Digoxin suppresses HIV-1 replication by altering viral RNA processing.

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 Tra2ß, 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.

Halogenated analogs of 1'-acetoxychavicol acetate, Rev-export inhibitor from Alpinia galanga, designed from mechanism of action.

In the course of search for the robust analogs of 1'-acetoxychavicol acetate (ACA, 1), the Rev-export inhibitor from the medicinal plant Alpinia galanga, we clarified formation of the quinone methide intermediate ii to be essential for exerting the inhibitory activity of 1. Based on this mechanism of action, the rational design from the MO calculation of the conclusive activation energy to ii resulted in the four halogenated analogs with more potent activity than ACA (1). In particular, the difluoroanalog 20d exhibited approximately four-fold potent activity as compared with 1.

Inhibition of HIV-1 multiplication by a modified U7 snRNA inducing Tat and Rev exon skipping.

The HIV-1 regulatory proteins Tat and Rev are encoded by multiply spliced mRNAs that differ by the use of alternative 3' splice sites at the beginning of the internal exon. If these internal exons are skipped, the expression of these genes, and hence HIV-1 multiplication, should be inhibited. We have previously developed a strategy, based on antisense derivatives of U7 small nuclear RNA, that allows us to induce the skipping of an internal exon in virtually any gene. Here, we have successfully applied this approach to induce a partial skipping of the Tat, Rev (and Nef) internal exons. Three functional U7 constructs were subcloned into a lentiviral vector. Two of them strongly reduced the efficiency of lentiviral particle production compared to vectors carrying either no U7 insert or unrelated U7 cassettes. This defect could be partly or fully compensated by coexpressing Rev from an unspliced mRNA in the producing cell line. Upon stable transduction into CEM-SS or CEM T-lymphocytes, the most efficient of these constructs inhibits HIV-1 multiplication. Although the inhibition is not complete, it is more efficient in combination with another mechanism inhibiting HIV multiplication. Therefore, this new approach targeting HIV-1 regulatory genes at the level of pre-mRNA splicing, in combination with other antiviral strategies, may be a useful new tool in the fight against HIV/AIDS.

Inhibition of multidrug-resistant HIV-1 by interference with cellular S-adenosylmethionine decarboxylase activity.

S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine biosynthesis, can be specifically inhibited by the experimental drug SAM486A. The pharmaceutical interference with SAMDC activity results in the depletion of the intracellular pool of spermidine and spermine. In particular, low spermidine levels compromise hypusine modification and, thereby, activation of eukaryotic initiation factor 5A (eIF-5A), which is a cellular cofactor of the essential human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev. In the present study, we show that SAM486A efficiently suppresses HIV-1 replication, including the replication of viruses that are resistant to multiple reverse transcriptase and protease inhibitors. At drug concentrations that efficiently inhibit the formation of progeny viruses, no toxic effects of SAM486A on cellular metabolism are observed. It is demonstrated that the antiretroviral effect of SAM486A is based on the fact that Rev activity is severely compromised in drug-treated cells. Thus, inhibition of cellular SAMDC activity may provide a novel strategy to achieve suppression of otherwise drug-resistant viruses.

Defective rev response element (RRE) and rev gene in HAART treated AIDS patients with discordance between viral load and CD4+ T-cell counts.

BACKGROUND: Highly Active Antiretroviral Therapy (HAART) has emerged as one the most effective method for treating AIDS patients. However, after variable period of treatment, many AIDS patients on HAART show elevated PCR viral load without a corresponding decline in CD4+ T-cells. Our earlier studies have revealed that the viruses present in the plasma of such patients are not infectious. OBJECTIVE: The aim of this study is to characterize the changes in the regulatory genes of HIV-1, namely tat, rev and rev response element (RRE) isolated from the plasma of such AIDS patients and to assess their role in role in affecting viral infectivity, hence its contribution, in the 'contradictory phenomenon' of high viral load and high CD4+ T-cell counts. STUDY DESIGN: The viral RNA was isolated from the plasma of HAART patients when they exhibited high plasma viral load and high CD4+ T-cell counts. The target regulatory genes were amplified by RT-PCR and sequenced. Sequences were also obtained from the proviral DNA from the peripheral blood mononuclear cells (PBMCs) of the study subjects. The sequences were compared with the wild type viral sequence to look for the changes induced in them due to HAART regime. RESULTS AND CONCLUSION: Our data revealed that RRE was missing in the viral particles isolated from the plasma of all study subjects. In two patients, the second exon of the rev gene was missing thereby leading to defective Rev protein. In another patients, Rev synthesis was prematurely stopped due to G135T substitution in the amino terminal domain. No such changes were observed in the corresponding proviral DNA. These changes are likely to result in the assembly of non-infectious virus due to lack of envelope proteins. Absence of RRE and Rev protein also leads to transport and packaging of multiply spliced transcripts into the virions instead of complete genomic RNA.

A comparison of gag, pol and rev antisense oligodeoxynucleotides as inhibitors of HIV-1.

Sequences from the gag, pol and rev regions of the RF strain of HIV-1 (HIV-1RF) were chosen as targets for antisense phosphorothioate oligodeoxynucleotides (S-oligos). These sequences were the p18/p24 junction in gag, the active site of HIV protease in pol; a sequence from the first exon of the rev gene and S-oligodeoxycytidylic acid controls. Compounds were tested against HIV-1 in both acutely and chronically infected cells. The results show that these phosphorothioate analogues tested in acutely infected cells were active in the 0.1-2 microM range, were dependent on chain length but had no sequence specificity. To study the mechanism of action, the time of addition of S-oligos to acutely infected cells was delayed for up to 48 h post-infection. It was found that antiviral activity was lost when compounds were added to the cultures later than 10 h post-infection. With chronically infected cells only the antisense rev sequence showed activity at 30 microM and neither of the gag or pol antisense sequences has a significant effect on HIV replication at 50 microM. These results are consistent with previous in vitro studies which demonstrate that antisense S-oligodeoxynucleotides have several modes of action.