-1 replication in Jurkat cells. Although Jurkat-NB-ZSG1 cells were infected by HIV-1, no virus production could be detected and addition of phorbol ester 12-myristate 13-acetate (PMA) and JQ1 had no effect, while suberanilohydroxamic acid (SAHA) modestly stimulated virus production but at levels 300-fold lower than those seen in HIV-1-infected Jurkat-ZSG1 cells. Virus replication was not recovered by coculture of HIV-1-infected Jurkat-NB-ZSG1 cells with uninfected Jurkat cells. Latently infected Jurkat latent 6.3 and ACH2 cells treated with latency-reversing agents produced measurable viral capsid (CA), but little or none was made when they expressed NB-ZSG1. When Jurkat cells chronically infected with HIV-1 were transduced with lentiviral virus-like particles conveying NB-ZSG1, a >3-log reduction in CA production was observed. Addition of PMA increased virus CA production but at levels 500-fold lower than those seen in nontransduced Jurkat cells. Transcriptome sequencing analysis confirmed that HIV-1 mRNA was strongly inhibited by NB-ZSG1 but indicated that full-length viral mRNA was made. Analysis of HIV-1-infected Jurkat cells expressing NB-ZSG1 by chromatin immunoprecipitation assays indicated that recruitment of RNA polymerase II (RNAPII) and histone 3 lysine 9 acetylation were inhibited. The reduction of HIV-1 promoter-associated RNAPII and epigenetic changes in viral nucleosomes indicate that Nullbasic can inhibit HIV-1 replication by enforcing viral silencing in cells.IMPORTANCEHIV-1 infection is effectively controlled by antiviral therapy that inhibits virus replication and reduces measurable viral loads in patients below detectable levels. However, therapy interruption leads to viral rebound due to latently infected cells that serve as a source of continued viral infection. Interest in strategies leading to a functional cure of HIV infection by permanent viral suppression, which may be achievable, is growing. Here we show that a mutant form of the HIV-1 Tat protein, referred to as Nullbasic, can inhibit HIV-1 transcription in infected Jurkat T cell to undetectable levels. Analysis shows that Nullbasic alters the epigenetic state of the HIV-1 long terminal repeat promoter, inhibiting its association with RNA polymerase II. This study indicates that key cellular proteins and pathways targeted here can silence HIV-1 transcription. Further elucidation could lead to functional-cure strategies by suppression of HIV transcription, which may be achievable by a pharmacological method.

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Transduction of Cu,Zn-superoxide dismutase mediated by an HIV-1 Tat protein basic domain into mammalian cells

FEBS Letters ◽

10.1016/s0014-5793(00)02215-8 ◽

2000 ◽

Vol 485 (2-3) ◽

pp. 163-167 ◽

Cited By ~ 89

Author(s):

Hyeok Yil Kwon ◽

Won Sik Eum ◽

Hyun Woo Jang ◽

Jung Hoon Kang ◽

Jiyoon Ryu ◽

...

Keyword(s):

Superoxide Dismutase ◽

Mammalian Cells ◽

Tat Protein ◽

Basic Domain ◽

Hiv 1

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Protein Transduction Domain of HIV-1 Tat Protein Promotes Efficient Delivery of DNA into Mammalian Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m010625200 ◽

2001 ◽

Vol 276 (28) ◽

pp. 26204-26210 ◽

Cited By ~ 201

Author(s):

Akiko Eguchi ◽

Teruo Akuta ◽

Hajime Okuyama ◽

Takao Senda ◽

Haruhiko Yokoi ◽

...

Keyword(s):

Mammalian Cells ◽

Protein Transduction ◽

Protein Transduction Domain ◽

Tat Protein ◽

Efficient Delivery ◽

Hiv 1

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Inhibition of Cyclin-dependent Kinase 1 Induces Cytokinesis without Chromosome Segregation in an ECT2 and MgcRacGAP-dependent Manner

Journal of Biological Chemistry ◽

10.1074/jbc.m508007200 ◽

2005 ◽

Vol 280 (43) ◽

pp. 36502-36509 ◽

Cited By ~ 58

Author(s):

Fumihiko Niiya ◽

Xiaozhen Xie ◽

Kyung S. Lee ◽

Hiroki Inoue ◽

Toru Miki

Keyword(s):

Rna Interference ◽

Mammalian Cells ◽

Specific Inhibitor ◽

Small Gtpase ◽

Dominant Negative ◽

Cleavage Furrow ◽

Dependent Manner ◽

Cyclin Dependent Kinase ◽

Mitotic Spindles ◽

Initiation Mechanism

Cleavage furrow formation marks the onset of cell division during early anaphase. The small GTPase RhoA and its regulators ECT2 and MgcRacGAP have been implicated in furrow ingression in mammalian cells, but the signaling upstream of these molecules remains unclear. We now show that the inhibition of cyclin-dependent kinase (Cdk)1 is sufficient to initiate cytokinesis. When mitotically synchronized cells were treated with the Cdk-specific inhibitor BMI-1026, the initiation of cytokinesis was induced precociously before chromosomal separation. Cytokinesis was also induced by the Cdk1-specific inhibitor purvalanol A but not by Cdk2/Cdk5- or Cdk4-specific inhibitors. Consistent with initiation of precocious cytokinesis by Cdk1 inhibition, introduction of anti-Cdk1 monoclonal antibody resulted in cells with aberrant nuclei. Depolymerization of mitotic spindles by nocodazole inhibited BMI-1026-induced precocious cytokinesis. However, in the presence of a low concentration of nocodazole, BMI-1026 induced excessive membrane blebbing, which appeared to be caused by formation of ectopic cleavage furrows. Depletion of ECT2 or MgcRacGAP by RNA interference abolished both of the phenotypes (precocious furrowing after nocodazole release and excessive blebbing in the presence of nocodazole). RNA interference of RhoA or expression of dominant-negative RhoA efficiently reduced both phenotypes. RhoA was localized at the cleavage furrow or at the necks of blebs. We propose that Cdk1 inactivation is sufficient to activate a signaling pathway leading to cytokinesis, which emanates from mitotic spindles and is regulated by ECT2, MgcRacGAP, and RhoA. Chemical induction of cytokinesis will be a valuable tool to study the initiation mechanism of cytokinesis.

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Regulation of circular dorsal ruffles, macropinocytosis, and cell migration by RhoG and its exchange factor, Trio

Molecular Biology of the Cell ◽

10.1091/mbc.e16-06-0412 ◽

2017 ◽

Vol 28 (13) ◽

pp. 1768-1781 ◽

Cited By ~ 13

Author(s):

Alejandra Valdivia ◽

Silvia M. Goicoechea ◽

Sahezeel Awadia ◽

Ashtyn Zinn ◽

Rafael Garcia-Mata

Keyword(s):

Cell Migration ◽

Mammalian Cells ◽

Dorsal Surface ◽

Receptor Internalization ◽

Dependent Manner ◽

Cellular Functions ◽

Unique Type ◽

Exchange Factor ◽

Cellular Processes

Circular dorsal ruffles (CDRs) are actin-rich structures that form on the dorsal surface of many mammalian cells in response to growth factor stimulation. CDRs represent a unique type of structure that forms transiently and only once upon stimulation. The formation of CDRs involves a drastic rearrangement of the cytoskeleton, which is regulated by the Rho family of GTPases. So far, only Rac1 has been consistently associated with CDR formation, whereas the role of other GTPases in this process is either lacking or inconclusive. Here we show that RhoG and its exchange factor, Trio, play a role in the regulation of CDR dynamics, particularly by modulating their size. RhoG is activated by Trio downstream of PDGF in a PI3K- and Src-dependent manner. Silencing RhoG expression decreases the number of cells that form CDRs, as well as the area of the CDRs. The regulation of CDR area by RhoG is independent of Rac1 function. In addition, our results show the RhoG plays a role in the cellular functions associated with CDR formation, including macropinocytosis, receptor internalization, and cell migration. Taken together, our results reveal a novel role for RhoG in the regulation of CDRs and the cellular processes associated with their formation.

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Transduction of human catalase mediated by an HIV-1 TAT protein basic domain and arginine-rich peptides into mammalian cells

Free Radical Biology and Medicine ◽

10.1016/s0891-5849(01)00734-1 ◽

2001 ◽

Vol 31 (11) ◽

pp. 1509-1519 ◽

Cited By ~ 85

Author(s):

Li Hua Jin ◽

Jae Hoon Bahn ◽

Won Sik Eum ◽

Hyeok Yil Kwon ◽

Sang Ho Jang ◽

...

Keyword(s):

Mammalian Cells ◽

Tat Protein ◽

Basic Domain ◽

Hiv 1

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Anti-Vpr Activity of a Yeast Chaperone Protein

Journal of Virology ◽

10.1128/jvi.78.20.11016-11029.2004 ◽

2004 ◽

Vol 78 (20) ◽

pp. 11016-11029 ◽

Cited By ~ 18

Author(s):

Zsigmond Benko ◽

Dong Liang ◽

Emmanuel Agbottah ◽

Jason Hou ◽

Karen Chiu ◽

...

Keyword(s):

Heat Shock ◽

Fission Yeast ◽

Mammalian Cells ◽

Suppressive Effect ◽

Dependent Manner ◽

Protein Protein Interaction ◽

Chaperone Protein ◽

Genome Wide ◽

Multicopy Suppressors ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) exerts multiple effects on viral and host cellular activities during viral infection, including nuclear transport of the proviral integration complex, induction of cell cycle G2 arrest, and cell death. In this report, we show that a fission yeast chaperone protein Hsp16 inhibits HIV-1 by suppressing these Vpr activities. This protein was identified through three independent genome-wide screens for multicopy suppressors of each of the three Vpr activities. Consistent with the properties of a heat shock protein, heat shock-induced elevation or overproduction of Hsp16 suppressed Vpr activities through direct protein-protein interaction. Even though Hsp16 shows a stronger suppressive effect on Vpr in fission yeast than in mammalian cells, similar effects were also observed in human cells when fission yeast hsp16 was expressed either in vpr-expressing cells or during HIV-1 infection, indicating a possible highly conserved Vpr suppressing activity. Furthermore, stable expression of hsp16 prior to HIV-1 infection inhibits viral replication in a Vpr-dependent manner. Together, these data suggest that Hsp16 inhibits HIV-1 by suppressing Vpr-specific activities. This finding could potentially provide a new approach to studying the contribution of Vpr to viral pathogenesis and to reducing Vpr-mediated detrimental effects in HIV-infected patients.

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The Antiproliferative and Apoptotic Effects of Sirtinol, a Sirtuin Inhibitor on Human Lung Cancer Cells by Modulating Akt/β-Catenin-Foxo3A Axis

The Scientific World JOURNAL ◽

10.1155/2014/937051 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 9

Author(s):

Yao Fong ◽

Yin-Chieh Lin ◽

Chang-Yi Wu ◽

Hui-Min David Wang ◽

Li-Li Lin ◽

...

Keyword(s):

Lung Cancer ◽

Cancer Cells ◽

Mammalian Cells ◽

Specific Inhibitor ◽

Annexin V ◽

Nonsmall Cell Lung Cancer ◽

Human Lung Cancer ◽

Nonhistone Proteins ◽

Cellular Processes ◽

Phosphorylated Akt

Sirtuins, NAD+-dependent deacetylases, could target both histones and nonhistone proteins in mammalian cells. Sirt1 is the major sirtuin and has been shown to involve various cellular processes, including antiapoptosis, cellular senescence. Sirt1 was reported to be overexpressed in many cancers, including lung cancer. Sirtinol, a specific inhibitor of Sirt1, has been shown to induce apoptosis of cancer cells by elevating endogenous level of reactive oxygen species. In the study, we investigated the effect of sirtinol on the proliferation and apoptosis of nonsmall cell lung cancer (NSCLC) H1299 cells. The results of proliferation assay and colony formation assay showed the antigrowth effect of sirtinol. The annexin-V staining further confirmed the apoptosis induction by sirtinol treatment. Interestingly, the levels of phosphorylated Akt andβ-catenin were significantly downregulated with treating the apoptotic inducing doses. On the contrary, sirtinol treatment causes the significantly increased level of FoxO3a, a proapoptotic transcription factor targeted by Sirt1. These above results suggested that sirtinol may inhibit cell proliferation of H1299 cells by regulating the axis of Akt-β-catenin-FoxO3a. Overall, this study demonstrates that sirtinol attenuates the proliferation and induces apoptosis of NSCLC cells, indicating the potential treatment against NSCLC cells by inhibiting Sirt1 in future applications.

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C-Terminal HIV-1 Transframe p6* Tetrapeptide Blocks Enhanced Gag Cleavage Incurred by Leucine Zipper Replacement of a Deleted p6* Domain

Journal of Virology ◽

10.1128/jvi.00103-17 ◽

2017 ◽

Vol 91 (10) ◽

Cited By ~ 4

Author(s):

Fu-Hsien Yu ◽

Kuo-Jung Huang ◽

Chin-Tien Wang

Keyword(s):

Leucine Zipper ◽

Virus Assembly ◽

Virus Production ◽

Supporting Evidence ◽

Dependent Manner ◽

Dimer Formation ◽

Reading Frame ◽

Virus Maturation ◽

C Terminus ◽

Hiv 1

ABSTRACT HIV-1 protease (PR) functions as a homodimer mediating virus maturation following virus budding. Gag-Pol dimerization is believed to trigger embedded PR activation by promoting PR dimer formation. Early PR activation can lead to markedly reduced virus yields due to premature Gag cleavage. The p6* peptide, located between Gag and PR, is believed to ensure virus production by preventing early PR maturation. Studies aimed at finding supporting evidence for this proposal are limited due to a reading frame overlap between p6* and the p6gag budding domain. To determine if p6* affects virus production via the modulation of PR activation, we engineered multiple constructs derived from Dp6*PR (an assembly- and processing-competent construct with Pol fused at the inactivated PR C terminus). The data indicated that a p6* deletion adjacent to active PR significantly impaired virus processing. We also observed that the insertion of a leucine zipper (LZ) dimerization motif in the deleted region eliminated virus production in a PR activity-dependent manner, suggesting that the LZ insertion triggered premature PR activation by facilitating PR dimer formation. As few as four C-terminal p6* residues remaining at the p6*/PR junction were sufficient to restore virus yields, with a Gag processing profile similar to that of the wild type. Our study provides supporting evidence in a virus assembly context that the C-terminal p6* tetrapeptide plays a role in preventing premature PR maturation. IMPORTANCE Supporting evidence for the assumption that p6* retards PR maturation in the context of virus assembly is lacking. We found that replacing p6* with a leucine zipper peptide abolished virus assembly due to the significant enhancement of Gag cleavage. However, as few as four C-terminal p6* residues remaining in the deleted region were sufficient for significant PR release, as well as for counteracting leucine zipper-incurred premature Gag cleavage. Our data provide evidence that (i) p6* ensures virus assembly by preventing early PR activation and (ii) four C-terminal p6* residues are critical for modulating PR activation. Current PR inhibitor development efforts are aimed largely at mature PR, but there is a tendency for HIV-1 variants that are resistant to multiple protease inhibitors to emerge. Our data support the idea of modulating PR activation by targeting PR precursors as an alternative approach to controlling HIV-1/AIDS.

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