We previously observed a very high sensitivity to b12 neutralization in a pseudovirus isolated in an individual (patient GX93) from Guangxi autonomous region in China (Wang S, et al., 2011). To understand whether the extraordinary sensitivity to b12 neutralization is common in the quasispecies from this individual and the mechanism underlying this phenomenon, the SGA method was employed to amplify the full-length env. In total, 24 different env genes were amplified and 24 corresponding Env-pseudotyped viruses were constructed. Phylogenetic analysis of the full-length env nucleotide sequences confirmed that all 24 isolates were grouped within CRF07_BC (Figure 1). The 24 full-length env were clustered into four groups (named as G1, G2, G3 and G4). The amino acid sequence length of the Env proteins was analyzed within and between each group. We found that the length of all the G1 Envs was 852, the Env length of G3 and G4 was 848. For the G2 group, the length of GX93.16 was 852, that of GX93.1, GX93.3 and GX93.10 were 857, and that of GX93.2 was 848.
Figure 1. Phylogenetic relationships of HIV-1 env sequences of the quasispecies with reference strains. Horizontal branch lengths are drawn to scale (the scale bar represents 0.02 nucleotide substitution per site), but vertical separation is for clarity only. Values at the node indicate the percentage of bootstraps in which the cluster to the right was found; only values of ≥80% are shown.
The 24 Env-pseudotyped viruses cloned from the same individual showed divergent sensitivity to b12 neutralization. According to the sensitivity, the 24 strains were divided into four groups (named as S1, S2, S3 and S4) (Table 1). The ID50 values of the viruses in groups S1, S2, S3 and S4 were > 25 μg/mL, 1 μg/mL–25 μg/mL, 0.1 μg/mL–1 μg/mL, and ＜0.1 μg/mL, respectively. The groups S1, S2, S3 and S4 contained 4 strains (GX93.1–GX93.4), 3 strains (GX93.5–GX93.7), 10 stains (GX93.8–GX93.17) and 7 strains (GX93.18–GX93.24), respectively. In the virus quasispecies, a large variation in the sensitivity for b12 neutralization was observed, ranging from 0.014 μg/mL for GX93.24 to more than 25 μg/mL for GX93.1. The ID50 difference between the highest and the lowest sensitive strains was more than 1700-fold.
pseudovirus clone length of gp160 Amino acid residues corresponding to HXB2 position ID50 of b12
Group strain 22 182 276 334 346 386 466 474 698 778 786 S1 GX93.1 857 L V N S R N T N I A G > 25 GX93.2 848 * * * * * * N * * * * > 25 GX93.3 857 * * * * * * * * * * * > 25 GX93.4 848 * * * * * * * * * * * > 25 S2 GX93.5 852 * * * * * * * * * * * 5.356 GX93.6 852 * * * * * * * * * * * 2.859 GX93.7 852 * * * * * * * * * * * 2.255 S3 GX93.8 848 * * * * * * * * * * * 0.734 GX93.9 852 * * * * * * * * * * * 0.454 GX93.10 857 * * * * * * * * * * * 0.325 GX93.11 852 * * * * * * * * * * * 0.280 GX93.12 852 * * * * * * * * * * * 0.258 GX93.13 852 * * * * * * * * * * * 0.230 GX93.14 852 * * * * * * * * * * * 0.188 GX93.15 852 * * * * * * * * * * * 0.172 GX93.16 852 * * * * * * * * * * * 0.167 GX93.17 852 * * * * * * G * V T R 0.117 S4 GX93.18 848 F L S N S D G D V T R 0.092 GX93.19 848 F L D N S D G D V T R 0.080 GX93.20 848 F L S N S D G D V T R 0.040 GX93.21 848 F L S N S D G D V T R 0.020 GX93.22 848 F L S N S D G D V T R 0.015 GX93.23 848 F L D N S D G D V T R 0.015 GX93.24 848 F L S N S D G D V T R 0.014 *Amino acids which were identical to the GX93.1 in the same position are designated with a dot.
Table 1. Length of gp160 and amino acid substitutions in Env-pseudotyped virus coinciding with variety of sensitivity to b12 neutralization.
To identify amino acid key sites in the gp160 protein that may play a role in the sensitivity to b12 neutralization, full-length Env protein sequences from quasispecies alignment was associated to the ID50 values for b12 neutralization. Eleven amino acid substitutions were found in the most b12-sensitive strains: L22F in the signal peptide, V182L in variable region 2 (V2), N276S in constant region 2 (C2), S334N in C3, R346S in C3,N386D in C3, T/N466G in V5, N474D in C5, I698V in transmembrane (TM) domain, T778A in cytoplasmic tail (CT) and G786R in CT. For the sensitivity groups, all the eleven substitutions were observed in strains belonging to the most sensitive group S4, and were all absent in the resistant groups S1 and S2. For group S3, nine of the ten strains had no such substitutions. Only the most sensitive isolates in this group had four substitutions.
To determine whether the identified sites play a role in the variation of the sensitivity to b12 neutralization, two pseudovirus strains were selected as templates to generate the mutations. To verify the function of the identified sites, we made site-directed mutagenesis in two directions, from resistant to sensitive and from sensitive to resistant. The sensitive mutations were introduced into GX93.6 (Figure 2A) and the resistant mutations were introduced into the template GX93.24 (Figure 2B). Eleven mutant viruses were produced for both of the virus strains. From the results, we found that the mutations in 182, 276 and 346 resulted in changes to the sensitivity of b12 neutralization. The L182V mutation decreased the sensitivity of GX92.23 to b12 nearly tenfold (0.143 μg/mL vs 0.015 μg/mL for ID50), while V182L increased the sensitivity of GX93.6 more than twofold (1.339 μg/mL vs 2.853 μg/mL for ID50). The D276N mutation reduced the sensitivity more than twofold in GX93.23 (0.35 μg/mL vs 0.15 μg/mL), while N276D increased the b12 sensitivity by nearly twofold in GX93.6 (1.564 μg/mL vs 2.853 μg/mL). For the mutation in position 346, the serine to arginine decreased the sen-sitivity of GX93.23 more than twofold (0.032 μg/mL vs 0.015 μg/mL), and the reverse mutation in GX93.6 increased the sensitivity more than two fold (1.179 μg/mL vs 2.835 μg/mL).
Figure 2. Effect of mutations in gp120 relating to the variation of b12 neutralization sensitivity. Eleven corresponding site-directed mutageneses were introduced into GX93.6 and GX93.23 to produce 11 mutants for each strain. For GX93.6, the aim of the mutagenesis was to enhance the sensitivity of the resistant strain (A). For GX93.23, the aim of the mutagenesis was to reduce the sensitivity of the sensitive strain (B). The results are presented with the (ID50 for mutants)/(ID50 for parental strain).
When the three-site mutageneses were introduced into the same strain, no additive effect was observed. The enhancement of combined three-site mutations was similar to the single V182L mutagenesis. In order to further determine the effect of V182L mutation on b12 neutralization susceptibility, this mutagenesis was introduced to a more resistant strain GX93.2 (ID50 >25 μg/mL). The pseudovirus containing the GX93.2–V182L Env was more sensitive to b12 than the parental pseudovirus, with an ID50 value of 21.35 μg/mL. In further studies using the 18 CRF07/08 BC Envpseudotyped viruses constructed previously (Chong H, et al., 2008), we found that the amino acid residues at position 182 were all valine. The V182L was also introduced to BC17 (ID50 = 13.23 μg/mL) and this produced a twofold enhanced sensitivity to b12, with an ID50 value of 5.85 μg/mL. However, when the V182L mutation was introduced to the CRF01_AE strain GX74.20 (Nie J, et al., 2010), no influence on b12 neutralization susceptibility was observed.