Background:  Many CXCR4-using HIV are classified as dual-tropic (DUAL) because they have the ability to use both CCR5 and CXCR4 co-receptors to enter target cells in vitro. The in vivo target cell specificity of DUAL viruses remains poorly defined. In this study, we analyzed a panel of DUAL envelope clones for their ability to use CCR5 and CXCR4 on cells expressing both co-receptors.

Methods:  We cloned gp160 envelope genes derived from different patient samples into an expression vector. Tropism of pseudotyped virus was determined by measuring luciferase activity (relative light units) following infection of U87/CD4⁺CCR5⁺ or U87/CD4⁺CXCR4⁺ cells (Trofile assay, Monogram Biosciences). Inhibition of infection by small molecule CCR5^– (Merck) and CXCR4^– (AnorMED) inhibitors was examined on U87/CD4⁺CCR5⁺CXCR4⁺ cells. Envelope genes were also inserted into an NL43 infectious clone and evaluated for their ability to promote syncytia in MT2 cells.

Results:  We identified 20 DUAL Env clones that exhibited a broad range of infectivity on U87/CD4⁺CCR5⁺ (range 10³ to 10⁶ relative light units) or U87/CD4⁺CXCR4⁺ (range 10³ to 10⁶ relative light units) cells and were loosely grouped into 3 categories (R5X4, R5~X4, and X4R5). We used inhibition of infection on U87/CD4⁺/CCR5⁺/CXCR4⁺ cells by specific co-receptor inhibitors to test whether DUAL clones use co-receptors preferentially when both CCR5 and CXCR4 were present on the cell surface. DUAL clones that efficiently use both CCR5 and CXCR4 (R5~X4) were not well inhibited by either CCR5- or CXCR4-inhibitors, indicating that infection by these viruses is not restricted to only CCR5 or only CXCR4. Infection by X4R5 DUAL clones was blocked by a CXCR4 inhibitor, but not a CCR5 inhibitor; conversely, infection by R5X4 DUAL clones was blocked by a CCR5 inhibitor, but not a CXCR4 inhibitor. Use of the CXCR4 co-receptor by R5X4 DUAL viruses was confirmed by syncytia formation on MT2 cells when the envelopes were transferred into an NL43 infectious clone. 

Conclusions:  Dual-tropic variants display a broad range of ability to use CCR5 or CXCR4 for infection in vitro. The variation in relative use of CCR5 or CXCR4 observed among dual-tropic variants suggests distinct entry pathways that might have an effect on virus pathogenesis and patient treatment response to regimens including co-receptor inhibitors.