Our results show that R5 envelopes vary by up to 1,000-fold in their capacity to confer infection of blood-derived primary macrophages. Thus, highly macrophage-tropic envelopes were found frequently in the brain but infrequently in semen, blood, and lymph node samples. The lack of macrophage tropism for the majority of the envelopes amplified from lymph node, blood, and semen is striking and contrasts with a widely held view that R5 primary isolates are generally macrophage tropic (2
In this study, PCR-amplified envelopes were coexpressed with an Env−
pNL4.3 construct in 293T cells to produce pseudovirions capable of just a single round of infection. A concern is that such pseudovirions may not represent replication-competent viruses present in vivo and may underestimate macrophage infection. In control experiments, we have shown that different patient envelopes assemble onto pseudovirions with slightly different efficiencies. However, these different assembly efficiencies did not correlate with the extent of macrophage infectivity (data not shown). Figure shows that full-length infectious chimeric clones (based on pNL4.3) that carry different brain- and lymph node-derived envelopes conferred the same macrophage-tropic and non-macrophage-tropic phenotypes as the corresponding envelope-positive pseudovirions. Virus produced from 293T cells following transfection of these infectious clones conferred at least 50-fold higher infectivity than pseudovirions. Nevertheless, ratios of infectivity for macrophages compared to HeLa TZM-BL cells were up to 1,000-fold lower for non-macrophage-tropic envelopes compared to highly macrophage-tropic envelopes from the brain. Thus, the experiments shown in Fig. strongly support the relevance of the data generated using envelope-positive pseudovirions but show that the non-macrophage-tropic envelopes are not negative for macrophage infection, just very inefficient. It was also possible that virions budding from 293T cells may lack the necessary adhesion molecules required for macrophage infection. However, Bounou et al. reported that virions produced from 293T cells that express ICAM-1 attached to primary macrophages with similar efficiencies to virions lacking ICAM-1 (4
). Bounou et al. concluded that attachment was conferred by CD4-independent and adhesion molecule-independent mechanisms that likely involve envelope sugars and the macrophage mannose receptor. This is supported by Nguyen and Hildreth, who showed that the macrophage mannose receptor was the predominant mode of attachment to macrophages by HIV particles (25
We also evaluated whether brain and lymph node envelopes conferred infection of primary CD4+ T-cell and PBMC cultures. Figure demonstrates that replication-competent clones carrying non-macrophage-tropic lymph node envelopes (NA420 LN85 and NA118 LN33) conferred high levels of infectivity for both CD4+ T cells and PBMCs. These results confirm that non-macrophage-tropic envelopes amplified from immune tissue are fully competent for replication in primary CD4+ T cells and are not compromised for infection of primary cell types generally.
The determinants of the distinct R5 tropisms are not yet fully defined. The presence of N283 is likely to play a major role in the macrophage tropism of envelopes in the brain. Dunfee et al. (13
) have modeled N283 onto the gp120 structure (21
) and shown that N283 more readily forms a hydrogen bond with Q40 on CD4 and presumably helps to stabilize the gp120-CD4 interaction. Regardless, macrophage-tropic envelopes amplified from pediatric patients and from adult semen did not carry N283, indicating that this residue was not the sole determinant for macrophage infection. In addition, NA20 LN14 did carry N283 and clustered phylogenetically with NA20 brain envelopes yet failed to infect macrophages. Thus, the impact of N283 on macrophage infection can be overcome by other determinants in envelope. For example, Walter et al. reported that sequences within the V1V2 loops modulated macrophage infectivity conferred by chimeric envelopes derived from macrophage-tropic R5 BAL and non-macrophage-tropic X4 NL4.3 HIV-1 isolates (39
). The V1V2 loops sit over gp120 sites involved in binding coreceptors (42
) but may also occlude the CD4 binding site (28
). It is also possible that glycosylation groups (12
), other variable loops, or other structural alterations may occlude the CD4 binding site. These protective strategies may reduce the efficiency of gp120-CD4 binding even in the presence of N283.
Neutralizing antibodies that target the CD4 binding site on gp120 may drive evolution of envelope conformations that protect this site. Since the brain usually contains lower levels of neutralizing antibodies compared to immune tissue, this environment may allow the evolution of variants that interact efficiently with CD4 and can replicate in macrophage lineage cells resident in the brain. Viral replication in PBMC cultures (in the absence of neutralizing antibodies) during isolation may also select for variants proficient for macrophage infection; however, they may not be representative of HIV-1 quasispecies in vivo.
In this study, we investigated envelopes from multiple samples and patients rather than multiple envelopes from a few samples. To this end, we have studied 53 envelopes derived from 22 samples of 11 patients. For most samples, we were able to study only one to three envelope clones and therefore have not extensively sampled the viral population present. It is therefore possible that highly macrophage-tropic variants are present at lower frequencies outside the brain, and this will require a more intensive investigation to evaluate.
The remarkable variation in R5 tropism shown here is likely to have a profound impact on pathogenesis, as also suggested by Gray et al. (16
), and on the efficiency of transmission, depending on the importance of macrophages or cells that express low amounts of CD4 as targets for infection in the new host.