In silico Analysis of HIV-1 Env-gp120 Reveals Structural Bases for Viral Adaptation in Growth-Restrictive Cells

Abstract

Variable V1/V2 and V3 loops on human immunodeficiency virus type 1 (HIV-1) envelope-gp120 core play key roles in modulating viral competence to recognize two infection receptors, CD4 and chemokine-receptors. However, molecular bases for the modulation largely remain unclear. To address these issues, we constructed structural models for a full-length gp120 in CD4-free and -bound states. The models showed topologies of gp120 surface loop that agree with those in reported structural data. Molecular dynamics simulation showed that in the unliganded state, V1/V2 loop settled into a thermodynamically stable arrangement near V3 loop for conformational masking of V3 tip, a potent neutralization epitope. In the CD4-bound state, however, V1/V2 loop was rearranged near the bound CD4 to support CD4 binding. In parallel, cell-based adaptation in the absence of anti-viral antibody pressures led to the identification of amino acid substitutions that individually enhance viral entry and growth efficiencies in association with reduced sensitivity to CCR5 antagonist TAK-779. Notably, all these substitutions were positioned on the receptors binding surfaces in V1/V2 or V3 loop. In silico structural studies predicted some physical changes of gp120 by substitutions with alterations in viral replication phenotypes. These data suggest that V1/V2 loop is critical for creating a gp120 structure that masks co-receptor binding site compatible with maintenance of viral infectivity, and for tuning a functional balance of gp120 between immune escape ability and infectivity to optimize HIV-1 replication fitness.

Keywords: MD simulation; V1/V2 loop; V3 loop; adaptive mutation; homology modeling.

FIGURE 1

Molecular dynamics (MD) simulation of a full-length, glycosylated HIV-1_JRFL gp120 monomer. (A) Schematic representation of molecular modeling and MD simulation. Molecular model for a full-length, glycosylated gp120 monomer of HIV-1 R5-tropic virus JRFL in a CD4-free state was constructed by assembling of HIV-1 gp120 parts and homology modeling. PDB codes of the structures used for modeling are indicated on the left. Thermodynamically and physically refined model was subjected to MD simulation using the PMEMD module in the AMBER 10 program package as described for simulations of HIV-1/SIV gp120 outer domains (Naganawa et al., 2008; Yokoyama et al., 2012; Kuwata et al., 2013; Yuan et al., 2013). (B) Time course of RMSD between the initial model and models at given times of MD simulation. RMSD values were calculated with trajectories at every 2 fs of MD simulation using the ptraj module in Amber 10. (C) Time course of distances between residues in V2 and V3 (red line), residues in V2 and β20-β21 (green line), and residues in V3 and β20-β21 (blue line) in gp120. (D) Structures of a full-length, glycosylated gp120 monomer at 20, 30, 40, and 50 ns of MD simulations. V1/V2 and V3 regions are highlighted by red and blue colors, respectively. (E) Distances between the variable loops and core. The distances between the Cα of P117 at core neighboring V1 base and Cα of G307 at the tip of V3 loop (D_117-307), Cα of L121 at core neighboring V1 base and the Cα of R163 at V2 loop (D_121-163), and Cα of L172 at V2 loop and the Cα of Q324 at V3 base (D_172-324) were calculated with the gp120 model at 50 ns simulation and x-ray crystal structure (4TVP) to quantitatively compare relative 3-D locations of the V1/V2 and V3 loops on the core. Amino acid numbers are based on those of the JRFL Env protein (GenBank accession no. U63632).

FIGURE 2

Model for a full-length HIV-1_JRFL gp120 in a CD4-free state. (A) Full-length gp120 monomer model. The structure at 50 ns of MD simulation in Figure 1D is shown. Glycans are removed for clear view of the outer domain. A red asterisk indicates the location of CD4-binding loop. (B) Full-length gp120 trimer model. The model was constructed by superposing CD4-free monomer model on the x-ray crystal structure of Env gp140 protein (PDB code: 4TVP; Pancera et al., 2014). Side and top views are shown on the left and on the right, respectively. V1/V2 and V3 regions are highlighted by red and blue colors, respectively.

FIGURE 3

Model for a full-length HIV-1_JRFL gp120 in a CD4-bound state. (A) Full-length gp120 monomer model. Molecular model for a soluble CD4-bound full-length gp120 monomer was constructed as described in section “Materials and Methods.” (B) Full-length gp120 trimer model. The model was constructed by superposing CD4-bound monomer model on the Env structure derived from cryo-EM analysis (PDB code: 3DNO; Liu et al., 2008). Side and top views are shown on the left and on the right, respectively. V1/V2 and V3 regions are highlighted by red and blue colors, respectively.

FIGURE 4

Genetic information on HIV-1 gp120 adaptive mutations in the present study. (A) Location of HIV-1 adaptive mutations in Env-gp120. Amino acid sequences in V1 to V3 regions of HIV-1 R5-tropic 562 virus (Nomaguchi et al., 2013a) carrying the SF162 env gene (GenBank accession no. EU123924; Kawamura et al., 1994) are aligned with those of R5-tropic JRFL clone (GenBank accession no. U63632) and of X4-tropic HXB2 clone (GenBank accession no. K03455). Assignment of V1, V2, and V3 regions is based on gp120 structure: V1/V2 (PDB code: 3U4E), V3 (PDB code: 2QAD). (B) Frequency of authentic (light blue) and replaced (red) amino acid residues. Others (green) represent amino acid residues other than authentic and replaced ones. Naturally occurring amino acid residues at specific positions, where adaptive 562-gp120 mutations are located, were investigated in an HIV-1 subtype B population from different geographic regions in the world (19,419 sequences), and are graphically shown. The 19,419 sequences were obtained from the HIV Sequence Database (http://www.hiv.lanl.gov/content/sequence/HIV/mainpage.html).

FIGURE 5

Growth properties of 562 derivative viruses carrying various adaptive mutations in gp120. (A) Growth kinetics of variant viruses carrying a single adaptive mutation. Virus samples were prepared from 293T cells transfected with indicated proviral clones, and inoculated into HSC-F cells. Virus replication was monitored by RT activity released into the culture supernatants. Data of left and right panels were obtained from the same experiment, and the same result for 562 is separately shown as a control. Infection condition: 2 × 10⁵ RT units/2 × 10⁵ cells. (B) Growth properties of slowly growing variant viruses carrying a single adaptive mutation. Experiment was performed as described above. Infection condition: 4 × 10⁶ RT units/10⁶ cells. (C) Growth kinetics of variant viruses carrying two adaptive mutations. Experiments were performed as above. LF, L124F; NK, N132K; GR, G150R; FL, F174L; SG, S304G; IV, I307V; GR, G310R. Infection conditions: left, 2 × 10⁵ RT units/2 × 10⁵ cells; right, 5 × 10⁴ RT units/1 × 10⁵ cells.

FIGURE 6

Effects of growth-enhancing mutations in gp120 on viral entry. (A) Entry efficiency into HSC-F cells of 562 and 562 carrying indicated single substitutions. Virus samples were prepared as in Figure 5, and entry assays were performed as described in section “Materials and Methods.” Values obtained for ΔEnv construct (NL-Kp) were subtracted from those for test samples. Entry efficiency of each virus relative to that of 562 is presented. (B) Co-receptor usage of various viruses. Infection of HSC-F cells with viruses was performed as described above, and infected cells were cultured in the absence or presence (1 μM) of antagonists (CXCR4 antagonist AMD3100 or CCR5 antagonist TAK-779). Virus replication was monitored by RT activity released into the culture supernatants. Viral yields in test cultures relative to those on the peak day in cultures without antagonists were determined. 5R and 562 served as controls. (C) Sensitivity of 562 and its mutants to TAK-779. Virus samples prepared as above were inoculated into HSC-F cells pretreated with the indicated concentration of TAK-779. Virus replication was monitored by RT activity released into the culture supernatants. Viral yields in test cultures relative to those on the peak day in cultures without TAK-779 were determined and presented as % inhibition. Representative results from three independent experiments are shown.

FIGURE 7

The 3-D locations in a full-length gp120 of adaptive mutations. Adaptive amino acid substitutions are highlighted by colored globules on the gp120 models in CD4-free (A) and CD4-bound (B) states. Amino acid residues in V1/V2 and V3 regions are highlighted by red and blue colors, respectively. For details of the two models, see Figures 2A and 3A.

FIGURE 8

Effects of amino acid substitutions on the stability and affinity of gp120-CD4 complex. Full-length 562 gp120-CD4 complex model was constructed as described in section “Materials and Methods,” and used for in silico mutagenesis (Nomaguchi et al., 2013b). Changes in the stability (A) and affinity (B) scores by single amino acid substitutions were computed by using the Protein Design application in MOE as described in section “Materials and Methods.” Bars indicate standard deviations of the score (n = 3).

FIGURE 9

Effects of S304G mutation on molecular dynamics of V3 loop. Molecular models for 562 and 562 S304G V3 loops were constructed by homology modeling and subjected to MD simulations as described in section “Materials and Methods.” (A) Time course of RMSD between the initial model and models at given times of MD simulation. (B) Superposition of V3 structures obtained during 10–20 ns of MD simulations. (C) Distribution of RMSF in V3 loop. RMSF values that represent atomic fluctuations of the main chains of individual amino acids were calculated with 10,000 snapshots from 10 to 20 ns of each MD simulation. (D) Distributions of frequencies of β-sheet and turn structures in V3 loop that were formed during MD simulations. RMSD, RMSF, and frequencies were calculated using the ptraj module in Amber (Case et al., 2005). Red arrows in (B), (C), and (D) show the position of S304G mutation. Red and blue lines in (C) and (D) represent data on 562 and 562 S304G clones, respectively.