Generation of V3 mutant chimeric viruses. (A) Three different chimeric viruses were generated: NL4-3-V3_A1-92RW009 contains the V3 region of the primary, CCR5-tropic isolate A1-92RW009 in the NL4-3 background. NL4-3-gp120_B5-91US056 contains the gp120 region derived from the primary, CCR5-tropic isolate B5-91US056 in the NL4-3 background, and NL4-3-gp160_Yu-2 is an NL4-3 construct with the full-length envelope coding sequence of the CCR5-tropic isolate Yu-2. Site-directed mutagenesis was performed at positions 318 and 319 to change the wild-type amino acids (A1-92RW009, YA; B5-91US056, YA; Yu-2, YT) into the relevant polymorphisms (318Y/319A, 318Y/319T, 318Y/319R, 318R/319A, and 318R/319T). Sequence of the 35-amino-acid V3 loop is shown, and the crown sequence is boxed. The amino acids at 318 and 319 are indicated in boldface. Viruses containing wild-type sequences are named in boldface. (B) The X-ray crystal structure of the V3 loop derived from Huang et al. (19) is presented as a ribbon and a space-filling model. The crown GPGR of the subtype B JR-FL strain is depicted in this structure (purple). In JR-FL, position 319 is a threonine. Since a tyrosine or arginine at position 318 and an alanine or threonine at position 319 are natural polymorphisms in both subtype A and B, energy minimizations were performed with the T319A substitutions in the JR-FL structure and using the GROMOS96 algorithm within the DeepView/Swiss-PdbViewer v3.7 (assuming a uniform dielectric constant inside the protein). The alanine was easily accommodated without perturbing the minimal energy predicted by the model.

Coreceptor tropism and pseudovirus infection efficiency. Equivalent particle numbers of pseudovirus, as determined by limiting-dilution RT assay, were used to infect U87-CD4, U87-CD4/CXCR4, or U87-CD4/CCR5 cells. Cells were incubated with pseudovirus for 48 h, the cells were lysed, and the luciferase activity was determined. Pseudoviruses produced without envelope served as a negative control for infection, while treatment of cells with 100 μM 3TC served as a specificity control for luciferase activity. Values are presented as relative light units, and error bars represent the standard deviations from two independent experiments performed in triplicate.

Total relative fitness values for each chimeric virus. Total relative fitness was calculated by adding the relative fitness value (w) of the chimeric virus from each competition with a reference panel virus.

Correlation between entry inhibitor sensitivity and total relative fitness for V3 crown mutant chimeric viruses. Coefficients were determined by using the PPMC.

Analysis of envelope incorporation by V3 mutant chimeric viruses. To ensure equivalent incorporation of the envelope protein in the chimeric viruses, equal TCID₅₀ of replication competent virus was pelleted at 38,000 rpm and subjected to Western blot. (A) 10^3.5 infectious units of each virus were pelleted and resuspended in 100 μl of SDS lysis buffer. A 10-μl portion of this sample, or 316 infectious units, was diluted serially 1:5 for each NL4-3-V3_A1-92RW009 chimeric virus and probed for gp120 (B13 MAb), gp41 (Chessie 8 MAb), and p24 (24-4 MAb). (B) 316 infectious units of each NL4-3-gp120_B5-91US056 and NL4-3-gp160_Yu-2 V3 crown mutant chimeric virus were probed for p24 and gp120 content.

Replicative fitness of V3 polymorphisms in three envelope contexts. Each chimeric virus was competed in pairwise dual infection in PHA/IL-2-activated PBMC against a panel of CCR5-tropic primary isolate viruses (B2-92BR017, Yu-2, B5-91US056, C5-97ZA003, A1-92RW009, and B10-92BR003). Relative fitness values were calculated as described in Materials and Methods. Plots are of fitness difference values and are indicative of fold difference replication capacity. Fitness differences are plotted on a logarithmic scale, and bars that fall above the midline (W_D = 1) indicate competitions in which the chimeric virus was the winner, whereas bars falling below the midline indicate competitions in which the primary reference isolate was the winner. Bars that lie near the midline (W_D = 1) indicate competitions in which both viruses were of nearly equivalent replicative fitness. All competitions were performed in duplicate. (A) Fitness order of full-length viruses used in the reference panel. (B) Fitness difference values of NL4-3-V3_A1-92RW009 chimeric viruses with V3 polymorphisms against the reference panel. (C) Fitness difference values of NL4-3-gp120_B5-91US056 chimeric viruses with V3 polymorphisms against the reference panel. (D) Fitness difference values of NL4-3-gp160_Yu-2 chimeric viruses with V3 polymorphisms against the reference panel.

Sensitivity of 318/319 polymorphisms to entry inhibitors. U87-CD4/CCR5 cells were incubated with 10-fold dilutions of PSC-RANTES (A), ENF (B), TAK-779 (C), or 3TC (D) and then exposed to virus for 24 h. After the input virus was washed out, the supernatant samples were analyzed for RT activity at 4, 6, and 8 days postinfection. Drug susceptibility curves (data not shown) were plotted to determine IC₅₀ values using the probit algorithm. IC₅₀ values and standard deviations for each respective drug and virus are plotted in the panels. Asterisks indicate cutoff for significance of P < 0.01 (paired comparison t test).