The Kashanchi lab has recently identified two Baf subunits of the most highly recognized chromatin remodeling proteins, the SWI/SNF (switching-defective-sucrose non-fermenting) complexes as highly regulated in HIV-1 and HTLv-1 infected cells compared to uninfected counterparts. In the presence of HIV-1 infection, Baf53 elutes off of a size exclusion column in a different sized complex and is predominantly phosphorylated. Baf-53 containing complexes appear to be transcriptionally suppressive, in that knocking down Baf53 increases HIV-1 viral gene expression. In the presence of Tat, cdk9/Cyclin T is able to phosphorylate Baf53 in vitro, which suggests that the phosphorylated form negates the transcriptionally suppressing mechanism and allows for viral transcription to proceed (Van Duyne R, et al., Varying modulation of HIV-1 LTR activity by Baf complexes. J Mol Biol 2011, 411:581-596).
The inhibition of the HIV-1 viral transactivator Tat is an attractive anti-retroviral strategy. Here, they identified the GSK-3 inhibitor 6BIO, via a high-throughput drug screening assay, as a Tat-dependent HIV-1 transcriptional inhibitor. In TZMBl cells containing an integrated HIV-1 LTR Luciferase, 6BIO inhibits LTR transcription with an IC50 of 40 nM. Screening the derivatives of 6BIO resulted in the identification of 6BIOder, with an IC50 of 0.03 nM against GSK-3β, 4.0 nM in primary macrophages, and 0.5 nM in astrocytes infected with HIV-1. These two compounds were able to inhibit HIV-1 transcription and act to exhibit protection against Tat-dependent neurotoxicity (Kehn-Hall K, et al Inhibition of Tat-mediated HIV-1 replication and neurotoxicity by novel GSK3-beta inhibitors. Virology 2011, 415:56-68).
In a recent review of the literature, the Kashanchi lab focuses on the role of cellular and, more importantly, viral small non-coding RNAs and how they affect viral gene expression. HIV-1 produces microRNAs from the TAR, Nef, and miR-H1 regions. Particularly, TAR miRNA has been shown to mediate the downregulation of viral and host gene expression by recruiting chromatin remodeling proteins and complexes as well as altering host cell cycle progression and apoptosis. Nef miRNA regulates viral and host gene expression. The miR-H1 region is significant in the study of HIV-1 associated neurological disorders (HAND) as well as the establishment of latency (Narayanan A, et al., Analysis of the roles of HIV-derived microRNAs. Expert Opin Biol Ther 2011, 11:17-29).
A manuscript explores the effect the miRNA machinery on the functional cdk inhibitors and how they control inhibition of HIV-1 transcription. Well known cdk inhibitors such as Roscovitine and Flavopiridol suppress their target cdk’s at low IC50s resulting in a decrease in HIV-1 transcription with low toxicity. Due to the integral involvement of the RNAi pathway in host/viral interactions, they suggest that these and future generations of cdk inhibitors may be affected by miRNA mechanisms. Indeed, a third generation derivative, known as CR8#13 which is very effective at inhibiting viral transcription, was not as effective in against HIV-1 infected cell lines that do not contain Dicer, an integral enzyme of the RNAi pathway. The addition of this drug, results in an increased level of 3’ TAR miRNA in HIV-1 LTR containing cells, resulting in a more dramatic recruitment of miRNA to the LTR, resulting in chromatin remodeling, alterations in RNA Pol II phosphorylation, and viral inhibition (Carpio L, et al., microRNA machinery is an integral component of drug-induced transcription inhibition in HIV-1 infection. J RNAi Gene Silencing 2010, 6:386-400).
Kashanchi lab has identified unique proteins that are differentially expressed in the serum of HIV-1 infected patients that are long-term non-progressors (LTNPs); people who are latently infected but do not progress to AIDS, despite the absence of treatment. Depletion of abundant serum proteins, followed by front-end purification, and mass spectropmetry allows for the analysis of altered proteins expression in this peripheral fluid. They focused on the cdk4/6 cell cycle inhibitor p16INK4A and found that the treamtnet of HIV-1 latently infected cell lines with this protein decreases viral production, despite the absence of endogenous expression (Van Duyne R, et al., The identification of unique serum proteins of HIV-1 latently infected long-term non-progressor patients. AIDS Res Ther 2010, 7:21).
In order for the HIV-1 proviral DNA to be transcribed, the virus recruited the SWI/SNF remodeling complex to the LTR to initiate transcriptional elongation. In the manuscript below, the Kashanchi lab shows for the first time that the PBAF complex is required for chromatin remodeling at nuc-1 and that the Baf200 subunit is required to ensure activation at the LTR level and for viral production. Productive HIV-1 infection results in Tat-activated transcription, which facilitates the removal of histone H2A and histone H2B at the LTR, again to allow for polymerase access. Interestingly, the BAF complex is only observed at the LTR, whereas the PBAF complex is also present at the Env region (Easley R, et al., Transcription through the HIV-1 nucleosomes: effects of the PBAF complex in Tat activated transcription. Virology 2010, 405:322-333).
In a landmark study, the Kashanchi lab showed that HIV-1 suppresses the expression and function of the RNAi pathway enzyme Dicer in cells. They also find that Dicer protein (but not the RNA) is completly absent in monocytes and contain only low levels in macrophages. The state of miRNA production in monocytes and macrophages differs compared to other HIV-1 tropic cells. MicroRNA chips experiments showed that a drop in miRNA production coincided with Dicer protein suppression in macrophages in both transfected and treated cells (Coley W, etal., Absence of DICER in monocytes and its regulation by HIV-1. J Biol Chem 2010, 285:31930-31943).
Some of the major fundings grants from Kashanchi lab:
An NIH R01 grant on Tat peptide derivatives (mimetics) inhibitors that could potentially inhibit virus replication both in cell culture and humanized animal models. The understanding of how HIV-1 Tat manipulates the transcription machinery will aid in designing better Tat inhibitors of mimetic nature.
An NIH R21 grant on the effect of chromatin remodeling complex SWI/SNF that influence HIV-1 transcription. Specifically the role of Tat- SWI/SNF (Tat-PBAF) complex in chromatin remodeling at the HIV-1 LTR in infected cell lines and PBMC infected cells are being investigated.
– An NIH R21 grant on the effect the HIV viral miRNA on the virus genome and host transcription machinery. The virus has a multi-step life cycle that revolves around the transcriptional control of the virus as regulated by interaction between the viral Tat protein and an RNA element known as the transactivation response (TAR) element. Kashanchi lab has recently demonstrated that TAR RNA is utilized by proteins involved in RNA interference (RNAi) and is processed into a viral microRNA (miRNA) that drives remodeling of the viral LTR, leading to heterochromatin formation (transcription silencing).
An NIH R21 grant on the effect of GSK-3 inhibitor BIO on neuroAIDS and its important implications in HAND. The short term goal of the research is to determine if BIO and derivatives could be used as a potential HAND therapeutic.
An NIH collaborative grant with the Washington DC CFAR on basic science related to HIV. The mission of the Basic Science Core is to develop, refine, and provide training and services to HIV/AIDS investigators in DC for the assays used to evaluate and quantify HIV replication and gene expression, characterize HIV disease using immunologic, genomics and proteomics approaches, and facilitate drug development by providing small animal models of HIV disease.
1. Narayanan, A., et al., Exosomes derived from HIV-1 infected cells contain TAR RNA. J Biol Chem, 2013.
2. Klase, Z.A., G.C. Sampey, and F. Kashanchi, Retrovirus infected cells contain viral microRNAs. Retrovirology, 2013. 10: p. 15.
3. Sampey, G.C., et al., Complex role of microRNAs in HTLV-1 infections. Front Genet, 2012. 3: p. 295.
4. Van Duyne, R., et al., Effect of mimetic CDK9 inhibitors on HIV-1-activated transcription. J Mol Biol, 2013. 425(4): p. 812-29.
5. Chung, M.C., et al., Bacillus anthracis-derived nitric oxide induces protein S-nitrosylation contributing to macrophage death. Biochem Biophys Res Commun, 2013. 430(1): p. 125-30.
6. Tonry, J.H., et al., In vivo murine and in vitro M-like cell models of gastrointestinal anthrax. Microbes Infect, 2013. 15(1): p. 37-44.
7. Narayanan, A., et al., Curcumin inhibits Rift Valley fever virus replication in human cells. J Biol Chem, 2012. 287(40): p. 33198-214.
8. Van Duyne, R., et al., Localization and sub-cellular shuttling of HTLV-1 tax with the miRNA machinery. PLoS One, 2012. 7(7): p. e40662.
9. Narayanan, A., et al., Use of ATP analogs to inhibit HIV-1 transcription. Virology, 2012. 432(1): p. 219-31.
10. Austin, D., et al., p53 Activation following Rift Valley fever virus infection contributes to cell death and viral production. PLoS One, 2012. 7(5): p. e36327.
11. Kehn-Hall, K., et al., Modulation of GSK-3beta activity in Venezuelan equine encephalitis virus infection. PLoS One, 2012. 7(4): p. e34761.
12. Al-Harthi, L. and F. Kashanchi, Mechanisms of HIV-1 latency post HAART treatment area. Curr HIV Res, 2011. 9(8): p. 552-3.
13. Tyagi, M. and F. Kashanchi, New and novel intrinsic host repressive factors against HIV-1: PAF1 complex, HERC5 and others. Retrovirology, 2012. 9: p. 19.
14. Duyne, R.V., et al., Humanized mouse models of HIV-1 latency. Curr HIV Res, 2011. 9(8): p. 595-605.
15. Van Duyne, R., et al., Varying modulation of HIV-1 LTR activity by Baf complexes. J Mol Biol, 2011. 411(3): p. 581-96.
16. Kehn-Hall, K., et al., Inhibition of Tat-mediated HIV-1 replication and neurotoxicity by novel GSK3-beta inhibitors. Virology, 2011. 415(1): p. 56-68.
17. Carpio, L., et al., microRNA machinery is an integral component of drug-induced transcription inhibition in HIV-1 infection. J RNAi Gene Silencing, 2010. 6(1): p. 386-400.