We present data to show that a single amino acid substitution in the V1 loop modulates macrophage tropism for heterologous HIV-1 clade B R5 envelopes. The structures of the V1 and V2 loops have not yet been solved. Various studies showed that the V1 and V2 loops influenced the binding of CD4bs MAbs, suggesting that these loops may help to shield the CD4bs (28
). The identification of V1V2 stem residues as contacts for CD4 also added to the possibility that the loops may lie in the recessed space accessed by CD4 on the unliganded trimer (23
). However, the more recent electron tomographic structure of the trimeric envelope strongly suggests that the bulk of the V1 and V2 loops sit on the apex of the trimeric envelope and thus some distance from the CD4bs (27
). We therefore believe that our data are consistent with a model where the V1 loop determinant modulates the conformation or exposure of the V3 loop, which in turn affects the function of the CD4bs priming the envelope for low CD4 use and macrophage infection. Moreover, it is striking that V1 residue 153 control of the V3 loop and CD4bs was active for envelopes derived from three of six clade B-infected subjects. This mechanism is thus likely to be operative for 50% of clade B envelopes.
Previously several determinants have been reported to have major effects on macrophage infection by HIV-1 R5 envelopes. These include residue 283 in the CD4bs (11
), V3 loop determinants and residues adjacent to the CD4 contact residues on the CD4 binding loop (11
). Thus, macrophage tropism can be modulated by changes within the CD4bs, which directly impact Env-CD4 affinity, or by changes that indirectly affect the CD4bs, presumably by affecting the exposure CD4 contact residues. The identification of a distal V1 loop determinant that also confers macrophage tropism is striking and indicates that multiple envelope sites may have the potential to modulate macrophage tropism. The change from a charged glutamic acid (E) to the uncharged glycine could have profound effect on the interaction of the V1 loop with other envelope residues due to the loss of potential ionic bonds. Our initial interpretation was that the V1 determinant acted by affecting the exposure of CD4 contact residues. However, shifts in b12 sensitivity (our best indicator of CD4bs exposure) following substitutions at residue 153 ranged from extremely minor to modest at best and were substantially smaller than for sCD4 or 447-52D. It is therefore unlikely that the effects of V1 residue 153 result entirely from increased exposure of CD4bs residues. Thus, changes in the capacity of the envelope to react to suboptimal levels of cell surface CD4 (without changes in Env-CD4 affinity) and trigger conformational changes and viral entry should be considered as an alternative mechanism (see below).
For envelopes where the substitution in V1 did not affect tropism, changes in sCD4, b12, and 447-52D sensitivity were noted for some but not other envelopes. For envelopes that showed no change in sCD4, 447-52D, and b12 sensitivity, it is possible that a putative connection between the V1 and V3 loops is absent or held by alternative mechanisms, so that residue 153 substitutions have no effect on the CD4bs. Alternatively, other determinants that affect the CD4bs and macrophage tropism may simply override any effects conferred by changes in the V1 loop determinant. This latter possibility could apply to the highly macrophage-tropic brain envelope FL5-2-209. This envelope was initially highly sensitive to sCD4, 447-52D, and b12 (IC50
s of 0.34, 2.0, and 2.5 μg/ml, respectively) but became substantially more resistant (IC50
s of 7.5, >50, and 11.8 μg/ml, respectively) when the G153E substitution was introduced, even though no changes in macrophage infection were observed. It is thus likely that alternative envelope residues confer macrophage infectivity and simply override the effects of V1 residue 153. It is noteworthy that an E153G substitution was previously reported to be responsible for increased syncytium formation following infection of brain microglial cultures by an HIV-1 R5 variant selected in vitro
The C98-18 envelope conferred modest levels of macrophage infection compared to the highly macrophage-tropic C98-15 and the non-macrophage-tropic P1114 envelopes. C98-18 carries the D167N substitution in the V2 loop but not the V1 loop E153G substitution present in the C98-15 envelope. An N167D substitution in C98-18 abrogated the modest levels of macrophage infection confirming the role of this residue (Fig. ). In addition, N167D conferred an increase in the sensitivity of C98-18 to sCD4, reducing the IC50 from 26.62 to 8.19 μg/ml, but had no affect on the resistance to the V3 loop MAb, 447-52D (data not shown). These observations are curious since substitutions at residue 167 had no effect on the macrophage infectivity of the other P1114 envelopes tested. Presumably, there are other determinants in the P1114 envelopes that influence whether residue 167 affects macrophage infectivity. Regardless, our data indicate that V2 residue 167 is a only minor player in the macrophage tropism of the P1114 envelopes.
Other studies have also reported single or limited substitutions in the envelope that affect macrophage infectivity and/or sensitivity to neutralizing antibodies. Lynch et al. reported that the introduction of a leucine residue to replace a highly conserved isoleucine (I309) in the V3 loop of clade C envelopes affected the exposure of the CD4bs and conferred an increase in macrophage replication, although this was modest (29
). Li et al. described an N197Q substitution proximal to V2, which removed a potential N-linked glycosylation site. N197Q conferred increases in sensitivity to sCD4 and b12. However, N197Q also conferred sensitivity to 17b, a CD4i MAb, indicating that more substantial structural changes had resulted that exposed sites involved in coreceptor binding (26
). Similarly, Zhu et al. reported on an adjacent substitution in the β3 strand, T198P (50
), while O'Rourke et al. described a D179N substitution in the LDV motif of V2 (35
), both of which conferred global neutralization sensitivity and exposure of the 17b epitope. Some of these substitutions may affect the interactions of the gp120 subunits on the trimer, perhaps altering the position of the V1V2 loops, which may interact at the apex. Such changes may also result in triggering some of the conformational changes (usually associated with CD4 binding) and exposure of CD4i epitopes.
Limited changes in the extracellular segment of gp41 have also been shown to affect neutralization sensitivity. Thus, two amino acid changes in the conserved HR1 and MPER regions in gp41 together conferred increased sensitivity to sCD4, broadly neutralizing gp120 and gp41 MAbs, and to human HIV-1+
). In addition, Shen et al. recently reported an L669S substitution in the MPER that conferred increased sensitivity to a range of gp41- and gp120-specific neutralizing MAbs. Presumably, these substitutions also at indirectly by influencing the arrangement of gp120 subunits on the monomer and thus affecting their sensitivity to neutralizing antibodies.
In contrast, the effects of V1 residue 153 are unique in conferring large shifts in envelope phenotype that are focused specifically on the V3 loop and CD4bs without major effects on other envelope sites, as estimated by envelope sensitivity to inhibitors that targeted other stages of HIV-1 entry, including Env-CCR5 interactions and gp41 conformational changes. All envelopes also remained relatively resistant to 17b, a CD4i MAb, indicating that extensive conformational changes that are usually associated with gp120-CD4 interactions had not occurred. The changes affected by V1 residue 153 thus contrast with the substitutions described above (26
Our data indicate that V1 residue 153 has strong affects on the V3 loop and the CD4bs, which are only partially explained by increased exposure of the CD4 contact residues. The location of the V3 loop on the unliganded envelope is not known but may sit close enough to restrict the approach of both CD4 and b12 (8
). It is also tempting to envisage that residue 153 modulates a gp120 conformational change to form a structure intermediate between the unliganded envelope and the CD4-bound form. Such an intermediate may be partially activated for the conformational changes required to form the coreceptor binding site and thus require less robust interactions with fewer CD4 molecules to trigger fusion and infection.
The determinant at residue 153 is proximal to sites in the V2 loop that are targeted by the recently described MAbs PG9 and PG16 (46
). In fact, these MAbs target determinants in both the V2 and the V3 loops. Since the substitutions in V1 described here also had effects on the V3 loop, we hypothesized that they would impact on PG9 and PG16 sensitivity. However, only modest effects on PG9 and PG16 sensitivity were observed. This result illustrates the robustness of the PG9 and PG16 epitope and is a reassuring result for vaccine research focused on this epitope.
The conservation of glutamic acid at position 153 across several clades is intriguing and implies that this site plays an important functional role for the envelope. Our data suggest that this role may involve an interaction with the V3 loop, which in turn affects the CD4bs. The decreased frequency of E153 among clade D and CRF02_AG envelopes and increased frequency of G153 is also striking. Whether the higher prevalence of G153 associates with increased macrophage tropism for these clades is not known. An E153G substitution for the one clade D envelope that we tested (CMT19 S531) had no affect on macrophage tropism. However, a more extensive survey would be needed to establish an influence of G153 on clade D or CRF02_AG envelopes. Of note, clade D viruses have been associated with faster disease progression (1
) and a higher frequency of dementia (40
). In addition, CXCR4 tropism was common among CRF02_AG viruses, a sign of a mature HIV epidemic (15
). It is thus possible that an increased frequency of G153 may simply reflect a high prevalence of R5 viruses with increased macrophage tropism late in disease (18
In summary, we describe here a novel determinant in the V1 loop that modulates macrophage tropism conferred by HIV-1 R5 envelopes. The V1 loop determinant appears to control the exposure or conformation of the V3 loop, which in turn impacts the CD4bs to prime the envelope for low CD4 use and macrophage infection. Our data also have relevance for vaccines that aim to target the CD4bs.