Background:  The proteolytic cleavage of HIV-1 Gag and Gag/Pol polyproteins, mediated by the viral aspartic protease, is of paramount importance for the viral maturation process. Information regarding the order and the rate of Gag/Pol processing events is largely limited to HIV-1 subtype B viruses. To date, no data are available for Gag/Pol processing in non-B subtypes.

Methods:  We obtained “near full-length” clones of the gag/pol genes for subtypes A, B, and C from the NIH Reagent Repository and we analyzed in trans Gag processing employing an in vitro transcription-translation system that uses ³⁵S-Met to visualize the intermediates by autoradiography. Site-directed mutagenesis technique was used to engineer desired mutations within Gag constructs. We followed in trans processing of Gag polyprotein by protease from the corresponding subtype, but we also mixed Gag polyproteins with proteases from other subtypes.

Results:  We observed that in trans processing of subtype A and C Gag polyproteins produced a p15 intermediate that migrates on a different position on a Tris/Glycine/SDS gel when compared with subtype B. When we combined Gag polyproteins with proteases from different subtypes we noticed that the pattern of cleavage was dependent on the Gag sequence and seemed not to be influenced by the protease added in trans. Furthermore, subtype B and C Gag proteins are cleaved with comparable efficiency by all 3 proteases, while subtype A Gag is processed at a slower rate. The rate of p24/p25 production is slower in subtype A Gag, independently of the protease subtype added in trans. Surprinsingly, densitometric analysis showed that the rate of p24/p25 production is slower even when subtype A Gag polyprotein is processed by subtype A protease. When we introduced S124V at MA/CA cleavage site, the production of p24/p25 increased by 3-fold when compared with initial subtype A Gag polyprotein, but did not reach the level of cleavage observed in subtype B.

Conclusions:  The pattern of cleavages of subtype A, B, and C Gag polyprotein, independent of the subtype of the protease added in trans, is noticeably similar, indicating that there are determinants in Gag that modulate the processing events. Additional proof is brought by the 3-fold increase in p24/p25 yield after the introduction of S124V mutation at P5 position of MA/CA cleavage site.