Effects of a mutated G-box on Gag production in cell culture. (A) A schematic of the four HIV-2 genomic RNAs produced in this study. The wild-type genomic RNA contains a wild-type C-box and a wild-type G-box. The C-box119 genomic RNA contains a mutated C-box and a wild-type G-box. The G-box119 genomic RNA contains a wild-type C-box and a mutated G-box. The double 119 genomic RNA contains a mutated C-box and a mutated G-box. The mutations are in bold lowercase letters. (B) A schematic of the three HIV-2 Gag protein isoforms that are synthesized in cell culture. The 58-, 51-, and 45-kDa isoforms are synthesized when translation initiates from methionine codons at positions 546, 744, and 897, respectively. (C) Western blot analysis of intracellular proteins from duplicate experiments where COS-7 cells were transfected with equimolar amounts of the wild-type, C-box119, G-box119, or CLS-119 proviral DNAs. (Lane 1) The mock-transfected sample. DNA templates used in each transfection are designated below each lane. The relative proportion of each isoform band was quantified and is graphically represented. The y-axis error bars represent the standard error of duplicate experiments. (D) Western blot analysis of virion proteins produced from duplicate experiments where COS-7 cells were transfected with equimolar amounts of the wild-type, C-box119, G-box119, or CLS-119 proviral DNAs. DNA templates used in each transfection are designated below each lane. The averaged level of capsid p27 protein in the COS-7 media was quantified (ELISA) and is indicated below the Western blot. The relative proportion of each isoform band was quantified and is graphically represented. The y-axis error bars represent the standard error of the duplicate experiments.
