FIG. 6.

HIV-1 PR targets RIG-I and interferes with IFN-β activation. (A) HIV-1 PR downregulates the IFNB promoter. HEK293 cells were transfected with an IFNB-pGL3 reporter plasmid, the pEGFP-C1 vector, or expression plasmids encoding ΔRIG-I and TBK1 along with increasing amounts (50 ng, 100 ng, 200 ng, and 500 ng) of the GFP-protease expression construct. IFNB promoter activity was measured at 24 h posttransfection. Values represent means ± SEM. (B) RIG-I is downregulated in the presence of HIV-I PR. HEK293 cells were cotransfected with expression vectors for Myc-RIG-I (2 μg) and increasing amounts (2 μg, 5 μg, and 10 μg) of GFP-protease as indicated. Cell lysates were subjected to Western blot analysis, and expression levels of RIG-I, protease, and β-actin were analyzed by immunoblotting with antibodies against Myc, GFP, and β-actin, respectively. (C) HIV-1 PR specifically targets RIG-I. HEK293 cells were cotransfected with plasmids expressing Myc-IRF-3, Flag-MDA5, and GFP-protease. Cell lysates were subsequently subjected to immunoblotting with anti-Myc, anti-Flag, anti-GFP, and anti-β-actin antibodies. (D) HIV-1 PR inhibits ΔRIG-I-mediated activation of IRF-3. HEK293 cells were transfected with 500 ng of Myc-IRF-3, 1 μg Myc-ΔRIG-I or GFP-TBK1, and 1 μg of GFP-protease expression plasmids as indicated. Whole-cell extracts were analyzed by immunoblotting for IRF-3 pSer-396, RIG-I, GFP-TBK1, GFP-protease, and β-actin. (E) HIV-1 PR downregulates IFN-stimulated gene expression. HEK293 cells were transfected with Myc-ΔRIG-I along with the GFP-protease expression plasmid. cDNA samples were subjected to real-time PCR analysis with primers specific for IFN-β, APOBEC3G, and β-actin. Results are presented as RQs. Values are means ± SEM.