Heterogeneous spectrum of coreceptor usage among variants within a dualtropic human immunodeficiency virus type 1 primary-isolate quasispecies

Abstract

Human immunodeficiency virus type 1 (HIV-1) variants that use the coreceptor CCR5 for entry (R5; macrophage tropic) predominate in early infection, while variants that use CXCR4 emerge during disease progression. Some late-stage variants use CXCR4 alone (X4; T-cell tropic), while others use both CXCR4 and CCR5 (R5X4; dualtropic). It has been proposed that dualtropic R5X4 strains are intermediates in the evolution from R5 to X4, and we hypothesized that a dualtropic primary-isolate quasispecies might contain variants that represent the spectrum of coreceptor use in vivo. We generated a panel of 35 functional full-length env clones from the primary-isolate quasispecies of a dualtropic prototype strain, HIV-1 89.6(PI). Thirty of the functional env clones (86%) were R5X4, four (11%) were R5, and one (3%) was predominantly X4. V3 to V5 sequences did not reveal clustering by coreceptor usage, and no specific sequence motif or V3 charge pattern corresponded to coreceptor utilization. Complete sequencing of seven functionally divergent Env proteins revealed > or =98.7% homology and conservation of structurally important domains. Chimeras between the R5X4 89.6 prototype and an R5 variant indicated that multiple regions contributed to the use of CXCR4, while chimeras with the X4 variant implicated a single residue in V4 in CCR5 use. These results confirm, at the molecular level, both that dualtropic variants are a predominant component of late-stage syncytium-inducing isolates and that variants restricted to each coreceptor coexist with dualtropic species in vivo. Coreceptor-restricted minority variants may reflect residual R5 species from earlier in disease as well as emerging X4 variants.

FIG. 1

Fusion and infection mediated by functionally divergent env clones within the 89.6_PI quasispecies. (A) Cloned env genes were analyzed for CCR5 and CXCR4-mediated fusion in a cell-cell fusion assay, along with the R5 and X4 prototypes JRFL and 3B, respectively. The env gene designated 89.6 is derived from the full-length infectious clone described previously (8, 11). Only data for the five variants with distinct R5 or X4 patterns and one representative R5X4 env variant are shown in this graph. Each envelope-coreceptor combination was tested a minimum of three times. (B) Pseudotype virions generated with one R5 and one X4 env variant were used to confirm coreceptor selectivity in infection. To generate pseudotype virions, env genes were cotransfected with an env-defective HIV-1 plasmid that carries the luciferase reporter gene in place of nef. U87 cells were transfected with CD4 and the indicated chemokine receptor, infected with equal amounts of each pseudotype virus based on p24 antigen content, and lysed 3 days later for measurement of luciferase expression. Pseudotype infections were tested in three independent experiments.

FIG. 2

Genetic relatedness of all 36 functional env clones from the 89.6_PI swarm, determined on the basis of the nucleotide sequences across a ∼600-bp V3-to-V5 region. The dendrogram was generated by using the Clustal method. Clones restricted to CCR5 or CXCR4 are indicated by R5 or X4 to the right of the clone number, while the rest of the env clones, for which no coreceptor usage designation is given, were R5X4.

FIG. 3

Amino acid alignment of functionally diverse env gene sequences. Full-length sequences were determined for the four R5 env clones (clones 10, 13, 14, and 23), one X4 env clone (clone 22), and one representative R5X4 env clone derived from this pool (clone 2). Sequences were aligned with that of the original R5X4 89.6 clone. R5 clones 13 and 14 had identical Env sequences.

FIG. 4

Coreceptor usage of chimeras generated between related env genes with distinct phenotypes. (A) Construction of recombinant env genes. Chimeras were made between the R5X4 89.6 env molecular clone and the R5 variant 14, and between 89.6 and the X4 variant 22. Overlap extension PCR was used to exchange the indicated regions, which introduced the amino acid changes noted. (B) Coreceptor selectivity of chimeric env variants. The env chimeras were tested for CCR5 and CXCR4 utilization based on cell-cell fusion.

FIG. 4

Coreceptor usage of chimeras generated between related env genes with distinct phenotypes. (A) Construction of recombinant env genes. Chimeras were made between the R5X4 89.6 env molecular clone and the R5 variant 14, and between 89.6 and the X4 variant 22. Overlap extension PCR was used to exchange the indicated regions, which introduced the amino acid changes noted. (B) Coreceptor selectivity of chimeric env variants. The env chimeras were tested for CCR5 and CXCR4 utilization based on cell-cell fusion.