Background
Despite the efficacy of ART, HIV-1 persistence in reservoirs, mostly formed by CD4 and macrophages hinders eradication. Nuclear import of pre-integration complex is assisted by proteins from mRNA processing pathways like CPSF6 (3´RNA-cleavage and polyadenylation factor 6) and SRSF2/SC35 (Serine/Arginine-rich splicing factor 2). Dasatinib and ponatinib are tyrosine kinase inhibitors (TKIs) that interfere with HIV-1 proviral integration in both CD4 and macrophages by preserving the antiviral activity of SAMHD1. Our objective was to determine if dasatinib and ponatinib may also interfere with proviral integration in MDMs via CPSF6 and/or SC35.
Methods
Blood samples were obtained from healthy donors (n=15). CD14+ cells were isolated from PBMCs and differentiated into monocyte-derived macrophages (MDMs). In vitro infection of MDMs with JR_FL_Renilla and DHIV3-GFP was monitored in presence/absence of dasatinib and ponatinib by time-lapse microscopy. Influence of TKIs on HIV-1 early and late reverse transcription (RT), formation of 2-LTR circles, and proviral integration was analyzed by ddPCR. Subcellular localization and expression levels of CPSF6 and SC35 were analyzed by confocal microscopy.
Results
1)Time-lapse of dasatinib- and ponatinib-treated MDMs infected with DHIV-3-GFP revealed that GFP was nearly undetectable for 50 hours post-infection and the scarcely detectable infected cells were observed by 22h after infection of untreated samples (p=0.0032). 2)No differences were observed in early and late RT in MDMs pretreated or not with dasatinib after infection with JR_FL_Renilla. However, 2-LTR circles and proviral integration were 3.9- (p=0.0259) and 1.6-fold (p=0.0467) lower in dasatinib-treated MDMs 48h post-infection, respectively. Similar results were obtained with ponatinib. 3)MDMs treated with dasatinib showed higher expression of CPSF6 in both nuclear (p<0.0001) and cytoplasmic regions (p<0.0001) than untreated MDMs, while ponatinib-treated MDMs also showed higher expression in both regions (p<0.0001) than untreated MDMs, and higher nuclear expression than dasatinib-treated MDMs (p=0.0110). 4)Ponatinib-treated MDMs had 5.8- and 5.4-fold lower expression of SC35 than dasatinib and untreated MDMs (p<0.0001).
Conclusions
Dasatinib and ponatinib modified the expression and subcellular localization of CPSF6 and SC35, which are essential for HIV-1 proviral integration and transcription in MDMs. This is a new potential mechanism of action for TKIs to interfere with HIV-1 infection.