Statistical correlation of nonconservative substitutions of HIV gp41 variable amino acid residues with the R5X4 HIV-1 phenotype

Abstract

Background: The interaction of the envelope glycoprotein of HIV-1 (gp120/gp41) with coreceptor molecules has important implications for specific cellular targeting and pathogenesis. Experimental and theoretical evidences have shown a role for gp41 in coreceptor tropism, although there is no consensus about the positions involved. Here we analyze the association of physicochemical properties of gp41 amino acid residues with viral tropism (X4, R5, and R5X4) using a large set of HIV-1 sequences. Under the assumption that conserved regions define the complex structural features essential for protein function, we focused our search only on amino acids in the gp41 variable regions.

Methods: Gp41 amino acid sequences of 2823 HIV-1 strains from all clades with known coreceptor tropism were retrieved from Los Alamos HIV Database. Consensus sequences were constructed for homologous sequences (those obtained from the same patient and having the same tropism) in order to avoid bias due to sequence overrepresentation, and the variability (entropy) per site was determined. Comparisons of hydropathy index (HI) and charge (Q) of amino acid residues at highly variable positions between coreceptor groups were performed using two non-parametrical tests and Benjamini-Hochberg correction. Pearson's correlation analysis was performed to determine covariance of HI and Q values.

Results: Calculation of variability per site rendered 58 highly variable amino acid positions. Of these, statistical analysis rendered significantly different HI or Q only for the R5 vs. R5X4 comparison at twelve positions: 535, 602, 619, 636, 640, 641, 658, 662, 667, 723, 756 and 841. The largest differences in particular amino acid frequencies between coreceptor groups were found at 619, 636, 640, 641, 662, 723 and 756. A hydrophobic tendency of residues 619, 640, 641, 723 and 756, along with a hydrophilic/charged tendency at residues 636 and 662 was observed in R5X4 with respect to R5 sequences. HI of position 640 covariated with that of 602, 619, 636, 662, and 756.

Conclusions: Variability and significant correlations of physicochemical properties with viral phenotype suggest that substitutions at residues in the loop (602 and 619), the HR2 (636, 640, 641, 662), and the C-terminal tail (723, 756) of gp41 may contribute to phenotype of R5X4 strains.

Fig. 1

Entropy at each amino acid position (S _k) of HIV-1 gp41 calculated from the whole set of sequences. Residue numbers correspond to positions in gp160 HXB2 strain. Residues statistically related to virus phenotype (Tables 2 and 3) are indicated with position number. FP, fusion peptide; HR1, heptad repeat region 1; HR2, heptad repeat region 2; MPER, membrane proximal external region; MSD, membrane spanning domain; MSD2, membrane spanning domain 2; MSD3, membrane spanning domain 3; ME, minor ectodomain; LLP-1, lentiviral lytic peptide 1; LLP-2, lentiviral lytic peptide 2; LLP-3, lentiviral lytic peptide 3

Fig. 2

Mean hydropathy index differences (∆HI) of all 58 variable positions included in Additional file 1: Table S1. (a) ∆HI between R5X5 and R5 sequences. Positions showing significant differences of HI (Q_FD = 0.05) between R5X4 and R5 viruses are indicated with red circles. Position 658, which exhibited difference in charge only (Table 3) is indicated with a red square. Positions with the largest differences in amino acid frequencies between coreceptor groups (see Fig. 3) are indicated with position number. (b) ∆HI between R5X5 and X4 sequences. (c) ∆HI between X4 and R5 sequences. Positive or negative differences in HI imply a hydrophobic or hydrophilic tendency, respectively, for R5X4 (a, b) or X4 (c) sequences. Note that positions that were significant in the R5X4-R5 comparison (a) where not significant for the comparisons shown in (b) and (c), and are presented to illustrate the diminution of the hydrophobic or hydrophilic tendency of the respective residues (white circles and squares)

Fig. 3

Amino acid distribution at positions statistically related to virus phenotype in the R5, X4 and R5X4 groups

Fig. 4

Three-dimensional representation of the trimeric gp41 protein ectodomain. Ribbon representation of the protein with the HR1 domain (positions 531–591) in light blue and the HR2 (positions 624–681) domain in blue. Positions relevant for the R5 or R5X4 tropism (see Fig. 2) are shown in green. The image was obtained from a consensus homology model generated with Prime software [49] from gp160 (Uniprot: Q70626, positions: 531–681) of HIV-1 group M subtype B (isolate LW123), and using two templates (PDB ID's: 2X7R and 1IF3) [47, 48]. The coordinates of this structure are available in the Additional file 4: Figure S1 (Gp41 coordinates - Homology model)