Fig. 3.

Effect of Env mutations on calmodulin co-immunoprecipitation. (A) Jurkat Tet-off cells stably transfected with wild-type env (wild-type) or the A835W env gene (A835W) were incubated in the presence of 2 μg/ml tetracycline (+Tet) to inhibit Env expression or in the absence of tetracycline (−Tet) for 48 h to induce expression of Env. Cells were metabolically labeled with ³⁵S-cysteine/methionine followed by immunoprecipitation with HIV⁺ patient serum. Immunoprecipitated proteins were separated by SDS-PAGE and visualized by autoradiography for gp160/120 (upper panel) or by Western blot for calmodulin (CaM) (lower panel). (B) H9 cells were infected as described in panel A, and, on day 11 post-infection, approximately 1 × 10⁶ cells were collected and lysed. Viral proteins were immunoprecipitated with HIV⁺ patient serum, separated by SDS-PAGE, and Western blotted for gp120 (top) and calmodulin (bottom). Shown is a representative Western blot of two independent experiments and is identical to results using cell lysates made on day 7 and 14 post-infection (data not shown). (C) H9 cells were infected with HIV-1 NL4-3 containing the env BamHI/XhoI fragments from HXB2. This region was either unchanged (HXB2) or changed as indicated (A835W, A838I, I842R). On day 8 post-infection, cells were lysed and viral proteins immunoprecipitated as described in Materials and methods. Shown is a representative Western blot (n = 3) and is similar to results obtained on days 5 and 12 post-infection (not shown). (D) Co-immunoprecipitation of calmodulin with envelope was quantified by densitometry. For each mutation of envelope tested, the band density corresponding to calmodulin was determined, and this value was expressed as a percentage of the band density from HXB2 Env-infected cells. Data shown are the means ± SEM, n = 6 for HXB2 and A835W and n = 3 for A838I, A838W and I842R. Asterisks indicate a significant decrease in calmodulin co-immunoprecipitation compared to HXB2 (P < 0.05).