Molecular Genetic Analysis of INI1/hSNF5 in HIV-1 Replication, HIV-1 latency and Cancer: Single molecule analysis to Organismal studies

INI1/hSNF5/SMARCB1/BAF47 is a component of the chromatin remodeling SWI/SNF complex. This complex influence replication of HIV-1 and SARS Corona Virus-2. INI1/SMARCB1 was discovered as a HIV-1 IN binding protein and is a tumor suppressor biallelically mutated/deleted in many human cancers. The goal of our laboratory is to determine how INI1 affects viral replication, tumor suppression, and in general cellular function.    

(i) RNA mimicry of IN-binding Rpt1 domain and novel strategies to inhibit acutely and latently infected HIV-1: Viruses coopt many cellular factors for its own replication. INI1/SMARCB1 is one such factors that is hijacked by HIV-1 to facilitate its replication. INI1/SMARCB1 plays multiple roles during HIV-1 replication including viral integration, transcription and post-transcriptional events. Interfering with IN and INI1 interaction leads to potent inhibitory effects. NMR structure of IN-binding Rpt1 domain of INI1 revealed that it structurally mimics TAR RNA and that this is important for IN binding and to facilitate particle production. Currently we are developing novel class of anti-HIV therapeutics by leveraging the structure of INI1 and its molecular mimicry to TAR RNA. Initial studies demonstrate that these novel therapeutics are able to inhibit both acute and latent HIV-1 replication. The goal is to complete these studies and then trasnlate it into clinic.     

(ii) RNA binding by INI1/SMARCB1 and its implications for viral and cellular transcription: We have identified a novel function of INI1, namely its ability to bind to HIV-1 TAR RNA and cellular 7SK RNA. Interestingly, TAR and 7SK mimic each other.  TAR RNA binds to HIV-1 tat protein and is required for transcriptional elongation.  7SK is a cellular long non-coding RNA (lnc) and has implications for enhancer-mediated functions during transcription. This novel function is likely to shed light on SMARCB1 role in transcription, tumor suppression.

(iii) Study of HIV-1 latency- Application of a novel single cell and single molecule RNA-FISH and IF method: A final hurdle to eradicate HIV-1 is the persistence of the virus in latent reservoirs, which are transcriptionally suppressed and low in number. While the current drugs control viremia, they are unable to eliminate the virus from the infected cells. To detect these rare reactivated latent cells in patient samples, we have developed novel Single Cell Single Molecule Immuno-fluorescence and RNA-FISH assay (SMIRA) in collaboration with Dr. Robert Singer. This novel assay will be applied to characterize latent reservoirs in various anatomical locations and the effect of various Latency Reversing Agents (LRA, that are in clinical trials) and drugs of abuse.

(iv) Mechanism of tumor suppression by INI1/hSNF5 and developing novel and effective therapeutic strategies to combat INI1-deficient tumors: By using a series of genetic systems developed in our laboratory (knock-out, knock-in mouse models, cell culture models), we are dissecting the mechanism of INI1-medaited tumor suppression and developing molecularly targeted therapies. Previously we discovered that INI1 harbors a masked nuclear export signal. Recently, in collaboration with international team of neuropathologists, we have found that mutations that affect the regulated nuclear export is a tumorigenic event that it offers a novel therapeutic target. In addition, we are investigating novel downstream targets of INI1/SMARCB1 and their effect on senescence and their therapeutic potential.

Yung, E., Sorin, M., Pal, A., Craig, E., Morozov, A., Delattre, O. Kappes J., Ott, D. and Kalpana, G.V. (2001) Inhibition of HIV-1 particle production by a dominant negative mutant of INI1/hSNF5. Nature Med. 7, 920-926.

Craig, E., Zhang, Z., Davies, K., and Kalpana, G.V. (2002) A masked NES in INI1/hSNF5 mediates hCRM1-dependant Nuclear Export: Implications for tumorigenesis. EMBO J. 21, 31-42. 

Yung, E., Sorin, M. Wang, E., Perumal, S., and Kalpana, G.V. (2004) Specificity of interaction of INI1/hSNF5 with retroviral integrases and its functional significance. J. Virol., 78, 2222-2231.

Tsikitis, M., Zhang, Z., Zagzag, D. and Kalpana, G.V. (2005) Genetic ablation of CyclinD1 abrogates the genesis of rhabdoid tumors that arise due to Ini1 loss. Proc. Natl. Acad. Sci. USA, 102, 12129-34.

Alarcon-Vargas, D., Zhang, Z., Agarwal, B., Challagulla, K., Mani, S.* and Kalpana, G.V.* (2006)  Targeting Cyclin D1, a downstream effector of INI1/hSNF5, in Rhabdoid tumors. Oncogene 25, 722�734.

Sorin, M. and Kalpana, G. V. (2006) Dynamics of host-virus interplay during HIV-1 replication. Curr HIV Res. 4, 117-30). 

Masha Sorin¹, Eric Yung¹, Xuhong Wu¹ and Ganjam Kalpana (2006) Replication of HIV-1 in cell lines defective for INI1/hSNF5 Retrovirology, 3:56.

Morozov, A.  Lee, S-J, Zhnag, ZK, Cimica, V., Zagzag, D. Kalpana, G. V. (2007) Induction of interferon signaling and mitotic spindle check-point by INI1/hSNF5:  Identification of potential therapeutic targets for rhabdoid tumors. Clin Cancer Res.13:4721-30. 

Smith ME, Cimica V, Chinni S, Challagulla K, Mani S, Kalpana GV. (2008) Rhabdoid tumor growth is inhibited by flavopiridol. Clin Cancer Res. 14(2):523-32.

 Das BC, Smith ME, Kalpana GV.(2008) Design, synthesis of novel peptidomimetic derivatives of 4-HPR for rhabdoid tumors. Bioorg Med Chem Lett. 18(14):4177-80. Epub 2008 May 29.

 Das S, Cano J, Kalpana GV.(2009) Multimerization and DNA Binding Properties of INI1/hSNF5 and Its Functional Significance. J Biol Chem. 284(30):19903-14. Epub 2009 Apr 27.

Das S, Kalpana GV. (2009) Reverse two-hybrid screening to analyze protein-protein interaction of HIV-1 viral and cellular proteins. Methods Mol Biol. 485:271-93.

Sorin M, Cano J, Das S, Mathew S, Wu X, Davies KP, Shi X, Cheng SW, Ott D, Kalpana GV. (2009) Recruitment of a SAP18-HDAC1 complex into HIV-1 virions and its requirement for viral replication. PLoS Pathog. 5(6).

Kalpana, G. V. and M. Smith (2009) �Development of targeted therapies for rhabdoid tumors based on the functions of INI1/hSNF5 tumor suppressor� (invited chapter for the monograph on Molecularly targeted Therapies for pediatric tumors Edited by Bob Arceci and Peter Houghton, in press).

Cimica V, Smith ME, Zhang Z, Mathur D, Mani S, Kalpana GV. (2010) Potent inhibition of rhabdoid tumor cells by combination of flavopiridol and 4OH-tamoxifen. BMC Cancer. 10:634. PMID: 21092078

Smith ME, Cimica V, Chinni S, Jana S, Koba W, Yang Z, Fine E, Zagzag D, Montagna C, Kalpana GV. (2011). Therapeutically targeting Cyclin D1 in primary tumors arising from loss of Ini1. Proc Natl Acad Sci U S A. 2011 Jan 4;108(1):319-24. Epub 2010 Dec 20. PMID: 21173237

Cano, J. and Kalpana, G. V. (2011) Inhibition of early stages of HIV-1 assembly by INI1/hSNF5 transdominant negative mutant S6. J Virol.  85(5):2254-65. Epub 2010 Dec 15.  PMID: 21159874

Lee, S-J., Cimica, V., Zagzag, D., and Kalpana, G. V. Aurora A Is a Repressed Effector Target of the Chromatin Remodeling Protein INI1/hSNF5 Required for Rhabdoid Tumor Cell Survival. Cancer Res. 71(9):3225-35. Epub 2011 Apr 26. PMID: 21521802

Mathew, S., Nguyen, M., Wu, X., Pal, A., Shah, V. B., Aiken, C., and Kalpana, G. V. INI1/hSNF5-interaction defective HIV-1 IN mutants exhibit impaired particle morphology, reverse transcription and integration in vivo. Retrovirology. 10:66. [Epub ahead of print 2013] PMID:23799881

La Porte, A. and Kalpana, G. V. (2015) “Quantification of HIV-1 Gag localization within virus producer cells” Methods Mol Biol. 1354:165-74. PMID: 26714711

La Porte, A., Cano, J., Wu, X., Mitra, D., Kalpana, G. V. (2016) An essential role of INI1/hSNF5 chromatin remodeling protein in HIV-1 post-transcriptional events and Gag/GagPol stability. J Virol. 2016 Oct 14;90(21):9889-9904 (PMID:27558426 PMCID:PMC5068538)

Prasad VR, Kalpana GV. 2017. FISHing out the hidden enemy: advances in detecting and measuring latent HIV-infected cells. mBio 8:e01433-17. https://doi.org/10.1128/mBio.01433-17

Ajasin DO, Rao VR, Wu X, Ramasamy S, Pujato M, Ruiz AP, Fiser A, Bresnick AR, Kalpana GV, Prasad VR. CCL2 mobilizes ALIX to facilitate Gag-p6 mediated HIV-1 virion release. Elife. 2019 Jun 7;8:e35546. doi: 10.7554/eLife.35546. PMID: 31172941; PMCID: PMC6592687.

Dixit, U., Bhutoria, S., Wu, X. et al. INI1/SMARCB1 Rpt1 domain mimics TAR RNA in binding to integrase to facilitate HIV-1 replication. Nat Commun 12, 2743 (2021). https://doi.org/10.1038/s41467-021-22733-9

Pathak, R., Zin, F., Thomas, C. et al. Inhibition of nuclear export restores nuclear localization and residual tumor suppressor function of truncated SMARCB1/INI1 protein in a molecular subset of atypical teratoid/rhabdoid tumors. Acta Neuropathol (2021). https://doi.org/10.1007/s00401-021-02328-w