Background:  We previously described R5 envelopes (env) amplified from uncultured tissue that conferred distinct tropisms. R5 env from brain were highly macrophage-tropic, while lymph node env infected macrophages inefficiently. Macrophage-tropism correlated with the use of low CD4 levels for infection. A N283 in the CD4 binding site was associated with highly mac-tropic brain env. Highly mac-tropic brain env may have adapted for replication in resident macrophages and microglia. However, low concentrations of neutralizing antibodies in brain tissue protected by the blood brain barrier may play a role. Moreover, brain env from 2 individuals were sensitive to neutralization by the CD4 binding site monoclonal antibody b12 while lymph node env were resistant. Here, we map env determinants conferring mac-tropism and b12 sensitivity.

Methods:  NA420 env B33 (brain) is highly mac-tropic and b12 sensitive, while LN40 (lymph node) is non-mac-tropic and b12 resistant. The role of N283 was investigated by constructing B33 N283T and LN40 T283N. Chimeric B33 env were constructed containing non-overlapping LN40 env regions, C2-C3 and C3-C5. Mutant envelopes were expressed on reporter viruses to analyze mac-tropism and b12 sensitivity.

Results:  N283T reduced B33 macrophage infection by <10-fold. In contrast, T283N conferred LN40 with substantial macrophage infection but still less than B33. Macrophage infectivity studies using B33/LN40 chimeric envelopes carrying T283 showed that 2 distinct gp120 regions (C2-C3 and C3-C5) contained determinants that influenced mac-tropism. N/T283 substitutions in B33 or LN40 had no effect on b12 sensitivity. In contrast, B33 carrying the LN40 C3-C5 region was completely resistant to b12. In addition, B33 carrying the LN40 C2-C3 region was also partially resistant to b12.

Conclusions:  Mutation of env residue 283 only partially modulated mac-tropism in either B33 or LN40. Further determinants of efficient macrophage infection were mapped to 2 distinct regions of gp120 (C2-C3 and C3-C5). In contrast, mutation of residue 283 had no effect on the sensitivity of B33 or LN40 to b12. Determinants in LN40 C3-C5 region conferred complete resistance to b12, while further determinants in LN40 C2-C3 region conferred partial resistance. Thus, b12 resistance partially mapped to gp120 regions that affect macrophage infectivity. These results only support a partial role for neutralizing antibodies in modulating macrophage-tropism of HIV-1 R5 envelopes.