Scripps Research Logo

Projects at the Valente Lab

Block and Lock strategy for HIV-1 Cure
HIV-1 Tat activates viral transcription and limited Tat transactivation correlates with latency establishment. We postulated a “block-and-lock” functional cure approach based on properties of the Tat inhibitor didehydro-Cortistatin A (dCA). HIV-1 transcriptional inhibitors could block ongoing viremia during antiretroviral therapy (ART), locking the HIV promoter in persistent latency. We investigated this hypothesis in human CD4+ T cells isolated from aviremic individuals. Combining dCA with ART accelerates HIV-1 suppression and prevents viral rebound after treatment interruption, even during strong cellular activation. We show that dCA mediates epigenetic silencing by increasing nucleosomal occupancy at Nucleosome-1, restricting RNAPII recruitment to the HIV-1 promoter. The efficacy of dCA was studied in the bone marrow-liver-thymus (BLT) mouse model of HIV latency and persistence. Adding dCA to ART-suppressed mice systemically reduces viral mRNA in tissues. Moreover, dCA significantly delays and reduces viral rebound levels upon treatment interruption. Altogether, this work demonstrates the potential of block-and-lock cure strategies.

block-and-lock

Figure – Tat inhibitors are amenable to functional cure approaches, which aim to reduce residual viremia during ART and limit viral rebound during treatment interruption. Using didehydro-Cortistatin A (dCA), Kessing et al. demonstrate the concept in human CD4+ T cells from aviremic individuals and in the bone marrow-liver-thymus mouse model of HIV latency.

Mode of action of a novel HIV-1 Tat inhibitor
HIV anti-retroviral therapy is based on the administration of drugs in combination, in order to minimize development of mutations that can confer single-drug resistance to the virus. The viral protein Tat, a potent activator of HIV gene expression, is a potential antiviral target.
When integrated into the host genome, the provirus becomes a eukaryotic transcriptional unit. The 5’ terminal region of all HIV mRNAs forms an identical stem-bulge-loop structure called the Transactivation Responsive (TAR) element. Tat binds to TAR and activates transcription from the HIV LTR promoter. Basal transcription from the integrated HIV LTR is low and Tat and host factors substantially increase the mRNA production from the integrated viral genome. Mutations in the Tat sequence usually affect its function and HIV replication, indicating a strong requirement for its conservation. Therefore, Tat is a potential therapeutic target and anti-Tat drugs would be expected to have a synergistic effect with other inhibitors.
We have recently discovered a promising anti-Tat drug candidate. This compound inhibits Tat-mediated trans-activation of the integrated HIV provirus by binding specifically to the TAR-binding domain of Tat. As a consequence, the compound causes a reduction in cell-associated HIV-1 viral RNA and capsid p24 antigen production in acutely and chronically infected cells, at a half maximal effective concentration (EC50) of 0.7 pM to 2.5 nM, depending on the multiplicity of infection (MOI)). This molecule reduces both transcriptional initiation/elongation from the viral promoter and alters the nucleolar localization of Tat (Figure 3). Termination of compound treatment does not result in immediate virus rebound as the HIV promoter is transcriptionally silenced. It acts on both HIV-1 and HIV-2, and displays high bioavailability. With a therapeutic index in cultured cells of over 8000, this novel compound could define a novel class of HIV anti-viral drugs.
We are currently further defining the mechanism of action of this molecule that appears to be an excellent tool to further explore the mechanism of the Tat-TAR transcriptional activation and also a promising anti-HIV compound.

Research image

Figure 3 – Tat inhibitor alters Tat-Flag nucleolar localization. A. Confocal microscopy analysis of the sub-cellular localization of transfected wild type Tat flag tagged (Tat-F-86-F) or Tat-flag mutated in the basic domain (TAR binding domain) and where indicated treated (24h) or not with compound (CO). Flag epitope recognized with anti-flag and AlexaFluor 568 anti-IgG. Transfections performed in HeLa-CD4 cells. Magnification 300X.

Identify additional host factors that inhibit viral replication
Our long-term goal is to expand our knowledge, in any way possible, of the HIV life cycle: to determine who are the helper host partners, who are the interfering proteins or pathways, and what is absolutely necessary for replication. With this information we will be in an advantageous position to tackle this virus. The basic principle of the approach is that using retroviruses, large libraries of encoded objects (cDNAs, peptides, RNAs), among which are desired objects capable of exerting a dominant phenotype on target cells, can be transferred to target mammalian cells. By applying appropriate genetic selections one can isolate those cells expressing the object of interest from among a background of unwanted cells. Now that we have established the methodology (1,2,4), we are screening additional retroviral libraries.

Identification of inhibitors of HIV capsid dimerization

We have developed an ultra high throughput screening strategy to identify novel potent inhibitors of HIV particle assembly. We will focus on the capsid protein (CA), the main component of the HIV virion, which dimerization is the first step in assembly of the viral particle. No anti-HIV drugs have yet been clinically developed against this target. Methods to measure capsid multimerization have however been recently used to identify proof-of-principle peptides and small compound inhibitors which block viral assembly and HIV replication, including HIV strains resistant to drugs targeting viral enzymes. We designed an ultra High-Throughput Screening-amenable assay (uHTS), and companion screens and counter-screens to identify potent and selective inhibitors of capsid assembly. By combining transfer-of-energy biochemical screens with secondary assays in HIV-infected cells, we will enable the discovery of novel compounds of high affinity. To demonstrate dimerization using alternately tagged CA proteins, we designed a set of complementary and orthogonal assays. The primary Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay will be optimized and validated in pilot screens before running a large MPLCN screen. Our HIV CA-specific AlphaScreen will serve as a companion assay to help characterize hit compounds for further biologic validation. Several counter-screens will serve to eliminate false-positive hits. CA-specific inhibitors will be differentiated by their capacity to inhibit interactions between the N-terminal and C-terminal portions of the CA protein, by using CA-based assays. Secondary cell-based HIV infectivity assays will be used to characterize non-cytotoxic specific high-affinity inhibitors of HIV replication. The stage of the HIV replication cycle targeted by the hit compounds will be determined. Our goal is to provide the scientific community with freely accessible robust assays and potent, diversely acting HIV capsid inhibitors active in nanomolar concentrations.

Scheme for HIV CA TR-FRET assay:

Fret assay

N86-hnRNPU blocks HIV-1 mRNA export: mechanism of action
We have previously reported that expression of N86-hnRNPU, an N-terminal fragment of hnRNPU, renders human cells highly resistant to HIV infection (1). Characterization of the impact of N86-hnRNPU on viral replication revealed a dramatic decrease in the levels of HIV-1 transcripts in the cytoplasm of infected cells and a concomitant increase of viral mRNA levels in the nucleus. HIV-1 expresses three classes of RNAs: unspliced RNA that serves as genomic RNA and encodes the Gag and Pol precursor proteins, singly spliced RNAs encoding the envelope and accessory proteins, and several multiply spliced RNAs encoding additional accessory proteins. Multiply spliced HIV-1 mRNAs are constitutively exported by the default cellular TAP/NXF1-mediated mRNA export pathway. Singly spliced and unspliced (collectively termed intron-containing) RNAs are unable to access the TAP pathway and are retained in the nucleus. Efficient export of intron-containing mRNAs requires the HIV-1 protein Rev, which bridges mRNAs harboring the highly structured Rev response element (RRE) to the CRM1 nuclear export receptor. All three types of viral transcripts are subject to N86-hnRNPU-mediated inhibition, suggesting that the fragment blocks a step in export common to both TAP and CRM1 or at a step upstream before the pathways diverge (Figure 1). A region 100nt upstream from the 3’ long terminal repeat (LTR) and the 3’LTR itself of HIV-1 is the target of N86-hnRNPU mediated restriction (1). As expected, the scarcity of viral transcripts in the cytoplasm of N86-hnRNPU expressing cells results in a significant reduction in viral replication. 

Research image

Figure 1 – Export of HIV-1 mRNAs. Following expression, fully spliced mRNAs are exported in a TAP dependent manner. Intron containing RNAs contain an element called the rev responsive element (RRE) to which the viral protein Rev binds to. Rev associated with CRM1 to mediate export of the unspliced messages. N86-hnRNPU prevents the export of all three classes of RNAs.

We now wish to understand how N86-hnRNPU manipulates HIV mRNA export, in order to gain insights into the mechanism of replication of HIV-1 and potentially better understand cellular mRNA export pathways.

N91-eIF3f manipulation of HIV-1 mRNA maturation
By means of a genetic screen we identified the N-terminal 91 amino acids of the eukaryotic initiation factor 3 subunit f (N91-eIF3f) as a potent inhibitor of HIV-1 replication (2). Cells over-expressing N91-eIF3f or full-length eIF3f severely restrict the replication of the virus, by specifically targeting the 3’ end of the viral mRNA. Proviruses are formed normally but viral mRNA levels are reduced in the nucleus and cytoplasm. Restoration of viral gene expression is achieved upon addition of a heterologous polyadenylation signal downstream from the 3’ LTR. We showed that the 3’ end cleavage of the HIV-1 mRNA precursor is specifically reduced in N91-eIF3f expressing cells, suggesting a previously unsuspected role of eIF3f in 3‘ end processing of HIV-1 mRNAs. eIF3f mediates this restriction of HIV-1 expression through the previously unsuspected involvement of a set of factors that includes eIF3f, the splice factor SR-protein 9G8 and the cyclin dependent kinase 11 (CDK11) (2,3) (Figure 2). 

Research image

Figure 2 - Proposed model for N91-eIF3f inhibition of HIV-1 mRNA 3’ end cleavage. Schematic drawing of the pre-mRNA 3’ end processing complexes and cis elements. CPSF recognizes the poly(A) signal, whereas CstF recognizes the downstream sequence element (DSE). The CFIm preferentially binds to the sequence UGUAN (N=A>U≥C/G), and enhances the binding of CPSF. The HIV-1 3’ LTR contains a sequence related to the consensus-binding site of 9G8. 9G8 directly interact in vivo and in vitro with CDK11. CDK11 interacts with eIF3f, and therefore CDK11 may serve to link eIF3f and mRNA 3’ end processing. 9G8 also specifically interacts with the CFIm, and is thought to bridge regulation of alternative splicing and 3’ end processing. Overexpression of eIF3f or N91-eIF3f may disrupt the eIF3f-CDK11-9G8 interactions, and, in turn, would prevent the proper modification of 9G8/CFIm/CPSF required for efficient HIV poly(A) site recognition. Poly(A) polymerase (PAP), cyclin dependent kinase CDK11, SR protein  9G8.

We are currently defining the precise mechanism by which N91-eIF3f inhibits HIV-1 RNA cleavage. Once we have a better understanding of the specific interactions and stoichiometry between CDK111, eIF3f and 9G8 that are required to observe reduced maturation of the viral RNA, we will design  high throughput Alpha Screens which will potentially help us identify compounds that block processing of HIV-1 mRNA.



1-Valente, S. & Goff, S.P. (2006) Inhibition of HIV-1 Gene Expression by a Fragment of hnRNP U. Molecular Cell 23:597-605.

2-Valente, S.T., Gilmartin, G.M., Mott, C., Falkard, B., & Goff, S.P. (2009) Inhibition of HIV-1 replication by eIF3f. Proc Natl Acad Sci U S A 106:4071-4078.

3-Valente, S.T., Gilmartin, G.M., Venkataraman, K., Arriagada, G., & Goff, S.P. (2009) HIV-1 mRNA 3’ end processing is distinctively regulated by eIF3f, CDK11, and splice factor 9G8. Molecular Cell 23;36(2):279-89

4-Valente, S.T. & Goff, S.P. (2009) Somatic Cell Genetic Analyses to Identify HIV-1 Host Restriction Factors. Methods Molecular Biology 485:235-255.

___________

Home
___________

Projects
___________

Lab Members
___________

Publications
___________

Funding
___________

Positions
___________

Links
___________

Lab Photos
___________

Life in Jupiter
___________

In The Press
___________

Support Us
___________

Contacts
___________