Patterns of chemokine receptor fusion cofactor utilization by human immunodeficiency virus type 1 variants from the lungs and blood

Abstract

Human immunodeficiency virus type 1 (HIV-1) infection is highly compartmentalized, with distinct viral genotypes being found in the lungs, brain, and other organs compared with blood. CCR5 and CXCR4 are the principal HIV-1 coreceptors, and a number of other molecules support entry in vitro but their roles in vivo are uncertain. To address the relationship between tissue compartmentalization and the selective use of entry coreceptors, we generated functional env clones from primary isolates derived from the lungs and blood of three infected individuals and analyzed their use of the principal, secondary, orphan, and virus-encoded coreceptors for fusion. All Env proteins from lung viruses used CCR5 but not CXCR4, while those from blood viruses used CCR5 or CXCR4 or both. The orphan receptor APJ was widely used for fusion by Env proteins from both blood and lung viruses, but none used the cytomegalovirus-encoded receptor US28. Fusion mediated by the secondary coreceptors CCR2b, CCR3, CCR8, and CX3CR1 and orphan receptors GPR1, GPR15, and STRL33 was variable and heterogeneous, with relatively broad utilization by env clones from isolates of one subject but limited use by env clones from the other two subjects. However, there was no clear distinction between blood and lung viruses in secondary or orphan coreceptor fusion patterns. In contrast to fusion, none of the secondary or orphan receptors enabled efficient productive infection. These results confirm, at the level of cofactor utilization, previous observations that HIV-1 populations in the lungs and blood are biologically distinct and demonstrate diversity within lung-derived as well as blood-derived quasispecies. However, the heterogeneity in coreceptor utilization among clones from each isolate and the lack of clear distinction between lung- and blood-derived Env proteins argue against selective coreceptor utilization as a major determinant of compartmentalization.

FIG. 1

Replication of primary isolates in PBL and MDM. Four-day-old PHA- and IL-2-stimulated PBL cultures and 1-week-old MDM cultures were infected overnight with each isolate by using 25 ng of p24^gag antigen, washed, and sampled periodically for p24 antigen levels in supernatants. Data are representative of duplicate infections with cells from different donors.

FIG. 2

Fusion mediated by the primary-isolate env clones and the principal coreceptors CCR5 and CXCR4. Effector 293T cells were infected with the recombinant vaccinia virus vP11T7gene1, which expresses the T7 polymerase, and transfected with T7-driven env clones. These were then mixed with QT6 cells that were cotransfected with CD4, the indicated coreceptor or control vector, and a plasmid encoding luciferase under control of the T7 promoter. Cell-cell fusion was measured 16 h later on the basis of luciferase expression and is expressed as the fold increase in the luciferase level for the indicated cofactor in conjunction with CD4 compared with CD4 alone. Data represent the means of at least three independent experiments for each env-coreceptor combination.

FIG. 3

Fusion mediated by the primary-isolate env clones and the secondary chemokine receptor coreceptors CCR2b, CCR3, CCR8, and CX₃CR1. Fusion assays were performed as described in the legend to Fig. 2. Data represent the means of at least three independent experiments for each env-coreceptor combination.

FIG. 4

Fusion mediated by the primary-isolate env clones and the orphan receptors GPR1, GPR15, STRL33, and APJ. Fusion assays were performed as described in the legend to Fig. 2. Data represent the means of at least three independent experiments for each env-coreceptor combination.

FIG. 5

env clone sequence relationship. Sequencing of each clone was carried out on a 500-bp region that included the hypervariable V3 to V5 domains. The sequences were used to confirm the independence of env clones and to determine relationships between clones from each patient by using the University of Wisconsin GCG program.

FIG. 6

Coreceptor-mediated productive infection. U87-CD4 cells expressing CCR5 or CXCR4 were infected with 20 ng of p24^gag antigen as described in Materials and Methods, washed, and sampled on day 4. Bars represent levels of p24 antigen in supernatant relative to the dualtropic prototype 89.6, which uses both CCR5 and CXCR4 for infection.