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Fig. 4 | Retrovirology

Fig. 4

From: HIV-1 protease with leucine zipper fused at N-terminus exhibits enhanced linker amino acid-dependent activity

Fig. 4

Effects of C-terminal p6* residue substitutions on the capability of p6*-deleted Gag-Pol mutants to mediate virus maturation. a Schematic representations of HIV-1 Gag-Pol expression constructs with deletions of most p6* coding sequences. HIV-1 Gag domains, pol-encoded p6*, PR, RT and IN are indicated. All constructs contain a frame shift (fs) mutation forcing gag and pol into the same reading frame. Dashed lines denote deleted p6* regions. Remaining N-terminal and C-terminal p6* residues are indicated in boldface. Altered or foreign residues are in italics. b, c 293T cells were co-transfected with 10 μg of an HIV-1 Pr55gag expression plasmid (pGAG) and 1 or 10 µg (panel b) or 1 µg (panel c) of the designated Gag-Pol expression construct. At 48 h post-transfection, cells and supernatants were collected and analyzed by Western immunoblotting. Membrane-bound proteins were initially probed with anti-RT serum, stripped, and probed again with anti-p17MA and anti-p24CA monoclonal antibodies. Indicated are HIV-1 Gag-Pol, 66/51RT, Pr55gag, p41gag, p24gag and p17gag positions. d A single amino acid change blocked the capability of p6*-deleted Gag-Pol to confer virus infectivity. 293T cells were co-transfected with 10 µg of an HIV-1 Pr55gag expression vector (pGAG) and 1 µg of one of the designated constructs plus 5 µg of a VSV-G expression vector. At 48 h post-transfection, supernatants were collected, filtered, and used to infect HeLa cells. Infectivity for each Gag-Pol construct was determined as the ratio of mutant titers to wt Gag-Pol titers, normalized to virus-associated p24gag protein levels in parallel experiments. **p < 0.01; ***p < 0.001

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