Here, we have mapped envelope determinants of macrophage tropism by using two R5 envelopes that were amplified directly from patient tissues without culture (20
). Patient NA420's B33 was derived from brain tissue and showed high macrophage tropism, while LN40 from lymph node tissue infected macrophages inefficiently. Macrophage infectivity was previously shown to correlate with the capacity to infect cells via low levels of CD4 (20
), while Dunfee et al. reported that an asparagine at residue 283 in the C2 part of the CD4 binding site conferred a higher affinity for gp120-CD4 binding (8
). Nevertheless, our previous studies implicated additional unknown determinants (20
), which we sought to identify in the study reported here. Thus, determinants on the flanks of the CD4 binding loop and on the V3 loop were determined to modulate macrophage infection.
The CD4 binding loop is thought to be the most exposed and perhaps the first part of the CD4bs contacted by CD4 during HIV entry (4
). The substitutions identified on the flanks of the CD4 binding loop are either directly adjacent to CD4 contact residues or in close proximity (Fig. and ). We predict that these residues alter the exposure of CD4 contact residues on this loop, thus allowing better access to CD4 and a corresponding increase in affinity for B33. The affinity of the trimeric envelopes for CD4 is not straightforward to evaluate (5
). Here, we evaluated the sensitivity of infection to PRO 542, a tetrameric soluble CD4 construct based on immunoglobulin G. An increased affinity of the envelope for CD4 would be expected to result in increased sensitivity to PRO 542, although it is possible that PRO 542 inhibition may be conferred by additional mechanisms. Nevertheless, the B33T and LN40N mutants showed strong shifts in PRO 542 sensitivity toward resistance and sensitivity, respectively (compared to their wt counterparts), as expected for envelopes with altered CD4 binding affinities for gp120 subunits on the trimer. Overall, the capacity of B33, LN40, and a panel of mutants to infect macrophages or RC49 cells via low CD4 levels showed a highly significant correlation with sensitivity to PRO 542, consistent with modulation of env-CD4 affinity.
FIG. 6. The N-terminal flank of the CD4 binding loop is variable. A sequence alignment of HIV-1 R5 envelopes previously evaluated for macrophage infectivity (21) is shown.
FIG. 7. In the top panel, the proximity of macrophage tropism-determining residues flanking the CD4 binding loop to CD4 contact residues is shown. Residues Q363 and P364 (red) on the N-terminal flank of the CD4 binding loop are adjacent to CD4 contact residues (more ...)
The N-terminal flank of the CD4 binding loop is variable (Fig. ), and such residues have been shown to influence gp120-CD4 affinity (15
). These flanking residues are adjacent to the highly conserved SGGD-E CD4 contact residues on the apex of the CD4 binding loop. Such variation is consistent with a major impact on the exposure of these CD4 contact residues for this region and is suggestive of an immunity-mediated modulation. Recently, Sterjovski et al. reported that a potential glycosylation site (N362) in this flanking region conferred an increased envelope fusigenicity (25
). This observation seems counterintuitive since the presence of a glycan might be expected to shield the CD4 binding loop and reduce the efficiency of gp120-CD4 interactions. Nonetheless, Sterjovski's observations highlight the effect of this region on envelope-induced fusion. Our results also implicated residues in the V3 loop (H308 and F317 in non-macrophage-tropic LN40) as determinants of R5 macrophage tropism. How these V3 loop residues impact macrophage tropism is less clear, and it is possible that they influence a post-CD4 binding event during entry, e.g., binding to CCR5. However, B33 mutants carrying these residues were less sensitive to PRO 542, consistent with a change in env-CD4 affinity (not shown). The V3 loop extends 30 Å from the gp120-CD4 complex (13
). However, the position of the V3 loop on the unliganded envelope is not known, and it is possible that it lies close enough to the CD4 binding loop to influence its exposure. Lynch et al. also reported that a single amino acid change in the V3 loop of a clade C envelope conferred sensitivity to soluble CD4 inhibition (17
), consistent with a close association between V3 and the CD4bs.
Our approach in this study was to utilize HeLa RC49 cells as a surrogate for primary macrophages. RC49 cells express low levels of CD4 (like macrophages) and their use avoids the variation in sensitivity to HIV infection observed for macrophages from different donors. Once the envelope determinants that modulated infection of RC49 cells via low CD4 levels were identified, we tested a representative panel of envelopes, including B33, LN40, and critical mutants, with primary macrophages from several donors. The infectivity data for this panel of envelopes were similar for primary macrophages and RC49 cells. However, the V3 loop (HF) and CD4 binding loop N-terminal flank (QP) residues were sufficient to abrogate macrophage infectivity for B33, without a contribution from downstream residues (R373 and N386) which were required to maximally reduce infectivity for RC49 cells. The mechanistic basis for this difference between macrophage and RC49 infection is unclear but presumably relates to subtly different expression levels of CD4 and/or CCR5 in different cell surface environments. Nonetheless, the levels of infectivity for primary macrophages correlated tightly with those for RC49 cells (P = 0.0001). Finally, infectivity titers determined with different macrophage batches varied (reflecting donor variation in sensitivity to infection), although the overall pattern of infectivities remained the same, with B33 as the most macrophage-tropic and LN40 as the least.
Our data also provide further information on the role of N283 in macrophage tropism. A T283N substitution in LN40 conferred substantial levels of macrophage infection. The presence of asparagine at residue N283 thus overrules the involvement of additional residues identified here (in the V3 loop and on the flanks of the CD4 binding loop) in macrophage tropism. This could be due to the direct effect of N283 on g120-CD4 binding via the introduction (or improved stabilization) of a hydrogen bond between N283 and Q40 on CD4, as suggested by Dunfee et al. (8
). The role of the additional residues identified here presumably involves the modulation of exposure of the CD4 binding loop (as discussed above) affecting env-CD4 affinity without directly altering the CD4 contact residues. Thus, our results indicate that env-CD4 affinity may be modulated by two distinct mechanisms that involve either a direct effect on the CD4bs or an indirect effect that may affect its exposure.
Recently, we reported the determinants in B33 and LN40 envelopes that affect their different sensitivities to the CD4bs MAb b12 (7
). B33 is sensitive to b12, while LN40 is resistant. In that study, we showed that the LN40 residues Q363 and P364 on the flank of the CD4 binding loop in combination with the V3 loop residues H308 and F317 conferred a partial shift toward resistance to b12 for B33. However, complete B33 resistance was conferred by a combination of R373 and the glycan at N386. The side chain of R373 and the N386 glycan appear to combine to fill a proximal cavity on gp120 that is targeted by the organic ring on the side chain of W100 on b12 (7
). Thus, determinants for macrophage tropism and b12 resistance overlap, although different residues have distinct effects on each phenotype. Our data are consistent with immune modulation of the identified residues during selection of the LN40 envelope in the immune environment of the lymph node. Presumably, the selective force is neutralizing antibodies. In contrast, in brain tissue, where antibodies are usually restricted by the blood brain barrier, envelopes like B33 with a more exposed CD4 binding loop may evolve with an increased sensitivity to neutralization by CD4bs antibodies and an enhanced capacity to infect macrophages via low levels of CD4. Unfortunately, plasma from subject NA420 is not available to test directly whether LN40 is more resistant to neutralization than B33.
In summary, we have identified envelope determinants that modulate R5 macrophage tropism and that have evolved naturally in vivo. The determinants are located on the flanks of the CD4 binding loop but also involve residues in the V3 loop. We predict that they modulate exposure of the CD4 binding loop and accessibility of the CD4bs to CD4. Furthermore, the N-terminal flank of the CD4 binding loop is variable and such variation may contribute to a general mechanism for protecting CD4 contact residues on this loop from neutralizing antibodies. Our data have strong relevance for the design of envelope antigens, which will need to optimally present and expose residues involved in CD4 binding for the induction of neutralizing antibodies targeting this critical and conserved site.