Differential effect of CLK SR Kinases on HIV-1 gene expression: potential novel targets for therapy

Abstract

BackgroundRNA processing plays a critical role in the replication of HIV-1, regulated in part through the action of host SR proteins. To explore the impact of modulating SR protein activity on virus replication, the effect of increasing or inhibiting the activity of the Cdc2-like kinase (CLK) family of SR protein kinases on HIV-1 expression and RNA processing was examined.ResultsDespite their high homology, increasing individual CLK expression had distinct effects on HIV-1, CLK1 enhancing Gag production while CLK2 inhibited the virus. Parallel studies on the anti-HIV-1 activity of CLK inhibitors revealed a similar discrepant effect on HIV-1 expression. TG003, an inhibitor of CLK1, 2 and 4, had no effect on viral Gag synthesis while chlorhexidine, a CLK2, 3 and 4 inhibitor, blocked virus production. Chlorhexidine treatment altered viral RNA processing, decreasing levels of unspliced and single spliced viral RNAs, and reduced Rev accumulation. Subsequent experiments in the context of HIV-1 replication in PBMCs confirmed the capacity of chlorhexidine to suppress virus replication.ConclusionsTogether, these findings establish that HIV-1 RNA processing can be targeted to suppress virus replication as demonstrated by manipulating individual CLK function and identified chlorhexidine as a lead compound in the development of novel anti-viral therapies.

Characterization of novel inhibitors of HIV-1 replication that function via alteration of viral RNA processing and rev function

“…The parallel determination that activity of the SR proteins can be modulated by multiple kinases (SRPK1, SRPK2, CLK1, CLK2, CLK3, and CLK4) (22,23) opens up the possibility that modulation of kinase activity could induce changes in HIV-1 RNA splicing and, subsequently, replication. Recent experiments by our group have confirmed this hypothesis by demonstrating that overexpression of specific CLKs results in suppression of viral Gag and Env expression, a response that can be replicated with the use of the small molecule inhibitor, chlorhexidine, a known modulator of CLK function (24). …”

“…Screens were performed using the HeLa rtTA-HIV-Δ Mls cell line containing a doxycycline (Dox)-inducible Tet-On HIV-1 LAI strain provirus (25,26) as described in our previous study (24). Compounds tested were obtained from the ChemBridge Online Chemical Store (www.hit2lead.com).…”

“…For further analysis of HIV-1 protein expression, western blots were performed on cell lysates. The antibodies and conditions used for Tat, anti-tubulin and isotype-specific HRP-conjugated antibodies were used as described (24). Additional antibodies used include anti-p24 from hybridoma 183 and mouse anti-gp120 from hybridoma 902 (AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH), mouse anti-Rev (Abcam, #ab85529) and rabbit anti-GAPDH (Sigma, #9545).…”

“…RNA was extracted from cells, reverse transcribed and HIV-1 mRNA levels quantitated by qRT-PCR as described (24). The effect of drugs on HIV-1 splice site usage within the 2-kb MS RNA class was analysed by RT-PCR of cDNA as previously described (24).…”

Detained introns are a novel, widespread class of post-transcriptionally spliced introns

“…Three other Clk family members have interesting roles in specific contexts, including insulin response, splicing of exons involved in neuronal tauopathies, and the replication cycle of HIV (Duncan et al 1998;Hartmann et al 2001;Jiang et al 2009;Rodgers et al 2010;Wong et al 2011). These studies suggest that Clk kinases, through regulation of splicing, are positioned to channel multiple cellular signaling pathways in different cell types and disease states, including global stress responses.…”