Fig 2: Confocal immunofluorescence and Western blotting analyses of M-LPH-WT and -KO HepG2 cells using specific antibodies. (a) In M-LPH-WT cells, the anti-M-LPH antibody displayed faint nuclear and cytosolic staining, and a small number of punctate staining foci were observed within the cytoplasm. All of these foci were colocalized with a subset of mitochondria. DAPI and MitoTracker were used for nuclear and mitochondrial staining, respectively. Scale bar: 10 μm. (b) Subcellular fractionation/immunoblotting analysis of M-LPH-WT cells using specific antibodies against M-LPH. CE: cytosolic extract; ME: membrane extract; NE: nuclear soluble extract. Loading controls for CE, ME, and NE were Hsp90AA1, VDAC, and HDAC1, respectively. (c) M-LPH-KO resulted in a significant increase of γ-H2AX formation. (d) Western blot analysis of chromatin-bound extracts from MLPH-WT and -KO HepG2 cells. HIST1H1C was used as a loading control for nuclear protein. (e, f) Immunofluorescence detection showed that the protein level of TFAM was downregulated by M-LPH-KO (e), whereas that of TWNK was upregulated (f).

Fig 3: TRIM25 is required for KHNYN to inhibit HIV-1 with clustered CpG dinucleotides.(A–B) HeLa Control CRISPR cells or TRIM25-G1 CRISPR cells were transfected with 500 ng pHIV-1 or pHIV-1EnvCpG86-561 and 500 ng of pGFP-FLAG or 62.5 ng pKHNYN-1-FLAG or pKHNYN-2-FLAG plus 437.5 ng of pGFP-FLAG. Culture supernatants from the cells were used to infect TZM-bl reporter cells (A). The bar charts show the average value of three independent experiments normalized to the value obtained for HeLa Control CRISPR cells co-transfected with pHIV-1 and pGFP-FLAG. Data are represented as mean ± SD. *p<0.05 as determined by a two-tailed unpaired t-test. p-values for GFP verses KHNYN-1 and KHNYN-2 for HIV-1EnvCpG86-561 in Control cells are 8.95 × 10−4 and 5.42 × 10−4, respectively. p-values for GFP verses KHNYN-1 and KHNYN-2 for HIV-1EnvCpG86-561 in TRIM25-G1 CRISPR cells are 1.78 × 10−3 and 1.01 × 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 (B). (C) Lysates of Control and TRIM25 CRISPR HEK293T cells transfected with pGFP-FLAG, pKHNYN-1-FLAG or pKHNYN-2-FLAG were immunoprecipitated with an anti-FLAG antibody. Post-nuclear supernatants and immunoprecipitation samples were analyzed by immunoblotting for HSP90, KHNYN-FLAG, TRIM25 and ZAP. The blots are representative of two independent experiments. (D) Lysates of Control and ZAP CRISPR HEK293T cells transfected with pGFP-FLAG, pKHNYN-1-FLAG or pKHNYN-2-FLAG were immunoprecipitated with an anti-FLAG antibody. Post-nuclear supernatants and immunoprecipitation samples were analyzed by immunoblotting for HSP90, KHNYN-FLAG, TRIM25 and ZAP. * indicates a non-specific band. The blots are representative of two independent experiments.

Fig 4: 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).

Fig 5: The KH and NYN domains are necessary for KHNYN antiviral activity.(A) Schematic of KHNYN-1 and KHNYN-2 domains and mutations in the NYN domain. (B–C) HeLa cells were transfected with 500 ng pHIV-1 or pHIV-1EnvCpG86-561 and either 500 ng of pGFP-FLAG or 62.5 ng pKHNYN-1/2-FLAG and 437.5 ng of pGFP-FLAG. Culture supernatants were used to infect TZM-bl reporter cells to measure infectivity (B). The bar charts show the average values of three independent experiments normalized to the value obtained for HeLa cells co-transfected with pHIV-1 and pGFP-FLAG. Data are represented as mean ± SD. *p<0.05 as determined by a two-tailed unpaired t-test. p-values for KHNYN-1 versus ΔKH-KHNYN-1, D443N-KHNYN-1, and D524A/D525A-KHNYN-1 for HIV-1EnvCpG86-561 are 1.31 × 10−2, 5.22 × 10−3, and 1.26 × 10−3, respectively. p-values for KHNYN-2 versus ΔKH-KHNYN-2, D443N-KHNYN-2, and D524A/D525A-KHNYN-2 for HIV-1EnvCpG86-561 are 2.88 × 10−2, 3.26 × 10−2, and 2.56 × 10−4, respectively. Gag expression in the media as well as Gag, Hsp90, Env, Actin, KHNYN-1/2-FLAG and GFP-FLAG expression in the cell lysates was detected using quantitative immunoblotting (C). See also Figure 6—figure supplements 1 and 2.