Direct inhibition of CDK9 blocks HIV-1 replication without preventing T-cell activation in primary human peripheral blood lymphocytes

Abstract

HIV-1 transcription is essential for the virus replication cycle. HIV-1 Tat is a viral transactivator that strongly stimulates the processivity of RNA polymerase II (RNAPII) via recruitment of the cyclin T1/CDK9 positive transcription elongation factor, which phosphorylates the C-terminal domain (CTD) of RNAPII. Consistently, HIV-1 replication in transformed cells is very sensitive to direct CDK9 inhibition. Thus, CDK9 could be a potential target for anti-HIV-1 therapy. A clearer understanding of the requirements for CDK9 activity in primary human T cells is needed to assess whether the CDK9-dependent step in HIV-1 transcription can be targeted clinically. We have investigated the effects of limiting CDK9 activity with recombinant lentiviruses expressing a dominant negative form of CDK9 (HA-dnCDK9) in Peripheral Blood Lymphocytes (PBLs) and other cells. Our results show that direct inhibition of CDK9 potently inhibits HIV-1 replication in single-round infection assays with little to undetectable effects on RNAPII transcription, RNA synthesis, proliferation and viability. In PBLs purified from multiple donors, direct inhibition of CDK9 activity blocks HIV-1 replication/transcription but does not prevent T cell activation, as determined via measurement of cell surface and cell cycle entry and progression markers, and DNA synthesis. We have also compared the effects of HA-dnCDK9 to flavopiridol (FVP), a general CDK inhibitor that potently inhibits CDK9. In contrast to HA-dnCDK9, FVP interferes with key cellular processes at concentrations that inhibit HIV-1 replication with potency similar to HA-dnCDK9. In particular, FVP inhibits several T cell activation markers and DNA synthesis in primary PBLs at the minimal concentrations required to inhibit HIV-1 replication. Our results imply that small pharmacological compounds targeting CDK9 with enhanced selectivity could be developed into effective anti-HIV-1 therapeutic drugs.

Indirubin 3′-Monoxime, from a Chinese Traditional Herbal Formula, Suppresses Viremia in Humanized Mice Infected with Multidrug-Resistant HIV

“…7 Targeting of P-TEFb with the CDK inhibitor flavopiridol inhibits HIV expression, 8 but has unfavorable toxicities. [9][10][11] Indirubin, another CDK inhibitor, 12 is the active ingredient of the traditional Chinese herbal formula Dan Gui Long Hui Wan, used to treat chronic myelogenous leukemia and psoriasis. 13 We have previously demonstrated that indirubin-3¢-monoxime (IM), a derivative of indirubin with improved solubility, is an inhibitor of CDK9 and P-TEFb function that blocks HIV gene expression in the absence of toxicity.…”

Dual Myxovirus Screen Identifies a Small-Molecule Agonist of the Host Antiviral Response

“…As substantiated by recent evidence (4,5), many host requirements are conserved among related viruses, increasing the possibility for broad-spectrum antiviral activity. Likewise, the host-directed approach may contribute to reducing the frequency of viral escape from inhibition (6)(7)(8), since individual viral mutations are less likely to compensate for functional loss of a host factor or pathway required for viral replication.…”

Latent HIV-1 Infection Occurs in Multiple Subsets of Hematopoietic Progenitor Cells and Is Reversed by NF-κB Activation

“…HIV-1 Tat assists in this process by competitively displacing HEXIM1 to free P-TEFb from the inhibitory complex (4,64). Activated P-TEFb is then recruited to nascent HIV-1 transcripts, where it facilitates transcript elongation and thus reactivation of latent virus (38,63,70,76).…”