Involvement of both the V2 and V3 regions of the CCR5-tropic human immunodeficiency virus type 1 envelope in reduced sensitivity to macrophage inflammatory protein 1 alpha

To determine whether C-C chemokines play an important role in the phenotype switch of human immunodeficiency virus (HIV) from CCR5 to CXCR4 usage duri ng the course of an infection in vivo, macrophage inflammatory protein (MIP )-1 alpha-resistant variants were isolated from CCR5-tropic (R5) HIV-1 in v itro. The selected variants displayed reduced sensitivities to MIP-1 alpha (fourfold) through CCR5-expressing CD4-HeLa/ long terminal repeat-beta-gala ctosidase (MAGI/CCR5) cells. The variants were also resistant to other natu ral ligands for CCR5, namely, MIP-1 beta (>4-fold) and RANTES (regulated up on activation, normal T-cell expressed and secreted) (6-fold). The env sequ ence analyses revealed that the variants had amino acid substitutions in V2 (valine 166 to methionine) and V3 (serine 303 to glycine), although the sa me V3 substitution appeared in virus passaged without MIP-1 alpha. A single -round replication assay using a luciferase reporter HIV-1 strain pseudotyp ed with mutant envelopes confirmed that mutations in both V2 and V3 were ne cessary to confer the reduced sensitivity to MIP-1 alpha, MIP-1 beta, and R ANTES. However, the double mutant did not switch its chemokine receptor usa ge from CCR5 to CXCR4, indicating the altered recognition of CCR5 by this m utant. These results indicated that V2 combined with the V3 region of the C CR5-tropic HIV-1 envelope modulates the sensitivity of HIV-1 to C-C chemoki nes without altering the ability to use chemokine receptors.