Maraviroc binds to the pocket of extracellular loops of the cell surface CCR5 and prevents R5 HIV-1 from using CCR5 as a coreceptor for entry into CD4-positive cells. To evaluate the contribution of the V3 loop structure in gp120 to maraviroc resistance, we isolated maraviroc-resistant variants from the V3 loop library virus (HIV-1(V3Lib)) containing a set of random combinations of 0-10 polymorphic mutations in vitro. HIV-1(V3Lib) at passage 17 could not be suppressed even at 10 μM (>1400-fold resistance), while HIV-1(JR-FL) at passage 17 revealed an 8-fold resistance to maraviroc. HIV-1(V3Lib-P17) contained T199K and T275M plus 5 mutations in the V3 loop, I304V/F312W/T314A/E317D/I318V. The profile of pseudotyped virus containing I304V/F312W/T314A/E317D/I318V in V3 loop alone revealed a typical noncompetitive resistance, although T199K and/or T275M could not confer noncompetitive resistance. This type of library virus is useful for isolation of escape viruses from effective entry inhibitors.

HIV-1 infection in Chinese blood donors is genetically diverse and the subtype distribution reflects that from the high-risk populations. Our results support continuous molecular epidemiologic surveillance for HIV-1 in blood donors as a part of a comprehensive HIV control program.

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-1V3-M5 derived from HIV-1JR-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-1JR-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.

No abstract