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. 2020 Mar 16;16(3):e1008430. doi: 10.1371/journal.ppat.1008430

Fig 1. NOP2 inhibits HIV-1 replication.

Fig 1

(A) RNAi gene enrichment ranking (RIGER) method was applied to analyze screens performed using multiple orthologous RNAi reagents (MORRs). Genes were ranked in order of their RIGER scores (lowest → highest), from host dependency factors to host restriction factors. RIGER analysis of these screens recognized several known host restriction factors (CCNK, BRD4) as well as new ones, such as NOP2. (B) MAGI-HeLa cells were transiently transfected with the indicated siRNAs (siNT or siNOP2), and NOP2 knockdown was analyzed by immunoblotting. (C) MAGI-HeLa cells transfected with the indicated siRNAs were infected with HIV-1 IIIB viruses, followed by the immunostaining of p24 (green). Nuclei were stained with Hoechst 33342 (blue). The infection rate is calculated by dividing p24-expressing cells by total cells, and normalized to that of non-targeting siRNA (siNT). (D) MAGI-HeLa cells transfected with the indicated siRNAs were infected with HIV-1 NL4–3-Luc (dEnv) viruses. The relative luminometer units (RLU) of luciferase was measured and normalized total proteins, and normalized to that of non-targeting siRNA (siNT). (E) Jurkat cells were stably transduced with indicated shRNAs (shNT or shNOP2) in pAPM vector, and NOP2 knockdown was analyzed by immunoblotting. (F) Jurkat cells stably expressing shNOP2 or shNT were infected with HIV IIIB viruses. A portion of supernatant was harvested every 2 days until 12 days post-of-infection (dpi), and titrated using the TZM-bl cells. The RLU was measured, and normalized to that of non-targeting shRNA (shNT). (G) MAGI-HeLa cells were stably transduced with the indicated lentiviral vectors expressing V5-tagged FLAG peptide or NOP2 ORF (pLEX-FLAG or pLEX-NOP2), and protein expression of V5-NOP2 was analyzed by immunoblotting. (H) MAGI-HeLa cells stably transduced with pLEX-FLAG or pLEX-NOP2 were infected with HIV-1 NL4–3-Luc (dEnv) viruses. The RLU was measured, and normalized to that of pLEX-FLAG. (I) Jurkat cells were stably transduced with the indicated vectors (pLEX-FLAG or pLEX-NOP2), and protein expression of V5-NOP2 was analyzed by immunoblotting. (J) Jurkat cells stably transduced with pLEX-FLAG or pLEX-NOP2 were infected with HIV-1 IIIB viruses. A portion of supernatant was harvested every 2 days until 14 dpi, and titrated using the TZM-bl cells. The RLU was measured, and normalized to that of pLEX-FLAG. Results were based on n = 3 experiments and presented as mean ± S.D., * p < 0.05; ** p < 0.01; *** p < 0.001, ANOVA.