Figure 9. Deletion of the KH-like domain or mutations in the NYN domain in KHNYN prevent reconstitution of CpG-dependent inhibition of HIV-1 infectious virus production in KHNYN CRISPR cells.
(A–B) HeLa KHNYN-G1 CRISPR clone B cells were transfected with 500 ng pHIV-1 or pHIV-1EnvCpG86-561 and 500 ng of pGFP-FLAG or 31.25 ng, 62.5 ng, 125 ng, 250 ng or 500 ng of pKHNYN-1-CRG1-FLAG or pKHNYN-2-CRG1-FLAG plus the amount of pGFP-FLAG required to make 500 ng total. Culture supernatants from the cells were used to infect TZM-bl reporter cells (A). Each point shows the average value of three independent experiments normalized to the value obtained for HeLa Control CRISPR cells co-transfected with pHIV-1 and pGFP. Data are represented as mean ± SD. Gag expression in the media as well as Gag, Hsp90, Env, Actin, KHNYN-FLAG and GFP-FLAG expression in the cell lysates was detected using quantitative immunoblotting (B). (C–D) HeLa KHNYN-G1 CRISPR clone B cells were transfected with 500 ng pHIV-1 or pHIV-1EnvCpG86-561, 468.75 ng of pGFP-FLAG and 31.25 ng of pKHNYN-1-CRG1-FLAG expressing either wild-type or mutant proteins. Culture supernatants from the cells were used to infect TZM-bl reporter cells (C). Each point shows the average value of seven independent experiments normalized to the value obtained for HeLa Control CRISPR cells co-transfected with pHIV-1 and pGFP. Data are represented as mean ± SD. *p<0.05 as determined by a two-tailed unpaired t-test. p-Values for HIV-1EnvCpG86-561 with KHNYN-1-CRG1 versus ΔKH-KHNYN-1-CGR1, D443N-KHNYN-1-CGR1 and D524A/D525A-KHNYN-1-CGR1 are 2.56 × 10−5, 1.95 × 10−4, and 2.12 × 10−4, respectively. Gag expression in the media as well as Gag, Hsp90, Env, Actin, KHNYN-FLAG and GFP-FLAG expression in the cell lysates was detected using quantitative immunoblotting (D).