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. 2022 Aug 1;96(16):e00526-22. doi: 10.1128/jvi.00526-22

FIG 2.

FIG 2

Riplet binds to ZAP to augment restriction of HIV-1 reporter virus. (A) Riplet interacts with ZAP via its PRY/SPRY domain. 293TrexhZAP cells were transiently transfected with DNAs overexpressing Riplet, Riplet mutants, or an empty vector control (EV), treated with doxycycline to induce ZAP expression as indicated (+/−), and lysed 48 h later. Riplet mutants included deletion of the RING domain (ΔRING), deletion of the P/SPRY domain (ΔP/SPRY), and dominant negative Riplet (RDN). (A, Left) In the input data, total proteins were analyzed by gel electrophoresis, blotted, and probed for myc-tagged ZAP (top) or Riplet (bottom). (A, Right) In the IP data, myc-tagged ZAP was recovered by immunoprecipitation using a mouse α-myc antibody, and bound proteins were analyzed by electrophoresis and probed for myc-tagged ZAP (top) or Riplet (bottom). (B) Riplet interaction with ZAP is RNase A-resistant. 293TrexhZAP cells were transfected with DNAs overexpressing Riplet or an EV control, treated with doxycycline or control DMSO to induce ZAP expression as indicated (+/−), and lysed 48 h later. Lysates were treated with RNase A (50 μg/mL) as indicated. (B, Left) In the input data, total proteins were analyzed by gel electrophoresis, blotted, and probed for myc-tagged ZAP with mouse α-myc antibody (top) or Riplet antibodies (bottom). (B, Right) In the IP data, myc-tagged ZAP was recovered by immunoprecipitation with mouse α-myc antibody, and bound proteins were analyzed by electrophoresis and probed for myc-tagged ZAP (top) or Riplet (bottom). Approximate molecular weights of major proteins estimated from size markers are indicated on left. (C) Riplet interacts with ZAP in pNL4.3-luc-infected cells. 293TrexhZAP cells transfected with an EV control or DNA overexpressing Riplet were either left uninfected or infected with a VSVG-pseudotyped pNL4.3-luc reporter virus or with a mock virus preparation lacking the VSVG envelope (mock virus). Cultures were treated with doxycycline or control DMSO to induce ZAP expression as indicated (+/−) and lysed 48 h later. (C, Left) In the input data, total proteins were analyzed by gel electrophoresis, blotted, and probed for myc-tagged ZAP with mouse α-myc antibody (top) or Riplet antibodies (bottom). (C, Right) In the IP data, myc-tagged ZAP was recovered by immunoprecipitation with mouse α-myc antibody, and bound proteins were analyzed by electrophoresis and probed for myc-tagged ZAP (top) or Riplet (bottom). Approximate molecular weights of major proteins estimated from size markers are indicated on left.