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Journal articles on the topic 'HIV-1 Cyclic Mutant'
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Sree Kanth, S., and M. Vijjulatha. "Tetrahydroxy Cyclic Urea-Potent Inhibitor for HIV-1 Protease Wild Type and Mutant Type—A Computational Design." E-Journal of Chemistry 5, no. 3 (2008): 584–92. http://dx.doi.org/10.1155/2008/154030.
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A series of novel tetrahydroxy cyclic urea molecules as HIV-1 protease inhibitors were designed using computational techniques. The designed molecules were compared with the known cyclic urea molecules by performing docking studies on six of wild type protein and three mutant protein varieties and calculating their ADME properties. A series of novel molecules were designed by substituting hydrogen at the P1/ P1′ positions with hydroxyl group increasing the bioavailability these had better ADME properties and binding affinity towards HIV-1 protease. The biological activity of these inhibitors were predicted by a model equation generated by the regression analysis between biological activity (log 1/Ki) of known inhibitors and there combined docking scores from six of the wild type protein docking. The synthetic studies are in progress.
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Nillroth, U., L. Vrang, P. O. Markgren, J. Hultén, A. Hallberg, and U. H. Danielson. "Human immunodeficiency virus type 1 proteinase resistance to symmetric cyclic urea inhibitor analogs." Antimicrobial Agents and Chemotherapy 41, no. 11 (November 1997): 2383–88. http://dx.doi.org/10.1128/aac.41.11.2383.
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Resistant virus was isolated from virus propagated in cell culture in the presence of the human immunodeficiency virus type 1 (HIV-1) proteinase inhibitor DMP 323, Ro 31-8959, or A-75925. The proteinase gene of resistant virus was sequenced, and key mutations (G48V, V82A, I84V, L90M, and G48V/L90M) were introduced into clones used for the expression, purification, and further characterization of the enzyme. The mutant enzymes were all less active than the wild-type enzyme, as judged by k(cat) and k(cat)/Km values. L90M had a lower Km than the wild type, whereas the G48V/L90M double mutant had an increased Km compared with that of the wild type, contributing to a 10-fold reduction in the k(cat)/Km. Vitality values were used to show that the enzyme of the I84V mutant is the enzyme most resistant to the two cyclic urea inhibitors DMP 323 and AHA 008. Virus with the same mutation is also resistant, although the double mutation L10F/I84V confers even greater resistance. All of these mutants are more resistant to DMP 323 than to AHA 008. The resistance of the I84V mutant may be attributed to a loss of van der Waals interactions with the inhibitor, since the larger amino acid side chain involved in the interaction is replaced by a smaller side chain. This is supported by the lower level of resistance to AHA 008 that was observed. The phenyl groups of AHA 008 should protrude deeper into the S1 and S1' subsites than those of the smaller compound DMP 323, reducing the loss of interaction energy. These results reveal that small structural modifications of inhibitors that do not affect the inhibitory effect on wild-type virus can influence the inhibition of resistant strains. This is of importance for optimizing drugs with respect to their potency and resistance.
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Avram, Speranta, Cristian Bologa, and Maria-Luiza Flonta. "Quantitative structure-activity relationship by CoMFA for cyclic urea and nonpeptide-cyclic cyanoguanidine derivatives on wild type and mutant HIV-1 protease." Journal of Molecular Modeling 11, no. 2 (February 16, 2005): 105–15. http://dx.doi.org/10.1007/s00894-004-0226-5.
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Thomas, Christian A., Ofra K. Weinberger, Benedikt L. Ziegler, Steven Greenberg, Ira Schieren, Samuel C. Silverstein, and Joseph El Khoury. "Human Immunodeficiency Virus-1 env Impairs Fcγ Receptor-Mediated Phagocytosis Via a Cyclic Adenosine Monophosphate-Dependent Mechanism." Blood 90, no. 9 (November 1, 1997): 3760–65. http://dx.doi.org/10.1182/blood.v90.9.3760.
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Abstract Human immunodeficiency virus (HIV)-1 expression in mononuclear phagocytes is associated with multiple functional defects, including phagocytosis. To assess Fcγ receptor (FcγR) function in cells expressing HIV-1, human promonocytic cells (U937) acutely or chronically infected with HIV-1, or stably transfected with a noninfectious reverse transcriptase (RT) defective HIV-1 provirus (Δpol), were treated with phorbol 12-myristate 13-acetate for 48 hours and tested for their ability to ingest sheep erythrocytes coated with IgG (E-IgG). HIV-1–infected or transfected U937 cells ingested 50% to 65% fewer E-IgG than controls despite normal surface expression of FcγRs. HIV-1 specifically impaired FcγR-mediated phagocytosis, as ingestion of complement-coated erythrocytes was unaffected. U937 cells transfected with an env deficient mutant of HIV-1 ingested E-IgG normally, suggesting that the expression of HIV-1 env was required for HIV-1 to inhibit FcγR-mediated phagocytosis. Expression of HIV-1 in U937 cells was associated with an increased accumulation of intracellular cyclic adenosine monophosphate (cAMP); addition of the adenylate cyclase inhibitor 2′,5′-dideoxyadenosine to these cells decreased intracellular cAMP levels to that of controls and restored FcγR-mediated phagocytosis. Addition of either interferon (IFN)-γ or an inhibitor of cAMP-dependent protein kinase A (KT 5720) to HIV-1–transfected U937 cells also restored FcγR-mediated phagocytosis. Expression of HIV-1 induces a specific defect of FcγR function in mononuclear phagocytes that correlates with increased levels of cAMP, and can be corrected by pharmacologic manipulation.
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Ala, Paul J., Edward E. Huston, Ronald M. Klabe, Denise D. McCabe, Jodie L. Duke, Christopher J. Rizzo, Bruce D. Korant, et al. "Molecular Basis of HIV-1 Protease Drug Resistance: Structural Analysis of Mutant Proteases Complexed with Cyclic Urea Inhibitors." Biochemistry 36, no. 21 (May 1997): 6556. http://dx.doi.org/10.1021/bi9750044.
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Ala, Paul J., Edward E. Huston, Ronald M. Klabe, Denise D. McCabe, Jodie L. Duke, Christopher J. Rizzo, Bruce D. Korant, et al. "Molecular Basis of HIV-1 Protease Drug Resistance: Structural Analysis of Mutant Proteases Complexed with Cyclic Urea Inhibitors†." Biochemistry 36, no. 7 (February 1997): 1573–80. http://dx.doi.org/10.1021/bi962234u.
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Garg, Rajni, and Barun Bhhatarai. "Possible allosteric interactions of monoindazole-substituted P2 cyclic urea analogues with wild-type and mutant HIV-1 protease." Journal of Computer-Aided Molecular Design 22, no. 10 (March 27, 2008): 737–45. http://dx.doi.org/10.1007/s10822-008-9210-y.
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Ala, Paul J., Edward E. Huston, Ronald M. Klabe, Prabhakar K. Jadhav, Patrick Y. S. Lam, and Chong-Hwan Chang. "Counteracting HIV-1 Protease Drug Resistance: Structural Analysis of Mutant Proteases Complexed with XV638 and SD146, Cyclic Urea Amides with Broad Specificities." Biochemistry 37, no. 43 (October 1998): 15042–49. http://dx.doi.org/10.1021/bi980386e.
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Ammosova, Tatyana, Marina Jerebtsova, Monique Beullens, Bart Lesage, Angela Jackson, Fatah Kashanchi, William Southerland, Victor R. Gordeuk, Mathieu Bollen, and Sergei Nekhai. "Nuclear Targeting of Protein Phosphatase-1 by HIV-1 Tat Protein." Journal of Biological Chemistry 280, no. 43 (August 29, 2005): 36364–71. http://dx.doi.org/10.1074/jbc.m503673200.
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Transcription of human immunodeficiency virus (HIV)-1 genes is activated by HIV-1 Tat protein, which induces phosphorylation of the C-terminal domain of RNA polymerase-II by CDK9/cyclin T1. We previously showed that Tat-induced HIV-1 transcription is regulated by protein phosphatase-1 (PP1). In the present study we demonstrate that Tat interacts with PP1 and that disruption of this interaction prevents induction of HIV-1 transcription. We show that PP1 interacts with Tat in part through the binding of Val36 and Phe38 of Tat to PP1 and that Tat is involved in the nuclear and subnuclear targeting of PP1. The PP1 binding mutant Tat-V36A/F38A displayed a decreased affinity for PP1 and was a poor activator of HIV-1 transcription. Surprisingly, Tat-Q35R mutant that had a higher affinity for PP1 was also a poor activator of HIV-1 transcription, because strong PP1 binding competed out binding of Tat to CDK9/cyclin T1. Our results suggest that Tat might function as a nuclear regulator of PP1 and that interaction of Tat with PP1 is critical for activation of HIV-1 transcription by Tat.
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Jadlowsky, Julie K., Masanori Nojima, Takashi Okamoto, and Koh Fujinaga. "Dominant negative mutant cyclin T1 proteins that inhibit HIV transcription by forming a kinase inactive complex with Tat." Journal of General Virology 89, no. 11 (November 1, 2008): 2783–87. http://dx.doi.org/10.1099/vir.0.2008/002857-0.
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Transcription of the human immunodeficiency virus type 1 (HIV) requires the interaction of the cyclin T1 (CycT1) subunit of a host cellular factor, the positive transcription elongation factor b (P-TEFb), with the viral Tat protein, at the transactivation response element (TAR) of nascent transcripts. Because of this virus-specific interaction, CycT1 may potentially serve as a target for the development of anti-HIV therapies. Here we report the development of a mutant CycT1 protein, containing three threonine-to-alanine substitutions in the linker region between two of the cyclin boxes, which displays a potent dominant negative effect on HIV transcription. Investigation into the inhibitory mechanism revealed that this mutant CycT1 interacted with Tat and the cyclin-dependent kinase 9 (Cdk9) subunit of P-TEFb, but failed to stimulate the Cdk9 kinase activity critical for elongation. This mutant CycT1 protein may represent a novel class of specific inhibitors of HIV transcription which could lead to development of new antiviral therapies.
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Fujinaga, Koh, Dan Irwin, Matthias Geyer, and B. Matija Peterlin. "Optimized Chimeras between Kinase-Inactive Mutant Cdk9 and Truncated Cyclin T1 Proteins Efficiently Inhibit Tat Transactivation and Human Immunodeficiency Virus Gene Expression." Journal of Virology 76, no. 21 (November 1, 2002): 10873–81. http://dx.doi.org/10.1128/jvi.76.21.10873-10881.2002.
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ABSTRACT The human cyclin T1 (hCycT1) protein from the positive transcription elongation factor b (P-TEFb) binds the transactivator Tat and the transactivation response (TAR) RNA stem loop from human immunodeficiency virus type 1 (HIV). This complex activates the elongation of viral transcription. To create effective inhibitors of Tat and thus HIV replication, we constructed mutant hCycT1 proteins that are defective in binding its kinase partner, Cdk9, or TAR. Although these mutant hCycT1 proteins did not increase Tat transactivation in murine cells, their dominant-negative effects were small in human cells. Higher inhibitory effects were obtained when hCycT1 was fused with the mutant Cdk9 protein. Since the autophosphorylation of the C terminus of Cdk9 is required for the formation of the stable complex between P-TEFb, Tat, and TAR, these serines and threonines were changed to glutamate in a kinase-inactive Cdk9 protein. This chimera inhibited Tat transactivation and HIV gene expression in human cells. Therefore, this dominant-negative kinase-inactive mutant Cdk9.hCycT1 chimera could be used for antiviral gene therapy.
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Mameli, Giuseppe, Satish L. Deshmane, Mohammad Ghafouri, Jianqi Cui, Kenneth Simbiri, Kamel Khalili, Ruma Mukerjee, Antonina Dolei, Shohreh Amini, and Bassel E. Sawaya. "C/EBPβ regulates human immunodeficiency virus 1 gene expression through its association with cdk9." Journal of General Virology 88, no. 2 (February 1, 2007): 631–40. http://dx.doi.org/10.1099/vir.0.82487-0.
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Transcriptional regulation of the human immunodeficiency virus type 1 (HIV-1) is a complex event that requires the cooperative action of both viral (e.g. Tat) and cellular (e.g. C/EBPβ, NF-κB) factors. The HIV-1 Tat protein recruits the human positive transcription elongation factor P-TEFb, consisting of cdk9 and cyclin T1, to the HIV-1 transactivation response (TAR) region. In the absence of TAR, Tat activates the HIV-1 long terminal repeat (LTR) through its association with several cellular factors including C/EBPβ. C/EBPβ is a member of the CCAAT/enhancer-binding protein family of transcription factors and has been shown to be a critical transcriptional regulator of HIV-1 LTR. We examined whether Tat–C/EBPβ association requires the presence of the P-TEFb complex. Using immunoprecipitation followed by Western blot, we demonstrated that C/EBPβ–cyclin T1 association requires the presence of cdk9. Further, due to its instability, cdk9 was unable to physically interact with C/EBPβ in the absence of cyclin T1 or Tat. Using kinase assays, we demonstrated that cdk9, but not a cdk9 dominant-negative mutant (cdk9-dn), phosphorylates C/EBPβ. Our functional data show that co-transfection of C/EBPβ and cdk9 leads to an increase in HIV-1 gene expression when compared to C/EBPβ alone. Addition of C/EBP homologous protein (CHOP) inhibits C/EBPβ transcriptional activity in the presence and absence of cdk9 and causes a delay in HIV-1 replication in T-cells. Together, our data suggest that Tat–C/EBPβ association is mediated through cdk9, and that phosphorylated C/EBPβ may influence AIDS progression by increasing expression of HIV-1 genes.
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Roth-Cross, Jessica K., Helen Stokes, Guohui Chang, Ming Ming Chua, Volker Thiel, Susan R. Weiss, Alexander E. Gorbalenya, and Stuart G. Siddell. "Organ-Specific Attenuation of Murine Hepatitis Virus Strain A59 by Replacement of Catalytic Residues in the Putative Viral Cyclic Phosphodiesterase ns2." Journal of Virology 83, no. 8 (January 28, 2009): 3743–53. http://dx.doi.org/10.1128/jvi.02203-08.
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ABSTRACT The Murine hepatitis virus (MHV) strain A59 ns2 protein is a 30-kDa nonstructural protein that is expressed from a subgenomic mRNA in the cytoplasm of virus-infected cells. Its homologs are also encoded in other closely related group 2a coronaviruses and more distantly related toroviruses. Together, these proteins comprise a subset of a large superfamily of 2H phosphoesterase proteins that are distinguished by a pair of conserved His-x-Thr/Ser motifs encompassing catalytically important residues. We have used a vaccinia virus-based reverse genetic system to produce recombinant viruses encoding ns2 proteins with single-amino-acid substitutions in, or adjacent to, these conserved motifs, namely, inf-ns2 H46A, inf-ns2 S48A, inf-ns2-S120A, and inf-ns2-H126R. All of the mutant viruses replicate in mouse 17 clone 1 fibroblast cells and mouse embryonic cells to the same extent as the parental wild-type recombinant virus, inf-MHV-A59. However, compared to inf-MHV-A59, the inf-ns2 H46A and inf-ns2-H126R mutants are highly attenuated for replication in mouse liver following intrahepatic inoculation. Interestingly, none of the mutant viruses were attenuated for replication in mouse brain following intracranial inoculation. These results show that the ns2 protein of MHV-A59 has an important role in virus pathogenicity and that a substitution of the histidine residues of the MHV-A59 ns2 His-x-Thr/Ser motifs is critical for virus virulence in the liver but not in the brain. This novel phenotype suggests a strategy to investigate the function of the MHV-A59 ns2 protein involving the search for organ-specific proteins or RNAs that react differentially to wild-type and mutant ns2 proteins.
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Fujinaga, Koh, Dan Irwin, Ran Taube, Fan Zhang, Matthias Geyer, and B. Matija Peterlin. "A Minimal Chimera of Human Cyclin T1 and Tat Binds TAR and Activates Human Immunodeficiency Virus Transcription in Murine Cells." Journal of Virology 76, no. 24 (December 15, 2002): 12934–39. http://dx.doi.org/10.1128/jvi.76.24.12934-12939.2002.
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ABSTRACT The transcriptional elongation of human immunodeficiency virus type 1 (HIV-1) is mediated by the virally encoded transactivator Tat and its cellular cofactor, positive transcription elongation factor b (P-TEFb). The human cyclin T1 (hCycT1) subunit of P-TEFb forms a stable complex with Tat and the transactivation response element (TAR) RNA located at the 5′ end of all viral transcripts. Previous studies have demonstrated that hCycT1 binds Tat in a Zn2+-dependent manner via the cysteine at position 261, which is a tyrosine in murine cyclin T1. In the present study, we mutated all other cysteines and histidines that could be involved in this Zn2+-dependent interaction. Because all of these mutant proteins except hCycT1(C261Y) activated viral transcription in murine cells, no other cysteine or histidine in hCycT1 is responsible for this interaction. Next, we fused the N-terminal 280 residues in hCycT1 with Tat. Not only the full-length chimera but also the mutant hCycT1 with an N-terminal deletion to position 249, which retained the Tat-TAR recognition motif, activated HIV-1 transcription in murine cells. This minimal hybrid mutant hCycT1-Tat protein bound TAR RNA as well as human and murine P-TEFb in vitro. We conclude that this minimal chimera not only reproduces the high-affinity binding among P-TEFb, Tat, and TAR but also will be invaluable for determining the three-dimensional structure of this RNA-protein complex.
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Nekhai, Sergei, Namita Kumari, Min Xu, Altreisha Foster, Sharmin Diaz, and Victor R. Gordeuk. "Ferroportin Q248H Mutation Protects From HIV-1 Infection in Vitro." Blood 120, no. 21 (November 16, 2012): 993. http://dx.doi.org/10.1182/blood.v120.21.993.993.
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Abstract Abstract 993 Ferroportin is the only iron exporter expressed in mammalian cells, and hepcidin produced by the liver binds to ferroportin leading to its internalization and degradation by lysosomes. We recently reported that expression of ferroportin in 293T cells transfected with HIV-1 LTR-LacZ and Tat expression vector led to decreased HIV transcription, possibly by reducing availability of intracellular iron, and that exposure to hepcidin restored HIV transcription1. The Q248H mutation in ferroportin has an allele frequency of 2.2–13.4% in African populations and is associated with a mild tendency to increased serum ferritin in the general population. The ferroportin Q248H mutation was reported to associate with lower hepcidin levels in HIV-1 infected Rwandese women2. We also recently showed that ferroportin Q248H mutant has reduced sensitivity to physiologic hepcidin concentrations. We expressed WT and Q248H mutant ferroportin in 293T cells that express very low levels of endogenous ferroportin. We also expressed ferroportin C326Y, a mutant that is not sensitive to hepcidin. We analyzed the effect of ferroportin Q248H on cellular Intracellular ferritin levels which reflect the amount of iron stored within the cells. 293T cells were transfected with ferroportin expressing vectors, incubated with ferric ammonium citrate as a source of iron, pretreated with cycloheximide to stop de-novo protein synthesis and then treated with 30 nM hepcidin. Ferritin levels increased significantly in the cells expressing WT ferroportin and treated with hepcidin (Fig.1A). In contrast, ferritin levels remained the same in untreated and hepcidin treated cells expressing ferroportin Q248H or C326Y (Fig.1A). This observation suggests continuing iron export by ferroportin Q248H with low dose hepcidin. HIV-1 transcription can be induced in 293T cells by co-expression of HIV-1 LTR reporter construct and HIV-1 Tat expression vector (Fig.1B, lane 2). HIV-1 Tat binds to TAR RNA located in the beginning of HIV-1 transcript and facilitates a recruitment of a host cell transcription elongation factor, CDK9/cyclin T1, inducing efficient elongation of HIV-1 transcription. Expression of ferroportin WT, Q248H or C326Y mutant inhibited Tat –induced HIV-1 transcription in comparison to non-relevant control (Fig.1B, lanes 3, 4, 6 and 8). Treatment with physiological hepcidin concentrations reversed the inhibition of Tat-induced HIV-1 transcription by WT but not the Q248H or C326Y mutant ferroportin (Fig.1B, lanes 5, 7 and 9). In this experiment, we utilized c-myc tagged ferroportin expression vectors as in our previous study1. We also obtained very similar results with EGFP-fused ferroportin expression, which also allowed an easier detection of reduction in ferroportin expression in the presence of hepcidin. Finally, we also isolated monocytes from two subjects, one with heterozygote and one with homozygote ferroportin Q248H. Monocytes were infected ex-vivo with pseudotyped HIV-1 virus expressing luciferase. HIV-1 replication was reduced in primary monocytes with heterozygote and homozygote ferroportin Q248H as compared to a control. Ferroportin glutamine 248 is located within the intracellular loop (residues 228–307), in close proximity to lysine residues 229–269 which ubiquitination promotes ferroportin internalization3. Future studies should address the details of ubiquitination of human ferroportin Q248H compared to WT ferroportin. An added protection value could be observed lower hepcidin expression levels in HIV-1 infected individuals with the ferroportin Q248H2. Further studies are needed to uncover a mechanism of this reduced hepcidin expression. Further molecular analysis is needed to understand the mechanism of ferroportin Q248H internalization. Taken together, our study shows that the ferroportin Q248H that has a reduced sensitivity to hepcidin may offer an additional protection from HIV-1. Acknowledgments. This work was supported NIH Research Grants SC1GM082325, R25 HL003679, 2G12RR003048, 8G12MD007597, K25GM097501 and 1P30HL107253. Disclosures: No relevant conflicts of interest to declare.
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Jadhav, Prabhakar K., Paul Ala, Francis J. Woerner, Chong-Hwan Chang, Sena S. Garber, Elizabeth D. Anton, and Lee T. Bacheler. "Cyclic Urea Amides: HIV-1 Protease Inhibitors with Low Nanomolar Potency against both Wild Type and Protease Inhibitor Resistant Mutants of HIV." Journal of Medicinal Chemistry 40, no. 2 (January 1997): 181–91. http://dx.doi.org/10.1021/jm960586t.
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Xie, Baode, Cédric F. Invernizzi, Stéphane Richard, and Mark A. Wainberg. "Arginine Methylation of the Human Immunodeficiency Virus Type 1 Tat Protein by PRMT6 Negatively Affects Tat Interactions with both Cyclin T1 and the Tat Transactivation Region." Journal of Virology 81, no. 8 (January 31, 2007): 4226–34. http://dx.doi.org/10.1128/jvi.01888-06.
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ABSTRACT Arginine methylation has been shown to regulate signal transduction, protein subcellular localization, gene transcription, and protein-protein interactions that ultimately alter gene expression. Although the role of cellular protein arginine methyltransferases (PRMT) in viral gene expression is largely unknown, we recently showed that the Tat protein of human immunodeficiency virus type 1 (HIV-1) is a substrate for one such enzyme, termed PRMT6. However, the mechanism by which arginine methylation impairs the transactivation potential of Tat and the sites of arginine methylation within Tat remain obscure. We now show that Tat is a specific in vitro and in vivo substrate of PRMT6 which targets the Tat R52 and R53 residues for arginine methylation. Such Tat methylation led to decreased interaction with the Tat transactivation region (TAR) of viral RNA. Furthermore, arginine methylation of Tat negatively affected Tat-TAR-cyclin T1 ternary complex formation and diminished cyclin T1-dependent Tat transcriptional activation. Overexpression of wild-type PRMT6, but not a methylase-inactive PRMT6 mutant, reduced levels of Tat transactivation of HIV-1 long terminal repeat chloramphenicol acetyltransferase and luciferase reporter plasmids in a dose-dependent manner. In cell-based assays, knockdown of PRMT6 resulted in increased HIV-1 production and faster viral replication. Thus, PRMT6 can compromise Tat transcriptional activation and may represent a form of innate cellular immunity in regard to HIV-1 replication. Finding a way of inhibiting or stimulating PRMT6 activity might help to drive quiescently infected cells out of latency or combat HIV-1 replication, respectively.
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Yik, Jasper H. N., Ruichuan Chen, Andrea C. Pezda, Craig S. Samford, and Qiang Zhou. "A Human Immunodeficiency Virus Type 1 Tat-Like Arginine-Rich RNA-Binding Domain Is Essential for HEXIM1 To Inhibit RNA Polymerase II Transcription through 7SK snRNA-Mediated Inactivation of P-TEFb." Molecular and Cellular Biology 24, no. 12 (June 15, 2004): 5094–105. http://dx.doi.org/10.1128/mcb.24.12.5094-5105.2004.
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ABSTRACT The HEXIM1 protein inhibits the kinase activity of P-TEFb (CDK9/cyclin T) to suppress RNA polymerase II transcriptional elongation in a process that specifically requires the 7SK snRNA, which mediates the interaction of HEXIM1 with P-TEFb. In an attempt to define the sequence requirements for HEXIM1 to interact with 7SK and inactivate P-TEFb, we have identified the first 18 amino acids within the previously described nuclear localization signal (NLS) of HEXIM1 as both necessary and sufficient for binding to 7SK in vivo and in vitro. This 7SK-binding motif was essential for HEXIM1's inhibitory action, as the HEXIM1 mutants with this motif replaced with a foreign NLS failed to interact with 7SK and P-TEFb and hence were unable to inactivate P-TEFb. The 7SK-binding motif alone, however, was not sufficient to inhibit P-TEFb. A region C-terminal to this motif was also required for HEXIM1 to associate with P-TEFb and suppress P-TEFb's kinase and transcriptional activities. The 7SK-binding motif in HEXIM1 contains clusters of positively charged residues reminiscent of the arginine-rich RNA-binding motif found in a wide variety of proteins. Part of it is highly homologous to the TAR RNA-binding motif in the human immunodeficiency virus type 1 (HIV-1) Tat protein, which was able to restore the 7SK-binding ability of a HEXIM1 NLS substitution mutant. We propose that a similar RNA-protein recognition mechanism may exist to regulate the formation of both the Tat-TAR-P-TEFb and the HEXIM1-7SK-P-TEFb ternary complexes, which may help convert the inactive HEXIM1/7SK-bound P-TEFb into an active one for Tat-activated and TAR-dependent HIV-1 transcription.
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Garber, Mitchell E., Timothy P. Mayall, Eric M. Suess, Jill Meisenhelder, Nancy E. Thompson, and Katherine A. Jones. "CDK9 Autophosphorylation Regulates High-Affinity Binding of the Human Immunodeficiency Virus Type 1 Tat–P-TEFb Complex to TAR RNA." Molecular and Cellular Biology 20, no. 18 (September 15, 2000): 6958–69. http://dx.doi.org/10.1128/mcb.20.18.6958-6969.2000.
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ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Tat interacts with cyclin T1 (CycT1), a regulatory partner of CDK9 in the positive transcription elongation factor (P-TEFb) complex, and binds cooperatively with CycT1 to TAR RNA to recruit P-TEFb and promote transcription elongation. We show here that Tat also stimulates phosphorylation of affinity-purified core RNA polymerase II and glutathioneS-transferase–C-terminal-domain substrates by CycT1-CDK9, but not CycH-CDK7, in vitro. Interestingly, incubation of recombinant Tat–P-TEFb complexes with ATP enhanced binding to TAR RNA dramatically, and the C-terminal half of CycT1 masked binding of Tat to TAR RNA in the absence of ATP. ATP incubation lead to autophosphorylation of CDK9 at multiple C-terminal Ser and Thr residues, and full-length CycT1 (amino acids 728) [CycT1(1–728)], but not truncated CycT1(1–303), was also phosphorylated by CDK9. P-TEFb complexes containing a catalytically inactive CDK9 mutant (D167N) bound TAR RNA weakly and independently of ATP, as did a C-terminal truncated CDK9 mutant that was catalytically active but unable to undergo autophosphorylation. Analysis of different Tat proteins revealed that the 101-amino-acid SF2 HIV-1 Tat was unable to bind TAR with CycT1(1–303) in the absence of phosphorylated CDK9, whereas unphosphorylated CDK9 strongly blocked binding of HIV-2 Tat to TAR RNA in a manner that was reversed upon autophosphorylation. Replacement of CDK9 phosphorylation sites with negatively charged residues restored binding of CycT1(1–303)-D167N-Tat, and rendered D167N a more potent inhibitor of transcription in vitro. Taken together, these results demonstrate that CDK9 phosphorylation is required for high-affinity binding of Tat–P-TEFb to TAR RNA and that the state of P-TEFb phosphorylation may regulate Tat transactivation in vivo.
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Labrosse, Béatrice, Anne Brelot, Nikolaus Heveker, Nathalie Sol, Dominique Schols, Erik De Clercq, and Marc Alizon. "Determinants for Sensitivity of Human Immunodeficiency Virus Coreceptor CXCR4 to the Bicyclam AMD3100." Journal of Virology 72, no. 8 (August 1, 1998): 6381–88. http://dx.doi.org/10.1128/jvi.72.8.6381-6388.1998.
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ABSTRACT The bicyclam AMD3100 is a potent and selective inhibitor of the replication of human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2). It was recently demonstrated that the compound inhibited HIV entry through CXCR4 but not through CCR5. Selectivity of AMD3100 for CXCR4 was further indicated by its lack of effect on HIV-1 and HIV-2 infection mediated by the CCR5, CCR3, Bonzo, BOB, and US28, coreceptors. AMD3100 completely blocked HIV-1 infection mediated by a mutant CXCR4 bearing a deletion of most of the amino-terminal extracellular domain. In contrast, relative resistance to AMD3100 was conferred by different single amino acid substitutions in the second extracellular loop (ECL2) or in the adjacent membrane-spanning domain, TM4. Only substitutions of a neutral residue for aspartic acid and of a nonaromatic residue for phenylalanine (Phe) were associated with drug resistance. This suggests a direct interaction of AMD3100 with these amino acids rather than indirect effects of their mutation on the CXCR4 structure. The interaction of aspartic acids of ECL2 and TM4 with AMD3100 is consistent with the positive charge of bicyclams, which might block HIV-1 entry by preventing electrostatic interactions between CXCR4 and the HIV-1 envelope protein gp120. Other features of AMD3100 must account for its high antiviral activity, in particular the presence of an aromatic linker between the cyclam units. This aromatic group might engage in hydrophobic interactions with the Phe-X-Phe motifs of ECL2 or TM4. These results confirm the importance of ECL2 for the HIV coreceptor activity of CXCR4.
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Mergenthaler, Philipp, Claudia Muselmann, Juliane Sünwoldt, Nickolay K. Isaev, Tadeusz Wieloch, Ulrich Dirnagl, Andreas Meisel, and Karsten Ruscher. "A Functional Role of the Cyclin-Dependent Kinase Inhibitor 1 (P21WAF1/CIP1) for Neuronal Preconditioning." Journal of Cerebral Blood Flow & Metabolism 33, no. 3 (January 9, 2013): 351–55. http://dx.doi.org/10.1038/jcbfm.2012.213.
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Hypoxic preconditioning is thought to rely on gene products regulated by hypoxia-inducible factor (HIF)-1. Here, we show that the HIF-1 target gene cyclin-dependent kinase inhibitor 1, p21 WAF1/CIP1, is essential for neuroprotection by hypoxic/aglycemic or erythropoietin preconditioning using wild-type and p21 WAF1/CIP1-deficient neurons. Furthermore, overexpression of wild-type p21 WAF1/CIP1 or phospho-mutants significantly increased cell death after hypoxia/aglycemia. Moreover, deferoxamine-induced endogenous tolerance did not involve p21 WAF1/CIP1 expression in cortical neurons. Our data suggest that balanced expression and potentially posttranslational regulation of p21 WAF1/CIP1 is required for hypoxic preconditioning.
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Oriolo, Andrea S., Flavia A. Wald, Gisella Canessa, and Pedro J. I. Salas. "GCP6 Binds to Intermediate Filaments: A Novel Function of Keratins in the Organization of Microtubules in Epithelial Cells." Molecular Biology of the Cell 18, no. 3 (March 2007): 781–94. http://dx.doi.org/10.1091/mbc.e06-03-0201.
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In simple epithelial cells, attachment of microtubule-organizing centers (MTOCs) to intermediate filaments (IFs) enables their localization to the apical domain. It is released by cyclin-dependent kinase (Cdk)1 phosphorylation. Here, we identified a component of the γ-tubulin ring complex, γ-tubulin complex protein (GCP)6, as a keratin partner in yeast two-hybrid assays. This was validated by binding in vitro of both purified full-length HIS-tagged GCP6 and a GCP6(1397-1819) fragment to keratins, and pull-down with native IFs. Keratin binding was blocked by Cdk1-mediated phosphorylation of GCP6. GCP6 was apical in normal enterocytes but diffuse in K8-null cells. GCP6 knockdown with short hairpin RNAs (shRNAs) in CACO-2 cells resulted in γ-tubulin signal scattered throughout the cytoplasm, microtubules (MTs) in the perinuclear and basal regions, and microtubule-nucleating activity localized deep in the cytoplasm. Expression of a small fragment GCP6(1397-1513) that competes binding to keratins in vitro displaced γ-tubulin from the cytoskeleton and resulted in depolarization of γ-tubulin and changes in the distribution of microtubules and microtubule nucleation sites. Expression of a full-length S1397D mutant in the Cdk1 phosphorylation site delocalized centrosomes. We conclude that GCP6 participates in the attachment of MTOCs to IFs in epithelial cells and is among the factors that determine the peculiar architecture of microtubules in polarized epithelia.
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Chaturvedi, Anuhar, Michelle Maria Araujo Cruz, Nidhi Jyotsana, Amit Sharma, Ramya Goparaju, Vishwas Sharma, Adrian Schwarzer, et al. "The Metabolite R-2-Hydroxyglutarate (R2HG) Collaborates with HoxA9 to Induce Monocytic Leukemia." Blood 124, no. 21 (December 6, 2014): 366. http://dx.doi.org/10.1182/blood.v124.21.366.366.
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Abstract Mutations in the metabolic enzymes IDH1 and IDH2 are frequently found in several tumors including glioma and acute myeloid leukemia (AML). Mutant IDH produces R-2-hydroxyglutarate (R2HG), which induces histone- and DNA-hypermethylation through inhibition of epigenetic regulators, thus linking metabolism to tumorigenesis. However, it is unknown whether R2HG alone is sufficient to recapitulate the biologic effects of mutant IDH1 in vivo. Recently, we have shown that IDH1mut cooperates with HoxA9 and induces a monocytic leukemia in mice. In order to evaluate the effects of R2HG independently of the mutated IDH1 protein and to determine whether the effects are specific to the R-enantiomer of 2HG, we treated mice transplanted with HoxA9 immortalised bone marrow cells with R2HG, S-2-hydroxyglutarate (S2HG), alpha-ketoglutarate (aKG) and phosphate buffered saline (PBS). The mice in the metabolite cohorts received an intraperitoneal dose of 1 mg per day. Mice treated with R2HG had higher engraftment levels at 16 and 20 weeks post transplantation than the mice treated with S2HG, αKG and PBS respectively (P<.01). High WBC counts (70±16 /nl) and lower platelet counts than in control mice were observed in the cohort receiving R2HG after 16 to 20 weeks of treatment, while the S2HG, αKG and PBS cohorts had normal blood counts even at 20 weeks (P<.05). Peripheral blood from R2HG treated mice revealed significantly more immature Mac1+Gr1- and less mature Mac1+Gr1+ cells at 12 and 16 weeks after treatment than S2HG, αKG and PBS treated mice (P<.001). In addition, the R2HG treated mice died with a median latency of 137 days post transplantation from monocytic leukemia, while mice treated with S2HG, αKG and PBS died with a median latency of 223, 202 and 184 days respectively (P<.001). Further, in order to assess whether R2HG alone was sufficient as a single hit to induce myeloproliferation, normal C57BL/6 mice (without HoxA9) were treated with R2HG, S2HG and PBS for eight months. No differences were observed for survival, blood counts, immunophenotype and frequencies of progenitor cells (lin-ckit+sca1+, CMP, GMP and MEP) between treatment groups and control. This data shows that the metabolite R2HG like the IDH1 mutant protein cooperates with HoxA9 to induce monocytic leukemia. We next compared mice receiving transplants of HoxA9+IDH1mut cells with mice receiving HoxA9 cells that were then treated with R2HG. Both cohorts developed monocytic leukemia, albeit with different kinetics. The Hoxa9+IDH1mut mice died with a median latency of 83 days while the R2HG cohort died with a median latency of 137 days post transplantation (P<0.001). Also, while the former cohort developed severe leukocytosis, anemia and thrombocytopenia at 12 weeks, the R2HG treated mice had high WBC counts and lower platelet counts than control mice at 16 to 20 weeks after treatment. The faster disease kinetics in IDH1mut mice could be attributed to a significantly lower proportion of cells in G0/G1 and higher proportion of cells in S phase when compared to cells from mice treated with R2HG at 9 weeks after transplantation (P<.001). This resulted from a marked downregulation of cyclin-dependent kinase inhibitors (Cdkn) 1A (p21), 1B (p27), 2A (p16), and 2B (p15) in HoxA9+IDH1mut cells as compared to HoxA9 cells treated with R2HG or PBS. In order to rule out an influence of differential R2HG levels on the disease progression between the two cohorts, levels of R2HG were quantified. IDH1mut expressing and R2HG treated bone marrow cells from mice had similarly high ratios of R2HG/S2HG. The quantified R2HG levels were also comparable to that of primary AML patient cells harbouring mutated IDH1. In unsupervised hierarchical clustering mutated cells clustered together with R2HG and separated from PBS treated mice. 69 of the top 100 enriched Gene Ontology gene sets of HoxA9+IDH1mut were also found in HoxA9+R2HG, suggesting largely redundant but also non-overlapping functions of the mutant IDH1 protein and the oncometabolite R2HG. In summary, we show that R2HG, similar to the mutant IDH1 protein, promotes leukemogenesis in cooperation with HoxA9, although with delayed kinetics. Our data proves that R2HG acts as an oncometabolite in vivo in a murine model of leukemogenesis. Disclosures No relevant conflicts of interest to declare.
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Simon-Crevatis, Karen A., Shailesh R. Metkar, Justin T. Hardcastle, Keith Jansson, Usman Shabon, Rajarshi Choudhury, Euan A. Stronach, Kevin G. Coleman, and Asli Muvaffak. "Abstract 6175: CDK8 inhibition potentiates the efficacy of niraparib in homologous recombination proficient cancer cell lines." Cancer Research 83, no. 7_Supplement (April 4, 2023): 6175. http://dx.doi.org/10.1158/1538-7445.am2023-6175.
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Abstract While cyclin dependent kinases (CDKs) were originally implicated in cell cycle regulation, several CDKs including CDK7, CDK8, and CDK9 play critical roles in transcription through regulation of RNA polymerase II (Pol II) activity. In addition to expected phosphosite targets associated with DNA-binding transcription factors, chromatin regulators, or other known regulators of pol II activity, a phospho-proteomics study on a CDK8/19 inhibitor (Cortistatin A) also identified DNA replication and repair proteins, i.e., namely BRCA1 and MDC1, as substrates for CDK8. RVU120, a first in-class CDK8/19 inhibitor in phase I clinical trial demonstrated preclinical efficacy in acute myeloid leukemia PDX models. A robust relationship has also been observed between the exposure to RVU120 and inhibition of pSTAT5, a pharmacodynamic marker, in solid tumor indications in a recent clinical trial. Additionally, preclinical data suggested RVU120 reduced expression of genes related to interferon-related DNA damage signature (IRDS) suggesting a potential role of CDK8 in DNA repair signaling. CDK8 loss was identified as a synthetic lethal hit in two internal, orthogonal screens that surveyed targets which can synergize with niraparib inhibition. Niraparib inhibits PARP enzymatic activity and promotes PARP trapping on ssDNA breaks which lead to replication stress-induced dsDNA breaks that require BRCA-dependent homologous recombination repair (HRR). Combination with other therapeutic agents is anticipated to improve the efficacy of niraparib in HR proficient (HRP) and BRCA1/2.WT tumors. This work describes our proof-of-concept data from in vitro validation of combination synergy response of RVU120 with niraparib in a panel of 129 HRR.WT and HRR mutant cancer cell lines across ovarian, breast, lung, and colorectal cancer indications. Our results indicated that the niraparib+RVU120 combination was synergistic, and a strong synergy response, i.e., a SS (Synergy Score) ≥ 5 was detected by Loewe modelling across the entire surface of the combination matrix in ~26% of Horizon Discovery OncoSignatureTM panel cell lines that were largely resistant to niraparib monotherapy. The study findings indicated the potential of CDK8/19 inhibitors to enhance niraparib activity in both HRR.WT and HRR mutant populations. The potential benefit of CDK8/19i+niraparib combination will be further explored in PARPi-resistant models. Molecular studies are underway to explore potential biomarkers associated with synergy response, and to analyze the downstream effects on DNA Damage Response. Citation Format: Karen A. Simon-Crevatis, Shailesh R. Metkar, Justin T. Hardcastle, Keith Jansson, Usman Shabon, Rajarshi Choudhury, Euan A. Stronach, Kevin G. Coleman, Asli Muvaffak. CDK8 inhibition potentiates the efficacy of niraparib in homologous recombination proficient cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6175.
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Auer, Franziska, Minhui Lin, Karin Nebral, Christoph G. W. Gertzen, Oskar A. Haas, Michaela Kuhlen, Holger Gohlke, et al. "Novel Recurrent Germline JAK2 G571S Variant in Childhood Acute B-Lymphoblastic Leukemia: A Double Hit One Pathway Scenario." Blood 132, Supplement 1 (November 29, 2018): 387. http://dx.doi.org/10.1182/blood-2018-99-115293.
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Abstract Introduction: Current studies demonstrate an involvement of germline predispositions in the development of approximately 5% of childhood leukemias (Zhang J et al.,N Engl J Med, 2015), although their actual contribution is believed to be much higher. Being able to understand tumor evolution starting from a predisposed cell, opens up a new avenue in the form of disease prevention rather than treatment. Here, we present a novel finding of a double hit - one pathway scenario, in which a rare (MAF<0.01) germline JAK2 variant (G571S), inherited paternally, and a newly described germline STAT3 variant (K370R), transmitted from the mother's side, act synergistically to induce Ph-like B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Methods: WES was carried out to identify predisposing germline variants. The cooperative functionality of the di-genic candidate mutations was experimentally tested using BaF3 oncogenic transformation assays, western blot and cell cycle analyses. In addition, we used structural homology calculations to visualize the cooperative impact of both mutations. Results: Utilizing Trio-calling based on WES we identified two concomitant germline SNVs in the JAK2/STAT3 pathway in a boy affected with BCP-ALL. The JAK2 variant, rs139504737, leads to an amino acid substitution from Glycine to Serine (p.G571S) (MAF<0.01). The second variant constitutes an extremely rare and so far for leukemia undescribed missense mutation in the STAT3 gene (c.1109A>G), causing an exchange of Lysine to Arginine (p.K370R). Structural modeling of STAT3 K370R showed that K370, which is an important site for acetylation, is located in a loop adjacent to the DNA binding site of STAT3. The substitution of Lys to Arg at position 370 leads to a strengthening of the beta-sheet due to simultaneous interaction of Arg with both E455 and E442. Moreover, in contrast to Lys, Arg cannot be acetylated, leading to a constitutively non-acetylated form of STAT3. JAK2 G571S on the other hand has a unique position affecting amino acid 571, which lies adjacent to the Y570 residue that downregulates kinase activity via autophosphorylation, indicating a potential functional mechanism of the G571S mutation by inhibiting Y570-directed negative feedback. To assess the cooperative oncogenic transforming potential of both variants, BaF3 depletion assays were carried out. In BaF3/CRLF2-IL-7Rwt cells, JAK2 G571S conferred IL-3 independent growth at similar rates as the well-known oncogenic JAK2 V617F mutation. Moreover, the combination of both JAK2 G571S and STAT3 K370R further increased the growth advantage significantly starting 2 days after IL-3 withdrawal (p=0.0226). Western Blot analyses revealed increased pSTAT5 levels, as well as high pSTAT-3 levels in the double mutant cells. We further observed that STAT3 K370R alone changed the phenotype of the in-vitro culture, with an accumulation of enlarged BaF3 cells. Surprisingly, this phenotype was reversed in cells expressing both STAT3 K370R and JAK2 G571S. Cell cycle analysis showed a significant increase of aneuploid cells (G2/M) (p=0.0009), while the G-1 phase was significantly decreased (p=0.0031) in STAT3 K370R expressing BaF3 cells compared to STAT3 WT cells. Again, this phenotype was drastically reduced in cells transfected with both mutations simultaneously (G1 phase p=0.0026; G2/M p=0.0032). Western Blot analyses confirmed increased p-CDC-2, p-Cyclin/Cyclin-B1, and Cyclin-A2 levels in BaF3 cells harboring both variants, suggesting that the cell cycle arrest observed in cells expressing STAT3 K370R is rescued by JAK2 G571S expression by enabling re-entering of the M-Phase. The link between the oncogenic capacity of JAK2 G571S and leukemia could further be strengthened by the identification of an additional B-precursor ALL patient from an independent family harboring the same JAK2 G571S germline mutation. The patient belongs to a previously described DS-ALL cohort (Bercovich D. et al., Lancet, 2008). In this patient with Down syndrome, we did neither identify the STAT3 K370R mutation nor CRLF2 activation. Thus, we hypothesize that the germline JAK2 G571S in combination with the constitutional trisomy 21 act in synergy. Conclusion: Taken together, we present evidence of an oncogenic potential of germline JAK2 G571S, which is significantly increased through additional expression of STAT3 K370R in a double-hit one-pathway scenario. Disclosures No relevant conflicts of interest to declare.
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St. Gelais, Corine, Sun Hee Kim, Victoria V. Maksimova, Olga Buzovetsky, Kirsten M. Knecht, Caitlin Shepard, Baek Kim, Yong Xiong, and Li Wu. "A Cyclin-Binding Motif in Human SAMHD1 Is Required for Its HIV-1 Restriction, dNTPase Activity, Tetramer Formation, and Efficient Phosphorylation." Journal of Virology 92, no. 6 (January 10, 2018). http://dx.doi.org/10.1128/jvi.01787-17.
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ABSTRACT Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) regulates intracellular deoxynucleoside triphosphate (dNTP) levels and functions as a retroviral restriction factor through its dNTP triphosphohydrolase (dNTPase) activity. Human SAMHD1 interacts with cell cycle regulatory proteins cyclin A2, cyclin-dependent kinase 1 (CDK1), and CDK2. This interaction mediates phosphorylation of SAMHD1 at threonine 592 (T592), which negatively regulates HIV-1 restriction. We previously reported that the interaction is mediated, at least in part, through a cyclin-binding motif (RXL, amino acids [aa] 451 to 453). To understand the role of the RXL motif in regulating SAMHD1 activity, we performed structural and functional analyses of RXL mutants and the effect on HIV-1 restriction. We found that the RXL mutation (R451A and L453A, termed RL/AA) disrupted SAMHD1 tetramer formation and abolished its dNTPase activity in vitro and in cells. Compared to wild-type (WT) SAMHD1, the RL/AA mutant failed to restrict HIV-1 infection and had reduced binding to cyclin A2. WT SAMHD1 and RL/AA mutant proteins were degraded by Vpx from HIV-2 but were not spontaneously ubiquitinated in the absence of Vpx. Analysis of proteasomal and autophagy degradation revealed that WT and RL/AA SAMHD1 protein levels were enhanced only when both pathways of degradation were simultaneously inhibited. Our results demonstrate that the RXL motif of human SAMHD1 is required for its HIV-1 restriction, tetramer formation, dNTPase activity, and efficient phosphorylation at T592. These findings identify a new functional domain of SAMHD1 important for its structural integrity, enzyme activity, phosphorylation, and HIV-1 restriction. IMPORTANCE SAMHD1 is the first mammalian dNTPase identified as a restriction factor that inhibits HIV-1 replication by decreasing the intracellular dNTP pool in nondividing cells, although the critical mechanisms regulating SAMHD1 function remain unclear. We previously reported that mutations of a cyclin-binding RXL motif in human SAMHD1 significantly affect protein expression levels, half-life, nuclear localization, and phosphorylation, suggesting an important role of this motif in modulating SAMHD1 functions in cells. To further understand the significance and mechanisms of the RXL motif in regulating SAMHD1 activity, we performed structural and functional analyses of the RXL motif mutation and its effect on HIV-1 restriction. Our results indicate that the RXL motif is critical for tetramer formation, dNTPase activity, and HIV-1 restriction. These findings help us understand SAMHD1 interactions with other host proteins and the mechanisms regulating SAMHD1 structure and functions in cells.
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Martinat, Charlotte, Arthur Cormier, Joëlle Tobaly-Tapiero, Noé Palmic, Nicoletta Casartelli, Bijan Mahboubi, Si’Ana A. Coggins, et al. "SUMOylation of SAMHD1 at Lysine 595 is required for HIV-1 restriction in non-cycling cells." Nature Communications 12, no. 1 (July 28, 2021). http://dx.doi.org/10.1038/s41467-021-24802-5.
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AbstractSAMHD1 is a cellular triphosphohydrolase (dNTPase) proposed to inhibit HIV-1 reverse transcription in non-cycling immune cells by limiting the supply of the dNTP substrates. Yet, phosphorylation of T592 downregulates SAMHD1 antiviral activity, but not its dNTPase function, implying that additional mechanisms contribute to viral restriction. Here, we show that SAMHD1 is SUMOylated on residue K595, a modification that relies on the presence of a proximal SUMO-interacting motif (SIM). Loss of K595 SUMOylation suppresses the restriction activity of SAMHD1, even in the context of the constitutively active phospho-ablative T592A mutant but has no impact on dNTP depletion. Conversely, the artificial fusion of SUMO2 to a non-SUMOylatable inactive SAMHD1 variant restores its antiviral function, a phenotype that is reversed by the phosphomimetic T592E mutation. Collectively, our observations clearly establish that lack of T592 phosphorylation cannot fully account for the restriction activity of SAMHD1. We find that SUMOylation of K595 is required to stimulate a dNTPase-independent antiviral activity in non-cycling immune cells, an effect that is antagonized by cyclin/CDK-dependent phosphorylation of T592 in cycling cells.
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Zhang, Shiyu, Andrew P. Holmes, Alexej Dick, Adel A. Rashad, Lucía Enríquez Rodríguez, Gabriela A. Canziani, Michael J. Root, and Irwin M. Chaiken. "Altered Env conformational dynamics as a mechanism of resistance to peptide-triazole HIV-1 inactivators." Retrovirology 18, no. 1 (October 9, 2021). http://dx.doi.org/10.1186/s12977-021-00575-z.
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Abstract Background We previously developed drug-like peptide triazoles (PTs) that target HIV-1 Envelope (Env) gp120, potently inhibit viral entry, and irreversibly inactivate virions. Here, we investigated potential mechanisms of viral escape from this promising class of HIV-1 entry inhibitors. Results HIV-1 resistance to cyclic (AAR029b) and linear (KR13) PTs was obtained by dose escalation in viral passaging experiments. High-level resistance for both inhibitors developed slowly (relative to escape from gp41-targeted C-peptide inhibitor C37) by acquiring mutations in gp120 both within (Val255) and distant to (Ser143) the putative PT binding site. The similarity in the resistance profiles for AAR029b and KR13 suggests that the shared IXW pharmacophore provided the primary pressure for HIV-1 escape. In single-round infectivity studies employing recombinant virus, V255I/S143N double escape mutants reduced PT antiviral potency by 150- to 3900-fold. Curiously, the combined mutations had a much smaller impact on PT binding affinity for monomeric gp120 (four to ninefold). This binding disruption was entirely due to the V255I mutation, which generated few steric clashes with PT in molecular docking. However, this minor effect on PT affinity belied large, offsetting changes to association enthalpy and entropy. The escape mutations had negligible effect on CD4 binding and utilization during entry, but significantly altered both binding thermodynamics and inhibitory potency of the conformationally-specific, anti-CD4i antibody 17b. Moreover, the escape mutations substantially decreased gp120 shedding induced by either soluble CD4 or AAR029b. Conclusions Together, the data suggest that the escape mutations significantly modified the energetic landscape of Env’s prefusogenic state, altering conformational dynamics to hinder PT-induced irreversible inactivation of Env. This work therein reveals a unique mode of virus escape for HIV-1, namely, resistance by altering the intrinsic conformational dynamics of the Env trimer.