Protease polymorphisms in HIV-1 subtype CRF01_AE represent selection by antiretroviral therapy and host immune pressure

Abstract

Background: Most of our knowledge about how antiretrovirals and host immune responses influence the HIV-1 protease gene is derived from studies of subtype B virus. We investigated the effect of protease resistance-associated mutations (PRAMs) and population-based HLA haplotype frequencies on polymorphisms found in CRF01_AE pro.

Methods: We used all CRF01_AE protease sequences retrieved from the LANL database and obtained regional HLA frequencies from the dbMHC database. Polymorphisms and major PRAMs in the sequences were identified using the Stanford Resistance Database, and we performed phylogenetic and selection analyses using HyPhy. HLA binding affinities were estimated using the Immune Epitope Database and Analysis.

Results: Overall, 99% of CRF01_AE sequences had at least 1 polymorphism and 10% had at least 1 major PRAM. Three polymorphisms (L10 V, K20RMI and I62 V) were associated with the presence of a major PRAM (P < 0.05). Compared to the subtype B consensus, six additional polymorphisms (I13 V, E35D, M36I, R41K, H69K, L89M) were identified in the CRF01_AE consensus; all but L89M were located within epitopes recognized by HLA class I alleles. Of the predominant HLA haplotypes in the Asian regions of CRF01_AE origin, 80% were positively associated with the observed polymorphisms, and estimated HLA binding affinity was estimated to decrease 19-40 fold with the observed polymorphisms at positions 35, 36 and 41.

Conclusion: Polymorphisms in CRF01_AE protease gene were common, and polymorphisms at residues 10, 20 and 62 most likely represent selection by use of protease inhibitors, whereas R41K and H69K were more likely attributable to recognition of epitopes by the HLA haplotypes of the host population.

Fig. 1. Observed CRF01_AE PRAMs and polymorphisms

(a). Proportion of PRAMs by time period, (b). Number of PRAMs by residue site and (c). CRF01_AE consensus sequence and observed residue diversity. Comparisons were made between two time periods (1990–2000 and 2001–2007). The frequencies of three of the polymorphisms increased significantly from 1990–2000 to 2001–2007, residues: 16 (11.8–32.2%), 37 (3.0–11.3%) and 63 (22.8–35.4%) (P < 0.05), which corresponded to an increase in protease inhibitors in the region (Roche, Bristol-Myers Squibb and Abbott, Thailand, personal communication). Sites where CRF01_AE consensus sequence differed from subtype B consensus are marked with filled grey boxes and the subscripts represent the number of sequences observed with these nonconsensus CRF01_AE amino acids. Protease inhibitors were first introduced into each country was as follows: Thailand and Singapore, 1996; Japan, 1997; China, 1998; Vietnam and Cambodia, 2000 (Roche, Bristol-Myers Squibb and Abbott, Thailand, personal communication).