Cysteine 95 and other residues influence the regulatory effects of Histidine 69 mutations on Human Immunodeficiency Virus Type 1 protease autoprocessing

Regulated autoprocessing of HIV Gag-Pol precursor is required for the production of mature and fully active protease. We previously reported that H69E mutation in a pseudo wild type protease sequence significantly (>20-fold) impedes protease maturation in an in vitro autoprocessing assay and in transfected mammalian cells. Interestingly, H69E mutation in the context of a laboratory adapted NL4-3 protease showed only moderate inhibition (approximately 4-fold) on protease maturation. There are six point mutations (Q7K, L33I, N37S, L63I, C67A, and C95A) between the NL4-3 and the pseudo wild type proteases suggesting that the H69E effect is influenced by other residues. Mutagenesis analyses identified C95 as the primary determinant that dampened the inhibitory effect of H69E. L63 and C67 also demonstrated rescue effect to a less extent. However, the rescue was completely abolished when H69 was replaced by aspartic acid in the NL4-3 backbone. Charge substitutions of surface residues (E21, D30, E34, E35, and F99) to neutral or positively charged amino acids failed to restore protease autoprocessing in the context of H69E mutation. Taken together, we suggest that residue 69 along with other amino acids such as C95 plus L63 and C67 to a less extent modulate precursor structures for the regulation of protease autoprocessing in the infected cell.