Fig 2: ZAP does not substantially inhibit a gammaretroviral vector(A) HEK293T CRISPR control, ZAP-ex4 ,or ZAP-ex6 producer cells were transfected with pMLV-CMV-GFP, pCMV-MLV Gag-Pol, and pVSV-G. Infectious titers were determined in transduced target cells (HEK293T CRISPR control, ZAP-ex4, or ZAP-ex6 cells) by flow cytometry for GFP-positive cells. (B and D) Intracellular MLV Gag expression from the packaging plasmid, GFP expression from the vector genome, and HSP90 and ZAP expression were determined by western blots of the producer cell lysates. Virus production was determined by western blots of the producer cell supernatant. (C) HEK293T cells were transfected with the pMLV-CMV-GFP genome and pCMV-MLV Gag-Pol packaging vector plus pVSV-G and pcDNA4, pZAP-L, or pZAP-S. Infectious titers of the vectors were determined by flow cytometry for GFP-positive HEK293T target cells. (A and C) The bar charts show the average values of three independent experiments. Data are shown as mean ± SD.

Fig 3: Binding of ZAP to WT, UpA-H, and CpG-H controls or 3' UTR mutant RNA transcripts. (A) WB of input long (L) and short (S) isoforms of ZAP (~100 kDa and ~70 kDa, respectively) and GAPDH in uninfected cell lysates detected by immunostaining with anti-ZAP polyclonal antibody or GAPDH polyclonal antibody. Cell lysates from replicon-transfected cells were incubated with anti-ZAP antibody or control antibody (IgG) and immobilized to columns with anti-rabbit IgG. The nitrocellulose membrane was damaged at positions indicated by the black arrowhead. The lower panel shows a WB for immunoprecipitated ZAP. (B) Quantitation of E7 viral RNA transcripts with WT, CpG-H, and UpA-H or 3' UTR mutant sequences by qPCR in immunoprecipitated ZAP (right column in each pair) or mock-precipitated control (left column). RNA was quantified by qPCR using conserved primers and probe from the 5' UTR (Table S2). Bar heights and error bars represent means and SDs for two biological replicates. The significance of differences from the WT construct is indicated as follows: ***, P = 0.0002; **, P = 0.0015; *, P < 0.015; ns, not significant (P > 0.05).

Fig 4: ZAP overexpression inhibits wild-type HIV-1 and HIV-1 containing introduced CpG dinucleotides. (A and B) HeLa cells were transfected with pHIV-1, pHIV-1env86–561CpG, pHIV-1pol795–1386CpG, pHIV-1-IRES-GFP, or pHIV-1-IRES-Renilla plus either pHA-Renilla, pHA-ZAP-S, or pHA-ZAP-L. Infectious-virus production was measured using TZM-bl reporter cells infected with cell culture supernatants. Gag expression in the media, as well as Gag and Hsp90 expression in the cell lysates, was detected using immunoblotting. The bar charts show the average values of the results of four independent experiments normalized to wild-type HIV-1 plus HA-Renilla. The numbers above the bars represent the fold decrease in relative infectious-virus production due to ZAP-S or ZAP-L overexpression. (C) Bar chart and representative Western blot for ZAP expression showing the average ZAP abundance for 15 independent transfections of p-HA-ZAP-S or pHA-ZAP-L in HeLa cells. The error bars represent standard deviations. *, P < 0.05 as determined by a two-tailed unpaired t test. The black asterisks compare the virus containing introduced CpGs in the control CRISPR cells to wild-type HIV-1 in the control CRISPR cells. The red asterisks compare the virus with HA-Renilla overexpression (black bars) to HA-ZAP overexpression (red bars).

Fig 5: KHNYN is a ZAP-interacting factor identified by yeast two-hybrid screening.A yeast two-hybrid screen for ZAP-S and ZAP-L interacting factors identified a region in KHNYN-1 and KHNYN-2. The selected interaction domain (SID) is the amino acid sequence shared by all prey fragments and is shown in magenta. A reciprocal yeast two-hybrid screen using KHNYN-2 as the bait identified a region in ZAP-S and ZAP-L. The SID is shown in red.