Date Published: March 28, 2013
Publisher: Public Library of Science
Author(s): Raymond W. Wong, Ahalya Balachandran, Mario A. Ostrowski, Alan Cochrane, Bryan R. Cullen.
To develop new approaches to control HIV-1 replication, we examined the capacity of recently described small molecular modulators of RNA splicing for their effects on viral RNA metabolism. Of the drugs tested, digoxin was found to induce a dramatic inhibition of HIV-1 structural protein synthesis, a response due, in part, to reduced accumulation of the corresponding viral mRNAs. In addition, digoxin altered viral RNA splice site use, resulting in loss of the essential viral factor Rev. Digoxin induced changes in activity of the CLK family of SR protein kinases and modification of several SR proteins, including SRp20 and Tra2β, which could account for the effects observed. Consistent with this hypothesis, overexpression of SRp20 elicited changes in HIV-1 RNA processing similar to those observed with digoxin. Importantly, digoxin was also highly active against clinical strains of HIV-1 in vitro, validating this novel approach to treatment of this infection.
Current highly active anti-retroviral therapies (HAARTs) have successfully delayed the progression of HIV-1-infected individuals to AIDS by targeting viral entry and all HIV-1 enzymes , . However, the clinical application of ARTs is being affected by the spread of drug resistant viral strains , , ; detection of drug resistant forms of HIV-1 in newly infected patients has increased ∼3-fold from 2000 to 2007 to 16% , . To overcome these hurdles, more drugs with better profiles, and especially, novel mechanisms of action, are necessary for continued success in combating HIV-1 , , . However, the majority of drugs currently undergoing clinical trials target the same enzymes/proteins for which drugs are already available , , , . In addition, the persistence of virus in reservoirs continues to be a challenge with standard HAART.
Despite the success of ART/HAART, there are many caveats with current HIV-1 therapies, including the emergence of drug resistant forms of HIV-1, high cost, and toxicity , , . New drugs with improvement in these profiles and novel mechanisms of action are necessary , , . A number of strategies have targeted HIV regulatory and accessory proteins to date, but most remain under development , . It is unclear whether disrupting cellular processes essential for HIV-1 replication can yield alternative therapies without significant cellular toxicity. However, a number of existing therapies for other human diseases (e.g. heart disease, cancer, and dementia) do work by altering host protein function and are well tolerated , , , . In this report, we demonstrate a novel and alternative use of the FDA-approved cardiovascular drug, digoxin, as an anti-HIV-1 therapeutic (summarized in Fig. 8). More importantly, digoxin was found to inhibit virus replication by a novel mechanism, inducing oversplicing of HIV-1 RNA (Figs. 3, S3, 4, and S4D)—a stage of the virus lifecycle not targeted by current HIV-1 inhibitors and under host cell control. Digoxin achieves this effect by altering the splicing of HIV-1 RNA, reducing accumulation of two classes of viral mRNA (US and SS; Figs. 3, S3, S4D) that encode structural proteins essential for new virion assembly (Gag, Gagpol, and Env; Fig. 1). In addition, digoxin selectively inhibits expression of the HIV-1 regulatory factor Rev through specific alteration of viral RNA splice site use without affecting the expression of other viral proteins (p16 Tat; Fig. 4). While digoxin induced a 73% reduction in Rev2/1 RNA accumulation, it also increased MS viral RNA levels ∼3 fold (Fig. 3). Combined, these alterations may not account for the complete loss of Rev protein observed, suggesting the possibility that digoxin may have effects beyond the changes in viral RNA processing detected. The loss of Rev further impairs expression of incompletely-spliced viral mRNAs (US and SS) by preventing Rev-mediated export of RNAs to the cytoplasm (Fig. 5A) for translation into respective viral structural proteins (Gag, Gagpol, and Env) and regulatory/accessory factors (p14 Tat, Vif, Vpr, and Vpu) (Figs. 1, 2, S2, 4D). Furthermore, the effects were achieved at concentrations of digoxin that did not impact HeLa, SupT1, and PBMC cell viability relative to control treatments (Figs. 1, 2, and S4). Rev expression in trans (Fig. 5B–C) only partially reversed the effects of digoxin, indicating that loss of Rev alone is not sufficient to explain the full effect of digoxin. Rather, in light of the demonstration that Rev acts primarily on newly synthesized viral RNA , the enhanced processing of the viral RNA induced by digoxin may result in the incompletely-spliced HIV-1 RNAs having too short a half-life to be engaged by Rev even when Rev is present. In summary (Fig. 8), digoxin selectively impairs HIV-1 replication at two levels: (1) through global alterations in the efficiency of HIV-1 RNA processing and (2) blocking export of incompletely-spliced viral RNAs to the cytoplasm.