Background:  HIV infects macrophages and microglia, which express low CD4 and CCR5 levels. Previous studies described HIV Env from brain that have enhanced macrophage/microglia tropism and increased ability to infect cells expressing low CD4 and CCR5 levels, suggesting that reduced CD4/CCR5 dependence may contribute to neurotropism. Here we sought to identify Env genetic determinants that contribute to neurotropism and HIV-associated dementia (HAD) by enhancing Env-CD4 interactions.

Methods:  Full-length Env were cloned from primary brain isolates from 2 HAD patients. Env with and without reduced CD4 dependence were compared to identify amino acid changes in or near the CD4 binding site that segregated with the ability to use low CD4 levels. Mutagenesis was used to investigate the role of particular amino acids in reduced CD4 dependence.

Results:  Brain-derived Env that had reduced CD4 dependence compared to the ADA Env had asparagines, rather than the consensus threonine, at position 283 in the C2 region. Based on the crystal structure of the gp120-CD4-17b complex, T283 contacts Q40 of CD4 via a hydrogen bond. If this residue is changed to N, there is the potential for additional hydrogen bonding to D477 of gp120, which may stabilize the interaction with CD4 or increase CD4 affinity by altering gp120 conformation. T283 is present in 80% of clade B Env, and a high ratio of synonymous to nonsynonymous substitutions indicates it may be a site of positive selection. In Env with reduced CD4 dependence, introducing an N283T mutation resulted in a 70% decrease in the ability to mediate fusion in cells expressing low CD4. The reciprocal T283N mutation resulted in a 50% increase in fusion with cells expressing low CD4, whereas a T283I mutation had no effect. The N283 variant was present at a frequency of 0.4 in the brain of 24 HAD patients. Furthermore, N283 was present among Env sequences in the database at a frequency of 0.19 in brain-derived Env (n = 390) compared with a frequency of 0.06 in blood-derived Env (n = 225), suggesting this genetic variant may be associated with brain infection.

Conclusions:  N283 is a single amino acid change in gp120 that contributes to reduced CD4 dependence and HIV neurotropism. This genetic variant is found more frequently in brain compared to non-brain tissues, suggesting N283 may confer a selective advantage for HIV replication in the brain. These results provide a better understanding of mechanisms that underlie HIV neurotropism.

Keywords: neurotropism; envelope glycoprotein; CD4