Deep sequencing reveals an association between HIV-1 subtype C mutations in gp41 MPER epitopes and mother-to-child transmission

Abstract

Background: Enhanced HIV-1 mother-to-child transmission (MTCT) by high maternal anti-gp41 antibody titer led to the hypothesis that transmitting mothers would have greater diversity in membrane-proximal external region (MPER) and mutated amino acid residues associated with resistance to gp41 antibodies. Methods: Pyrosequences of HIV-1 subtype-C gp41 heptad repeat-region 2 (HR2), MPER, and membrane-spanning domain (MSD) were generated from 2,000 plasma viral copies/subject from four mothers who transmitted via breast feeding (TM) and four non-transmitting mothers (NTM) in a matched case control study. A bioinformatic pipeline with rigorous quality controls generated ~50,000 quality pyrosequences/subject and provided 25-fold coverage of input virus populations. Population genetic algorithms clustered pyrosequences at 3% genetic distance to study biodiversity using rarefaction/Chao1. Frequency distribution of cluster sizes defined population structure. Consensus sequences constructed from bioclusters for each subject were aligned to an HIV-1 subtype-C consensus sequence to determine number and frequency of nonsynonymous substitutions at each position and to identify mutations by HIV Molecular Immunology Database. Groups were compared using paired t-test. Results: Sequences in MPER were more polymorphic than in HR2 or in MSD. TM had more diverse MPERs than NTM (p = 0.02). The number of clusters calculated from rarefaction curves was 62(+/-35) for TM vs 35(+/-28) for NTM. The Chao1-estimated maximum number of variants within populations was 106(+/-51) for TM vs 59(+/-65) for NT. Low fit viruses (