Viral requirements for 7SL packaging. (A) Schematic illustration of the utilized Gag variants. (Myr) myristate, (boxes) HIV-derived domains, (hatched box within CA) Major Homology Region, (lines) deleted regions, (ovals) heterologous protein domains: Z is a yeast-derived GCN4 zipper domain, and p2b contains Rous sarcoma virus late domain sequences (Accola et al. 2000). Schematic is based on information in (Accola et al. 2000); variants’ construction and other details are in (Accola et al. 2000). Minimal gag constructs were obtained from Dr. H. Gottlinger. (B) Env, genomic RNA, and Pol-deficient virus-like particle RNAs analyzed by a ribonuclease protection assay. All virus-like particles were produced by calcium phosphate transient transfection of 293T cells. Medium was harvested 48 h post-transfection, filtered, and concentrated by centrifugation through 20% sucrose. HIV-gpt (an HIV proviral clone with gpt in place of env) and pNL4–3.Luc.R–E– (HIV with frameshifts in env and vpr and luciferase replacing nef) were from the NIH AIDS Research and Reference Reagent Program. pCMVΔR8.2 (Naldini et al. 1996) encodes all HIV-1 proteins except Env. Rev-independent HIV Gag or Gag-pol encoding vectors (Huang et al. 2001) were supplied by Dr. Y. Huang. Ribonuclease protection was performed as in Figure 1. (C) Gag deletion variant virus-like particle RNAs analyzed by ribonuclease protection. Approaches are as above; variants’ structures are in panel A. All variants in panel B contain wild-type Gag. (D) Western blot of Gag proteins. Performed using anti-HIV-1 monoclonal anti-p24 (AS-55–11, Microbix Biosystems), a monoclonal anitibody that recognizes a CA N-terminal epitope. Expected protein sizes: ΔZwt6, 22 kD; Zwtp6, 50 kD; ΔZwt, 14 kD; ΔNCp1, 46 kD; Zwt, 44 kD; and ΔZwtp2b, 16 kD. Note that proteins missing the N-terminal half of CA are not visualized using this antibody.
