Concomitant Enhancement of HIV-1 Replication Potential and Neutralization-Resistance in Concert With Three Adaptive Mutations in Env V1/C2/C4 Domains

Abstract

HIV-1 Env protein functions in the entry process and is the target of neutralizing antibodies. Its intrinsically high mutation rate is certainly one of driving forces for persistence/survival in hosts. For optimal replication in various environments, HIV-1 Env must continue to adapt and evolve through balancing sometimes incompatible function, replication fitness, and neutralization sensitivity. We have previously reported that adapted viruses emerge in repeated and prolonged cultures of cells originally infected with a macaque-tropic HIV-1_NL4-3 derivative. We have also shown that the adapted viral clones exhibit enhanced growth potentials both in macaque PBMCs and individuals, and that three single-amino acid mutations are present in their Env V1/C2/C4 domains. In this study, we investigated how lab-adapted and highly neutralization-sensitive HIV-1_NL4-3 adapts its Env to macaque cells with strongly replication-restrictive nature for HIV-1. While a single and two mutations gave a significantly enhanced replication phenotype in a macaque cell line and also in human cell lines that stably express either human CD4 or macaque CD4, the virus simultaneously carrying the three adaptive mutations always grew best. Entry kinetics of parental and triple mutant viruses were similar, whereas the mutant was significantly more readily inhibited for its infectivity by soluble CD4 than parental virus. Furthermore, molecular dynamics simulations of the Env ectodomain (gp120 and gp41 ectodomain) bound with CD4 suggest that the three mutations increase binding affinity of Env for CD4 in solution. Thus, it is quite likely that the affinity for CD4 of the mutant Env is enhanced relative to the parental Env. Neutralization sensitivity of the triple mutant to CD4 binding site antibodies was not significantly different from that of parental virus, whereas the mutant exhibited a considerably higher resistance against neutralization by a CD4-induced epitope antibody and Env trimer-targeting V1/V2 antibodies. These results suggest that the three adaptive mutations cooperatively promote viral growth via increased CD4 affinity, and also that they enhance viral resistance to several neutralization antibodies by changing the Env-trimer conformation. In total, we have verified here an HIV-1 adaptation pathway in host cells and individuals involving Env derived from a lab-adapted and highly neutralization-sensitive clone.

Keywords: CD4; Env; Env structure; HIV-1; adaptive mutation; neutralization sensitivity; replication potential.

FIGURE 1

Proviral clones used in this study. (A) Genomes of HIV-1_NL4-3 and its derivative HIV-1mt clone (ScaVR). Sequences from HIV-1_NL4-3 (GenBank accession number AF324493) and SIVmac_MA239 (GenBank accession number M33262) are shown by white and black boxes, respectively. Amino acid mutations and their locations (Env domains in parentheses) in Env-gp120 of 5R and MN4 clones are indicated. CypA, cyclophilin A. (B) In vitro adaptation processes of HIV-1mt clones in HSC-F cells. Details for prolonged cultures of HSC-F cells infected with ScaVR and 5R (Genbank accession number AB266485) have been reported previously (Kamada et al., 2006; Nomaguchi et al., 2013a).

FIGURE 2

Growth kinetics of ScaVR and its Env-gp120 mutants. Viruses were prepared from 293T cells transfected with proviral clones indicated and inoculated into HSC-F, A2.01/HuCD4, and A2.01/CyMCD4 cells as described in Section “Materials and Methods.” Virus replication was monitored by RT activity released into culture supernatants. Representative data from at least two independent experiments are shown.

FIGURE 3

Surface CD4 expression on the cell lines used in this study. Various cell lines were stained with FITC-labeled CD4 antibody, and were subjected to flow cytometry analysis. Numbers in graphs indicate the mean fluorescence intensity of each sample. (A) Analysis of CD4-positive HSC-F cells. Negative control is shown by black line. (B) Analysis of A2.01 cell line and its derivatives. Parental A2.01 (CD4-negative), A2.01/CyMCD4, and A2.01/HuCD4 cells are shown by black, blue, and red lines, respectively.

FIGURE 4

Comparative characterization of ScaVR and ScaVR+138/275/427 viruses. (A) Effect of triple mutations in Env on Env expression level. Lysates were prepared from transfected 293T cells as indicated. Samples containing equal Gag-p24 amounts were analyzed by western blotting using anti-HIV rgp160 and anti-HIV-1 p24 antibodies. Representative data from two independent experiment are shown. (B) Effect of triple mutations in Env on viral infectivity. Viruses prepared from 293T cells transfected with indicated proviral clones were inoculated into TZM-bl cells. On day 2 post-infection, cells were lysed and used for luciferase assays. Viral infectivity is presented as RLU of ScaVR+3 relative to that of ScaVR. Mean values with standard errors (SE) are shown (n = 6). (C) Effect of triple mutations in Env on entry efficiency. Virus samples prepared from 293T cells transfected with indicated proviral clones were spin-inoculated into TZM-bl cells at 10°C for 1 h, and were then replaced with pre-warmed fresh media. At indicated time points, AMD3100 was added to culture media at final concentration of 1 μM. On day 2 post-infection, cells were lysed, and subjected to luciferase assays. Relative infectivity is presented by calculating RLU of each virus sample at the indicated time point relative to that of each virus sample at 120 min post-infection. Mean values ± SE are shown (n = 4). (D) Effect of triple mutations in Env on sCD4 sensitivity. Viruses were prepared from 293T cells transfected with proviral clones indicated, and were incubated with sCD4 at 37°C for 1 h. Virus samples were then inoculated into TZM-bl cells, and on day 2 post-infection, cell lysates were prepared for luciferase assays. Relative infectivity, obtained by calculating RLU of each virus sample incubated with sCD4 relative to that of each virus sample incubated without sCD4, is presented. Mean values ± SE are shown (n = 6).

FIGURE 5

Neutralization sensitivity assays for ScaVR and ScaVR+138/275/427 (ScaVR+3). Viruses were prepared from 293T cells transfected with indicated proviral clones. Virus samples were pre-incubated with each NAb at 37°C for 1 h, and were inoculated into TZM-bl cells. On day 2 post-infection, cells were lysed for luciferase assays. Relative infectivities were calculated by RLU values with indicated concentrations of NAb relative to that without NAb, and then relative inhibition rates (1.0 minus relative infectivity) were determined and are presented. IC₅₀ values are indicated in the upper panel. Mean values ± SE are shown (n = 6 or more). Representative graph data of neutralization sensitivity assays using NAbs are shown. b12, IgG1 b12; G54W, NIH45-46 G54W; 3BNC, 3BNC117; CH01, CH01 mAb.

FIGURE 6

Three-dimensional locations of Env-gp120 mutations in the MD-derived Env ectodomain (gp120 and gp41 ectodomain) bound with soluble CD4. HIV-1 Env ectodomain bound with CD4 was constructed for ScaVR and ScaVR+138/275/427 clones by the homology modeling method using the crystal structure of HIV-1 Env ectodomain with CD4 (PDB code: 5VN3) (Ozorowski et al., 2017). The models were subjected to MD simulations for 500 ns to obtain dynamic structures in solution (see Materials and Methods). Binding energies of the Env proteins to CD4 molecule were calculated using ensembles derived from last 5 ns after 500 ns MD simulations using MMPBSA.py program (Miller et al., 2012) in the AmberTools16 (AMBER 16, University of California, San Francisco). The Env-CD4 complex structure of ScaVR at 100 ns of MD simulation is shown with binding energies of ScaVR and ScaVR+138/275/427 Env proteins to CD4.