Natural Polymorphisms at gag cleavage sites and their potential impact on the substrate envelope structure of HIV-1 Subtype C.

Abstract

Limited studies have investigated the natural variations within the gag gene of HIV-1 subtype C, particularly at the cleavage sites (CSs), with most existing research focusing on subtype B. This study extended prior findings by comparing the natural variability at the CSs between HIV-1 subtypes B and C, extending the analysis from 5AA to 10AA and 15 AA on either side of the scissile bond, highlighting differences that may contribute to protease (PR)-substrate interactions and viral fitness. In addition, this study provided a more comprehensive understanding of how natural polymorphisms at the CSs (5AA) influence the substrate envelope, the substrate's consensus volume, which serves as a template that the PR uses to recognize and bind to a specific CS. The findings revealed distinct patterns of CS variability between subtypes B and C. Notably, subtype C sequences exhibited high variability at the P2/NC and P1/P6 CSs. The P2/NC CS showed the highest variability, with 100% of sequences in subtype C being polymorphic at this site. Furthermore, the study demonstrated that the increase in sequence length from 5AA to 15AA amplified the variability, particularly at the P2/NC and P1/P6 sites. While this was expected, it was interesting to note that the greatest variability was seen where the extended sites overlapped. This suggests that subtype C may have a more diverse and mutable PR CS profile. However, this requires further investigation. The structural analysis of the CSs showed that strong binding affinities were linked to extensive hydrogen bonding and π-alkyl interactions, often involving conserved residues, while unfavourable interactions such as steric clashes weakened binding. Subtype B generally had more diverse and distributed interactions, including extensive hydrophobic contacts (e.g., Val32, Ile50), salt bridges, and favourable hydrogen bonds involving the D25, Asp29, and Asp30 residues. Subtype C often formed fewer but stronger hydrogen bonds (shorter distances), with specific π interactions (e.g., with Val82), but also displayed unfavourable donor–donor clashes, especially in MA/CA and NC/P1 complexes. For P2/NC, subtype B had a wider interaction network, while subtype C relied on localized binding. Although subtype C sometimes showed slightly higher binding affinities (e.g., -8.3 kcal/mol), subtype B’s interactions were more varied and involved more structural and catalytic residues, suggesting potentially more stable binding overall. In conclusion, natural polymorphisms at the gag CSs impacted the structure of the substrate envelope of HIV-1 subtype C which could impact the cleavage by PR. These findings emphasize the importance of understanding the distinct mutation profiles of HIV-1 subtypes B versus C, which is important for the advancement of effective therapeutic strategies to combat HIV-1 globally.