Structure and dynamics of the gp120 V3 loop that confers noncompetitive resistance in R5 HIV-1(JR-FL) to maraviroc

Abstract

Maraviroc, an (HIV-1) entry inhibitor, binds to CCR5 and efficiently prevents R5 human immunodeficiency virus type 1 (HIV-1) from using CCR5 as a coreceptor for entry into CD4(+) cells. However, HIV-1 can elude maraviroc by using the drug-bound form of CCR5 as a coreceptor. This property is known as noncompetitive resistance. HIV-1(V3-M5) derived from HIV-1(JR-FLan) is a noncompetitive-resistant virus that contains five mutations (I304V/F312W/T314A/E317D/I318V) in the gp120 V3 loop alone. To obtain genetic and structural insights into maraviroc resistance in HIV-1, we performed here mutagenesis and computer-assisted structural study. A series of site-directed mutagenesis experiments demonstrated that combinations of V3 mutations are required for HIV-1(JR-FLan) to replicate in the presence of 1 µM maraviroc, and that a T199K mutation in the C2 region increases viral fitness in combination with V3 mutations. Molecular dynamic (MD) simulations of the gp120 outer domain V3 loop with or without the five mutations showed that the V3 mutations induced (i) changes in V3 configuration on the gp120 outer domain, (ii) reduction of an anti-parallel β-sheet in the V3 stem region, (iii) reduction in fluctuations of the V3 tip and stem regions, and (iv) a shift of the fluctuation site at the V3 base region. These results suggest that the HIV-1 gp120 V3 mutations that confer maraviroc resistance alter structure and dynamics of the V3 loop on the gp120 outer domain, and enable interactions between gp120 and the drug-bound form of CCR5.

Figure 1. Noncompetitive resistant HIV-1_V3-M5.

(A) Five amino acid substitutions in the V3 loop of HIV-1_V3-M5 (I304V/F312W/T314A/E317D/I318V). HIV-1_JR-FLan was created form HIV-1_JR-FL by incorporation of AflII and NheI. Incorporation of the NheI site led to amino acid substitutions Val³⁴²-Ile³⁴³ to Ala³⁴²-Ser³⁴³. HIV-1_JR-FLan was used as the parental virus. (B) Replication kinetics of HIV-1_V3-M5 in the presence or absence of 1 µM maraviroc in PM1/CCR5 cells. PM1/CCR5 cells (1×10⁵) were infected with 10 ng of p24 Gag for 3 h. Viral replication was monitored by measuring p24 Gag in the supernatant after infection. The analysis was repeated three times; the error bars represent the S.D. of three replicates from one representative experiment.

Figure 2. The effect of 1 µM of maraviroc on p24 Gag production in recombinant viruses containing one (A) and two or three (B) of the five amino acid substitutions.

PM1/CCR5 cells (1×10⁵) were infected with 10 ng p24 Gag for 3 h in the presence or absence of 1 µM maraviroc. On day 6 after infection, the amount of Gag in the supernatant was measured using HIV-1 p24 ELISA. The analysis was repeated three times; the error bars represent the S.D. of three replicates from one representative experiment. **, p<0.01. Statistical significant difference was calculated by t test.

Figure 3. The effect of 1 µM of maraviroc on p24 Gag production in recombinant viruses containing four of the five amino acid substitutions.

PM1/CCR5 cells (1×10⁵) were infected with 10 ng p24 Gag for 3 h in the presence or absence of 1 µM maraviroc. On day 6 after infection, the amount of Gag in the supernatant was measured using HIV-1 p24 ELISA. The analysis was repeated three times; the error bars represent the S.D. of three replicates from one representative experiment. **, p<0.01. Statistical significant difference was calculated by t test.

Figure 4. The effect of 1 µM of maraviroc on p24 Gag production in HIV-1_JR-FLan, HIV-1_T199K, HIV-1_V3-M5, HIV-1_V3-M5/T199K, HIV-1₂₃₄, and HIV-1_234/T199K.

PM1/CCR5 cells (1×10⁵) were infected with 10 ng p24 Gag for 3 h in the presence or absence of 1 µM maraviroc. On day 6 after infection, the amount of Gag in the supernatant was measured using HIV-1 p24 ELISA. The analysis was repeated three times; the error bars represent the S.D. of three replicates from one representative experiment. **, p<0.01. Statistical significant difference was calculated by t test.

Figure 5. The effect of 1 µM of maraviroc on p24 Gag production in recombinant viruses containing four amino acid substitutions plus T199K.

PM1/CCR5 cells (1×10⁵) were infected with 10 ng p24 Gag for 3 h in the presence or absence of 1 µM maraviroc. On day 6 after infection, the amount of Gag in the supernatant was measured by HIV-1 p24 ELISA. The analysis was repeated three times; the error bars represent the S.D. of three replicates from one representative experiment. *, p<0.05; **, p<0.01. Statistical significant difference was calculated by t test.

Figure 6. Maraviroc susceptibility of pseudotyped viruses derived from HIV-1_JR-FLan, HIV-1_V3-M5, HIV-1₂₃₄₅, and HIV-1₁₃₄₅, HIV-1₁₂₄₅, HIV-1₁₂₃₅, and HIV-1₁₂₃₄.

MAGIC-5 cells were infected with pseudotyped viruses in the absence or presence of 1 µM maraviroc. The analysis was repeated three times; the error bars represent the S.D. of three replicates from one representative experiment. **, p<0.01. Statistical significant difference was calculated by t test.

Figure 7. MD simulation of the HIV-1 gp120 outer domain.

(A) Superimposition of averaged structures obtained from 40,000 snapshots during the 10–20 ns of MD simulation. Grey and blue ribbons indicate the gp120 V3 of JR-FL_an and JR-FL_V3-M5, respectively. (B) Distribution of RMSF in the V3 region of gp120. The RMSF values indicate the atomic fluctuations of the main chains of individual amino acids during the 10–20 ns of MD simulations.