CCR5 and CXCR4 usage by non-clade B human immunodeficiency virus type 1 primary isolates

Abstract

CCR5 and CXCR4 usage has been studied extensively with a variety of clade B human immunodeficiency virus type 1 (HIV-1) isolates. The determinants of CCR5 coreceptor function are remarkably consistent, with a region critical for fusion and entry located in the CCR5 amino-terminal domain (Nt). In particular, negatively charged amino acids and sulfated tyrosines in the Nt are essential for gp120 binding to CCR5. The same types of residues are important for CXCR4-mediated viral fusion and entry, but they are dispersed throughout the extracellular domains of CXCR4, and their usage is isolate dependent. Here, we report on the determinants of CCR5 and CXCR4 coreceptor function for a panel of non-clade B isolates that are responsible for the majority of new HIV-1 infections worldwide. Consistent with clade B isolates, CXCR4 usage remains isolate dependent and is determined by the overall content of negatively charged and tyrosine residues. Residues in the Nt of CCR5 that are important for fusion and entry of clade B isolates are also important for the entry of all non-clade B HIV-1 isolates that we tested. Surprisingly, we found that in contrast to clade B isolates, a cluster of residues in the second extracellular loop of CCR5 significantly affects fusion and entry of all non-clade B isolates tested. This points to a different mechanism of CCR5 usage by these viruses and may have important implications for the development of HIV-1 inhibitors that target CCR5 coreceptor function.

FIG. 1.

Viral entry mediated by CXCR4 mutants. U87MG-CD4 cells transiently expressing wild-type CXCR4 or CXCR4 with alanine substitutions were infected with NLluc+env− reporter viruses pseudotyped with different non-clade B X4 envelope glycoproteins. The name and clade of each isolate are indicated in the first two columns and the mutated residues are indicated in the top row of each panel. Luciferase activity (RLU) was measured 48 h postinfection and standardized for CXCR4 expression levels (IDV). The coreceptor activity of each mutant is expressed as a percentage of wild-type coreceptor activity, calculated with the formula [(mutant RLU ÷ wild-type RLU) ÷ (mutant IDV ÷ wild-type IDV)] × 100% Shading indicates values lower than 20%. All values are means of three independent experiments, each performed in quadruplicate. Standard deviations were not more than 20% for each data set and are not shown for the sake of clarity.

FIG. 2.

Viral entry mediated by CCR5 mutants. U87MG-CD4 cells transiently expressing wild-type CCR5 or CCR5 with alanine substitutions were infected with NLluc+env− reporter viruses pseudotyped with different non-clade B R5 envelope glycoproteins. The name and clade of each isolate are indicated in the first two columns and the mutated residues are indicated in the top row of each panel. Luciferase activity (RLU) was measured 48 h postinfection and standardized for CCR5 expression levels (IDV). The coreceptor activity of each mutant is expressed as a percentage of wild-type coreceptor activity, calculated with the formula [(mutant RLU ÷ wild-type RLU) ÷ (mutant IDV ÷ wild-type IDV)] × 100%. Shading indicates values lower than 20%. All values are means of three independent experiments, each performed in quadruplicate. Standard deviations were not more than 20% for each data set and are not shown for the sake of clarity.

FIG. 3.

Alignment of amino acid sequences in the V3 and C4 regions of gp120. Amino acid sequences of the HIV-1 test isolates were obtained from GenBank and aligned. The first three columns show the name of the molecular clone, clade classification, and coreceptor tropism. The V3 crown is boxed. Residues in bold in the JR-FL sequence were previously shown to be important for gp120/CCR5 interactions. Net charges are on the far right.