FIG 5.

pp65/cGAS interaction. (A) HFFs were infected with wild-type, v65Rev, or v65Stop virus (MOI = 1) or left uninfected (mock) and subjected to IIF at 2 hpi. pp65 (green) and cGAS (red) were visualized using primary antibodies, followed by secondary-antibody staining in the presence of 10% HCMV-negative human serum. Nuclei were counterstained with TO-PRO-3 (blue). Images were generated by confocal microscopy; the far-right column shows 3D image reconstruction using the confocal z stacks. Digitally reconstructed 3D images were generated for at least 5 fields per condition; representative images are shown. (B) A PLA was performed to detect protein-protein interactions using fluorescence microscopy. The signal was detected as distinct fluorescent dots in the Texas Red channel when cells reacted with the indicated pairs of primary antibodies, followed by PLA to assess the interactions between pp65 and cGAS. (C) Coimmunoprecipitation from virus-infected or mock-infected cell lysates. HFFs were infected with wild-type, v65Rev, or v65Stop virus (MOI = 1) and harvested at 2 hpi. Immunoprecipitations were performed using antibodies against pp65 or without antibody as a negative control (CTRL). Immunoprecipitated proteins were detected by Western blotting analyses using antibodies against pp65, cGAS, and STING. Nonimmunoprecipitated whole-cell extracts (Input) were immunoblotted (IB) using anti-pp65, anti-cGAS, and anti-STING antibodies. (D) (Left) Immunoprecipitation was performed as described for panel C, except that the samples were split in two and half were treated with benzonase (1 U/μl) for 2 h on ice, followed by immunoprecipitation using antibodies against pp65. (Right) The mock cell lysate used in the IP depicted on the left was run on an ethidium bromide-stained (0.8%) agarose gel. −, IP in the absence of benzonase; +, IP in the presence of benzonase. (E) Mapping the region of pp65 required for its interaction with cGAS. Wild-type pp65 (pp65 Halo-WT) and serial deletion mutants of pp65 (pp65 Halo-ΔN and pp65 Halo-ΔC) were used to immunoprecipitate lysates of HFFs transiently expressing pp65 HaloTag. Interaction was detected by Western blotting using antibodies against cGAS.