A leading pharmacological strategy toward HIV cure requires "shock" or activation of HIV gene expression in latently infected cells with latency reversal agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs, we used fungal secondary metabolites as a source of bioactive molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA, the scaffold of the positive transcription elongation factor b (P-TEFb) inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex, to be significantly reduced upon GTX treatment of CD4+ T cells. GTX directly disrupted 7SK snRNP by targeting La-related protein 7 (LARP7), releasing active P-TEFb, which phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD), inducing HIV transcription.

Center of Expertise in Mycology of Radboud UMC CWZ Nijmegen Netherlands

Department of Biochemistry Erasmus MC University Medical Center Rotterdam PO Box 2040 3000 CA Rotterdam Netherlands

Department of Immunology Erasmus MC University Medical Center Rotterdam PO Box 2040 3000 CA Rotterdam Netherlands

Department of Internal Medicine Section of Infectious Diseases Erasmus University Medical Center PO Box 2040 3000 CA Rotterdam Netherlands

Department of Parasitology and Mycology Faculty of Medicine Mashhad University of Medical Sciences Mashhad Iran

Departments of Molecular Genetics and Radiation Oncology Erasmus University Medical Center PO Box 2040 3000 CA Rotterdam Netherlands

Erasmus MC Genomics Core Facility Department of Cell Biology Erasmus University Medical Center PO Box 2040 3000 CA Rotterdam Netherlands

Institute of Microbiology of the Czech Academy of Sciences Videnska 1083 CZ 14220 Prague 4 Czech Republic

Key Laboratory of Environmental Pollution Monitoring Disease Control Ministry of Education and Guizhou Talent Base of Microbes and Human Health School of Basic Medicine Guizhou Medical University Guiyang 550025 P R China

Microbiology Section Department of Experimental Diagnostic and Specialty Medicine School of Medicine University of Bologna Bologna Italy

Ministry of Health Directorate General of Health Services Ibri Oman

Westerdijk Fungal Biodiversity Institute Utrecht Netherlands

Zobrazit více v PubMed

Ho D. D., Neumann A. U., Perelson A. S., Chen W., Leonard J. M., Markowitz M., Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature 373, 123–126 (1995). PubMed

Perelson A. S., Essunger P., Cao Y., Vesanen M., Hurley A., Saksela K., Markowitz M., Ho D. D., Decay characteristics of HIV-1-infected compartments during combination therapy. Nature 387, 188–191 (1997). PubMed

Wei X., Ghosh S. K., Taylor M. E., Johnson V. A., Emini E. A., Deutsch P., Lifson J. D., Bonhoeffer S., Nowak M. A., Hahn B. H., Saag M. S., Shaw G. M., Viral dynamics in human immunodeficiency virus type 1 infection. Nature 373, 117–122 (1995). PubMed

Siliciano J. M., Siliciano R. F., The Remarkable Stability of the Latent Reservoir for HIV-1 in Resting Memory CD4+ T Cells. J. Infect. Dis. 212, 1345–1347 (2015). PubMed

Archin N. M., Liberty A. L., Kashuba A. D., Choudhary S. K., Kuruc J. D., Crooks A. M., Parker D. C., Anderson E. M., Kearney M. F., Strain M. C., Richman D. D., Hudgens M. G., Bosch R. J., Coffin J. M., Eron J. J., Hazuda D. J., Margolis D. M., Administration of vorinostat disrupts HIV-1 latency in patients on antiretroviral therapy. Nature 487, 482–485 (2012). PubMed PMC

Rasmussen T. A., Søgaard O. S., Clinical interventions in HIV cure research. Adv. Exp. Med. Biol. 1075, 285–318 (2018). PubMed

Spivak A. M., Planelles V., Novel latency reversal agents for HIV-1 cure. Annu. Rev. Med. 69, 421–436 (2018). PubMed PMC

Kim Y., Anderson J. L., Lewin S. R., Getting the "Kill" into "Shock and Kill": Strategies to eliminate latent HIV. Cell Host Microbe 23, 14–26 (2018). PubMed PMC

Battivelli E., Dahabieh M. S., Abdel-Mohsen M., Svensson J. P., Silva I. T. D., Cohn L. B., Gramatica A., Deeks S., Greene W. C., Pillai S. K., Verdin E., Distinct chromatin functional states correlate with HIV latency reactivation in infected primary CD4+ T cells. eLife 7, e34655 (2018). PubMed PMC

Ho Y.-C., Shan L., Hosmane N. N., Wang J., Laskey S. B., Rosenbloom D. I. S., Lai J., Blankson J. N., Siliciano J. D., Siliciano R. F., Replication-competent noninduced proviruses in the latent reservoir increase barrier to HIV-1 cure. Cell 155, 540–551 (2013). PubMed PMC

Bouchat S., Delacourt N., Kula A., Darcis G., Van Driessche B., Corazza F., Gatot J.-S., Melard A., Vanhulle C., Kabeya K., Pardons M., Avettand-Fenoel V., Clumeck N., De Wit S., Rohr O., Rouzioux C., Van Lint C., Sequential treatment with 5-aza-2′-deoxycytidine and deacetylase inhibitors reactivates HIV-1. EMBO Mol. Med. 8, 117–138 (2016). PubMed PMC

Jones R. B., O'Connor R., Mueller S., Foley M., Szeto G. L., Karel D., Lichterfeld M., Kovacs C., Ostrowski M. A., Trocha A., Irvine D. J., Walker B. D., Histone deacetylase inhibitors impair the elimination of HIV-infected cells by cytotoxic T-lymphocytes. PLOS Pathog. 10, e1004287 (2014). PubMed PMC

Zhao M., De Crignis E., Rokx C., Verbon A., van Gelder T., Mahmoudi T., Katsikis P. D., Mueller Y. M., T cell toxicity of HIV latency reversing agents. Pharmacol. Res. 139, 524–534 (2019). PubMed

Newman D. J., Cragg G. M., Natural products as sources of new drugs from 1981 to 2014. J. Nat. Prod. 79, 629–661 (2016). PubMed

Sanchez J. F., Somoza A. D., Keller N. P., Wang C. C. C., Advances in Aspergillus secondary metabolite research in the post-genomic era. Nat. Prod. Rep. 29, 351–371 (2012). PubMed PMC

Brakhage A. A., Regulation of fungal secondary metabolism. Nat. Rev. Microbiol. 11, 21–32 (2013). PubMed

Přichystal J., Schug K. A., Lemr K., Novák J., Havlíček V., Structural analysis of natural products. Anal. Chem. 88, 10338–10346 (2016). PubMed

Ne E., Palstra R.-J., Mahmoudi T., Transcription: Insights from the HIV-1 promoter. Int. Rev. Cell Mol. Biol. 335, 191–243 (2018). PubMed

Stoszko M., Ne E., Abner E., Mahmoudi T., A broad drug arsenal to attack a strenuous latent HIV reservoir. Curr. Opin. Virol. 38, 37–53 (2019). PubMed

Novák J., Sokolová L., Lemr K., Pluháček T., Palyzová A., Havlíček V., Batch-processing of imaging or liquid-chromatography mass spectrometry datasets and De Novo sequencing of polyketide siderophores. Biochim. Biophys. Acta. Proteins Proteom. 1865, 768–775 (2017). PubMed

Lassen K. G., Hebbeler A. M., Bhattacharyya D., Lobritz M. A., Greene W. C., A flexible model of HIV-1 latency permitting evaluation of many primary CD4 T-cell reservoirs. PLOS ONE. 7, e30176 (2012). PubMed PMC

Stanzani M., Orciuolo E., Lewis R., Kontoyiannis D. P., Martins S. L. R., John L. S. S., Komanduri K. V., Aspergillus fumigatus suppresses the human cellular immune response via gliotoxin-mediated apoptosis of monocytes. Blood 105, 2258–2265 (2005). PubMed

Suen Y. K., Fung K. P., Lee C. Y., Kong S. K., Gliotoxin induces apoptosis in cultured macrophages via production of reactive oxygen species and cytochrome c release without mitochondrial depolarization. Free Radic. Res. 35, 1–10 (2001). PubMed

Yamada A., Kataoka T., Nagai K., The fungal metabolite gliotoxin: Immunosuppressive activity on CTL-mediated cytotoxicity. Immunol. Lett. 71, 27–32 (2000). PubMed

Scharf D. H., Brakhage A. A., Mukherjee P. K., Gliotoxin--bane or boon? Environ. Microbiol. 18, 1096–1109 (2016). PubMed

Trautmann L., Kill: Boosting HIV-specific immune responses. Curr. Opin. HIV AIDS 11, 409–416 (2016). PubMed PMC

Stoszko M., De Crignis E., Rokx C., Khalid M. M., Lungu C., Palstra R.-J., Kan T. W., Boucher C., Verbon A., Dykhuizen E. C., Mahmoudi T., Small molecule inhibitors of BAF; A promising family of compounds in HIV-1 latency reversal. EBioMedicine 3, 108–121 (2016). PubMed PMC

Marian C. A., Stoszko M., Wang L., Leighty M. W., de Crignis E., Maschinot C. A., Gatchalian J., Carter B. C., Chowdhury B., Hargreaves D. C., Duvall J. R., Crabtree G. R., Mahmoudi T., Dykhuizen E. C., Small molecule targeting of specific BAF (mSWI/SNF) complexes for HIV latency reversal. Cell Chem. Biol. 25, 1443–55.e14 (2018). PubMed PMC

Quaresma A. J. C., Bugai A., Barboric M., Cracking the control of RNA polymerase II elongation by 7SK snRNP and P-TEFb. Nucleic Acids Res. 44, 7527–7539 (2016). PubMed PMC

Nguyen V. T., Kiss T., Michels A. A., Bensaude O., 7SK small nuclear RNA binds to and inhibits the activity of CDK9/cyclin T complexes. Nature 414, 322–325 (2001). PubMed

Uchikawa E., Natchiar K. S., Han X., Proux F., Roblin P., Zhang E., Durand A., Klaholz B. P., Dock-Bregeon A.-C., Structural insight into the mechanism of stabilization of the 7SK small nuclear RNA by LARP7. Nucleic Acids Res. 43, 3373–3388 (2015). PubMed PMC

Yang Z., Zhu Q., Luo K., Zhou Q., The 7SK small nuclear RNA inhibits the CDK9/cyclin T1 kinase to control transcription. Nature 414, 317–322 (2001). PubMed

Yik J. H. N., Chen R., Nishimura R., Jennings J. L., Link A. J., Zhou Q., Inhibition of P-TEFb (CDK9/Cyclin T) kinase and RNA polymerase II transcription by the coordinated actions of HEXIM1 and 7SK snRNA. Mol. Cell 12, 971–982 (2003). PubMed

Ott M., Geyer M., Zhou Q., The control of HIV transcription: Keeping RNA polymerase II on track. Cell Host Microbe 10, 426–435 (2011). PubMed PMC

Mousseau G., Valente S. T., Role of host factors on the regulation of Tat-Mediated HIV-1 transcription. Curr. Pharm. Des. 23, 4079–4090 (2017). PubMed PMC

Peterlin B. M., Price D. H., Controlling the elongation phase of transcription with P-TEFb. Mol. Cell 23, 297–305 (2006). PubMed

Jonkers I., Lis J. T., Getting up to speed with transcription elongation by RNA polymerase II. Nat. Rev. Mol. Cell Biol. 16, 167–177 (2015). PubMed PMC

Yukl S. A., Kaiser P., Kim P., Telwatte S., Joshi S. K., Vu M., Lampiris H., Wong J. K., HIV latency in isolated patient CD4+ T cells may be due to blocks in HIV transcriptional elongation, completion, and splicing. Sci. Transl. Med. 10, eaap9927 (2018). PubMed PMC

Mbonye U., Karn J., The molecular basis for human immunodeficiency virus latency. Annu. Rev. Virol. 4, 261–285 (2017). PubMed

Ramakrishnan R., Dow E. C., Rice A. P., Characterization of Cdk9 T-loop phosphorylation in resting and activated CD4+ T lymphocytes. J. Leukoc. Biol. 86, 1345–1350 (2009). PubMed PMC

Krueger B. J., Jeronimo C., Roy B. B., Bouchard A., Barrandon C., Byers S. A., Searcey C. E., Cooper J. J., Bensaude O., Cohen E. A., Coulombe B., Price D. H., LARP7 is a stable component of the 7SK snRNP while P-TEFb, HEXIM1 and hnRNP A1 are reversibly associated. Nucleic Acids Res. 36, 2219–2229 (2008). PubMed PMC

Abner E., Stoszko M., Zeng L., Chen H.-C., Izquierdo-Bouldstridge A., Konuma T., Zorita E., Fanunza E., Zhang Q., Mahmoudi T., Zhou M.-M., Filion G. J., Jordan A., A new quinoline BRD4 inhibitor targets a distinct latent HIV-1 reservoir for reactivation from other "Shock" drugs. J. Virol. 92, (2018). PubMed PMC

Chen H.-C., Martinez J. P., Zorita E., Meyerhans A., Filion G. J., Position effects influence HIV latency reversal. Nat. Struct. Mol. Biol. 24, 47–54 (2017). PubMed

Abner E., Jordan A., HIV "shock and kill" therapy: In need of revision. Antiviral Res. 166, 19–34 (2019). PubMed

Darcis G., Kula A., Bouchat S., Fujinaga K., Corazza F., Ait-Ammar A., Delacourt N., Melard A., Kabeya K., Vanhulle C., Van Driessche B., Gatot J.-S., Cherrier T., Pianowski L. F., Gama L., Schwartz C., Vila J., Burny A., Clumeck N., Moutschen M., De Wit S., Peterlin B. M., Rouzioux C., Rohr O., Van Lint C., An in-depth comparison of latency-reversing agent combinations in various In Vitro and Ex Vivo HIV-1 Latency Models Identified Bryostatin-1+JQ1 and Ingenol-B+JQ1 to Potently Reactivate Viral Gene Expression. PLOS Pathog. 11, e1005063 (2015). PubMed PMC

Hashemi P., Barreto K., Bernhard W., Lomness A., Honson N., Pfeifer T. A., Harrigan P. R., Sadowski I., Compounds producing an effective combinatorial regimen for disruption of HIV-1 latency. EMBO Mol. Med. 10, 160–174 (2018). PubMed PMC

Laird G. M., Bullen C. K., Rosenbloom D. I. S., Martin A. R., Hill A. L., Durand C. M., Siliciano J. D., Siliciano R. F., Ex vivo analysis identifies effective HIV-1 latency-reversing drug combinations. J. Clin. Invest. 125, 1901–1912 (2015). PubMed PMC

Rasmussen T. A., Lewin S. R., Shocking HIV out of hiding: Where are we with clinical trials of latency reversing agents? Curr. Opin. HIV AIDS 11, 394–401 (2016). PubMed

Rafati H., Parra M., Hakre S., Moshkin Y., Verdin E., Mahmoudi T., Repressive LTR nucleosome positioning by the BAF complex is required for HIV latency. PLOS Biol. 9, e1001206 (2011). PubMed PMC

Contreras X., Barboric M., Lenasi T., Peterlin B. M., HMBA releases P-TEFb from HEXIM1 and 7SK snRNA via PI3K/Akt and activates HIV transcription. PLOS Pathog. 3, 1459–1469 (2007). PubMed PMC

Kohoutek J., P-TEFb- the final frontier. Cell Div. 4, 19 (2009). PubMed PMC

Choi H. S., Shim J. S., Kim J.-A., Kang S. W., Kwon H. J., Discovery of gliotoxin as a new small molecule targeting thioredoxin redox system. Biochem. Biophys. Res. Commun. 359, 523–528 (2007). PubMed

Kwon-Chung K. J., Sugui J. A., What do we know about the role of gliotoxin in the pathobiology of Aspergillus fumigatus? Med. Mycol. 47, ( suppl. 1), S97–S103 (2009). PubMed PMC

Sakamoto H., Egashira S., Saito N., Kirisako T., Miller S., Sasaki Y., Matsumoto T., Shimonishi M., Komatsu T., Terai T., Ueno T., Hanaoka K., Kojima H., Okabe T., Wakatsuki S., Iwai K., Nagano T., Gliotoxin suppresses NF-κB activation by selectively inhibiting linear ubiquitin chain assembly complex (LUBAC). ACS Chem. Biol. 10, 675–681 (2015). PubMed

Wichmann G., Herbarth O., Lehmann I., The mycotoxins citrinin, gliotoxin, and patulin affect interferon-γ rather than interleukin-4 production in human blood cells. Environ. Toxicol. 17, 211–218 (2002). PubMed

Lewis R. E., Wiederhold N. P., Chi J., Han X. Y., Komanduri K. V., Kontoyiannis D. P., Prince R. A., Detection of gliotoxin in experimental and human aspergillosis. Infect. Immun. 73, 635–637 (2005). PubMed PMC

Gostinčar C., Zajc J., Lenassi M., Plemenitaš A., de Hoog S., Al-Hatmi A. M. S., Gunde-Cimerman N., Fungi between extremotolerance and opportunistic pathogenicity on humans. Fungal Diversity. 93, 195–213 (2018).

Spina C. A., Anderson J., Archin N. M., Bosque A., Chan J., Famiglietti M., Greene W. C., Kashuba A., Lewin S. R., Margolis D. M., Mau M., Ruelas D., Saleh S., Shirakawa K., Siliciano R. F., Singhania A., Soto P. C., Terry V. H., Verdin E., Woelk C., Wooden S., Xing S., Planelles V., An in-depth comparison of latent HIV-1 reactivation in multiple cell model systems and resting CD4+ T cells from aviremic patients. PLOS Pathog. 9, e1003834 (2013). PubMed PMC

Schmittgen T. D., Livak K. J., Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 3, 1101–1108 (2008). PubMed

Link A. J., LaBaer J., Trichloroacetic acid (TCA) precipitation of proteins. Cold Spring Harb. Protoc. 2011, 993–994 (2011). PubMed

Bartholomeeusen K., Xiang Y., Fujinaga K., Peterlin B. M., Bromodomain and extra-terminal (BET) bromodomain inhibition activate transcription via transient release of positive transcription elongation factor b (P-TEFb) from 7SK small nuclear ribonucleoprotein. J. Biol. Chem. 287, 36609–36616 (2012). PubMed PMC

Joosten R. P., Long F., Murshudov G. N., Perrakis A., The PDB_REDO server for macromolecular structure model optimization. IUCrJ. 1, ( Pt. 4), 213–220 (2014). PubMed PMC

Eichhorn C. D., Yang Y., Repeta L., Feigon J., Structural basis for recognition of human 7SK long noncoding RNA by the La-related protein Larp7. Proc. Natl. Acad. Sci. U.S.A. 115, E6457–E6466 (2018). PubMed PMC

Pettersen E. F., Goddard T. D., Huang C. C., Couch G. S., Greenblatt D. M., Meng E. C., Ferrin T. E., UCSF Chimera--a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004). PubMed

Trott O., Olson A. J., AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 31, 455–461 (2010). PubMed PMC

L. Schrödinger, The {PyMOL} Molecular Graphics System, Version 1.8 (2015).

Ten decadal advances in fungal biology leading towards human well-being