Abstract
The majority of HIV-1 strains enter CD4+ T cells using the CCR5 and/or CXCR4 co-receptor. However, we recently identified a transmitted/founder (T/F) virus (ZP6248) that efficiently used an alternative coreceptor GPR15, rather than commonly used CXCR4 and CCR5, to establish clinical infection. To understand which regions in the env gene were critical for the atypical coreceptor usage, we generated a set of V3 mutants and determined their infectivity in GHOST cells that expressed different coreceptors. When the variable loop 3 (V3) in YU2 was replaced with the ZP6248 V3 (YU2.6248V3), the chimera YU2.6248V3 infected GPR15+ cells but not CCR5+ cells. To determine which amino acids in V3 was responsible for this phenotype change, each of the eight amino acids that differed from the subtype B consensus V3 was substituted with alanine. The G306A and S322A mutations significantly reduced the replication capacity of YU2.6248V3 in GPR15+ cells, while all other alanine substitutions at positions 307, 314, 315, 316, 317 and 318 completely abrogated the infectivity of YU2.6248V3 in GPR15+ cells. The E314A mutation, as the E314G mutation reported before, also rendered the YU2.6248V3 infectious in CCR5+ cells, while none of other alanine mutants could infect CCR5+ cells. These results demonstrated that amino acids in ZP6248 V3 might form a unique conformation that was critical for the interaction with GPR15 while the amino acids at position 314 in the V3 crown of ZP6248 played a key role in interaction with both CCR5 and GPR15. The unique phenotypes of ZP6248 can serve as a model to understand how HIV-1 explores the diverse coreceptor reservoir through novel genetic variants to establish clinical infection.
Highlights
HIV-1 enters target cells by first binding to the primary receptor, CD4, and one of many co-receptors
The GPR15 tropism was determined by variable loop 3 (V3) in ZP6248 We have previously shown that GPR15 tropism of ZP6248 was retained when its env gene was substituted into a CCR5-tropic HIV-1 strain YU2 [19]
The E314G mutation allowed ZP6248 to efficiently use CCR5 for entry [19]. These results demonstrated that the majority of unique amino acids in the ZP6248 V3 crown might form a conformation that was critical for the interaction with GPR15 while the amino acid at position 314, in the context of the ZP6248 V3 sequence, played a critical role in efficient interaction with CCR5 and GPR15
Summary
HIV-1 enters target cells by first binding to the primary receptor, CD4, and one of many co-receptors. Inclusion of two additional mutations (G306 and/or S322), which were at the same positions as 11 and 25 in V3 and played a critical role in modulation of the CCR5 and CXCR4 usage, completely abrogated YU2.GPEK’s ability to replicate in CCR5+ and GPR15+ cell This result indicated that the amino acids unique for ZP6248 each alone were not sufficient to render a heterologous virus to gain the ability to use GPR15. The E314A mutant replicated in CCR5+ cells slightly better than YU2 but failed to grow in GPR15+ cells These results demonstrated that the different amino acids at position 314 played an important role in modulating usage of different coreceptors. Other studies showed that some viruses could infect target cells using GPR15
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