Role of the HIV type 1 glycoprotein 120 V3 loop in determining coreceptor usage

Macrophage (M)-tropic HIV-1 isolates use the beta-chemokine receptor CCR5 a s a coreceptor for entry, while T cell line-adapted (TCLA) strains use CXCR 4 and dual-tropic strains can use either CCR5 or CXCR4. To investigate the viral determinants involved in choice of coreceptor, me used a fusion assay (1) based on the infection of CD4(+) HeLa cells that express one or both co receptors with Semliki Forest virus (SFV) recombinants expressing the nativ e HIV-1 gp160 of a primary M-tropic isolate (HIV-1(BX08)), a TCLA isolate ( HIV-1(LAI)), Or a dual-tropic strain (HIV-1(MN)). We examined whether the V 3 region of these glycoproteins interacts directly with the corresponding c oreceptors by assaying coreceptor-dependent cell-to-cell fusion mediated by the different recombinants in the presence of various synthetic linear pep tides, Synthetic peptides corresponding to different V3 loop sequences bloc ked syncytium formation in a coreceptor-specific manner. Synthetic V2 pepti des mere also inhibitory for syncytium formation, but showed no apparent co receptor specificity, A BX08 V3 peptide with a D-320 --> R substitution ret ained no inhibitory capacity for BX08 Env-mediated cell-to-cell fusion, but inhibited LAI Env-mediated fusion as efficiently as the homologous LAI V3 peptide. The same mutation engineered in the BX08 env gene rendered it able to form syncytia on CD4(+)CXCR4(+)CCR5(-) HeLa cells and susceptible to in hibition by SDF-1 alpha and MIP-1 beta. Other substitutions tested (D-320 - -> Q/D-324 --> N or S-306 --> R) exhibited intermediate effects on corecept or usage. These results underscore the importance of the V3 loop in modulat ing coreceptor choice and show that single amino acid modifications in V3 c an dramatically modify coreceptor usage. Moreover, they provide evidence th at linear V3 loop peptides can compete with intact cell surface-expressed g p120/gp41 for CCR5 or CXCR4 interaction.