Non-macrophage-tropic human immunodeficiency virus type 1 R5 envelopes predominate in blood, lymph nodes, and semen: implications for transmission and pathogenesis

Abstract

Human immunodeficiency virus type 1 (HIV-1) R5 isolates that predominantly use CCR5 as a coreceptor are frequently described as macrophage tropic. Here, we compare macrophage tropism conferred by HIV-1 R5 envelopes that were derived directly by PCR from patient tissue. This approach avoids potentially selective culture protocols used in virus isolation. Envelopes were amplified (i) from blood and semen of adult patients and (ii) from plasma of pediatric patients. The phenotypes of these envelopes were compared to those conferred by an extended panel of envelopes derived from brain and lymph node that we reported previously. Our results show that R5 envelopes vary by up to 1,000-fold in their capacity to confer infection of primary macrophages. Highly macrophage-tropic envelopes were predominate in brain but were infrequent in semen, blood, and lymph node samples. We also confirmed that the presence of N283 in the C2 CD4 binding site of gp120 is associated with HIV-1 envelopes from the brain but absent from macrophage-tropic envelopes amplified from blood and semen. Finally, we compared infection of macrophages, CD4(+) T cells, and peripheral blood mononuclear cells (PBMCs) conferred by macrophage-tropic and non-macrophage-tropic envelopes in the context of full-length replication competent viral clones. Non-macrophage-tropic envelopes conferred low-level infection of macrophages yet infected CD4(+) T cells and PBMCs as efficiently as highly macrophage-tropic brain envelopes. The lack of macrophage tropism for the majority of the envelopes amplified from lymph node, blood, and semen is striking and contrasts with the current consensus that R5 primary isolates are generally macrophage tropic. The extensive variation in R5 tropism reported here is likely to have an important impact on pathogenesis and on the capacity of HIV-1 to transmit.

FIG. 1.

Phylogenetic analysis of the V1 to V3 sequences of envelopes investigated. V1 to V3 gp120 Env sequences were used to construct the phylogenetic tree by the neighbor-joining method. The sequence of the NL4.3 virus was used as an outgroup. The percentage of bootstrap repetitions (out of 1,000) in which the nucleotide sequences grouped together is represented as a number at the branch node, but only the bootstrap values of >74% are shown. The genetic distance was 2%. Envelopes from pediatric patients P-1206, P-1114, and P-1031 are designated by the letters S, C, and J, respectively.

FIG. 2.

Macrophage tropism of HIV-1 R5 envelopes amplified from patient tissues. Amplified envelopes were cloned into pSVIIIenv and expressed on HIV-1 particles. Infectivity was titrated on HeLa TZM-BL cells (HeLa/CD4/CCR5; JC53 clone [29]) and on primary macrophages. Infectivity was assessed by immunostaining in situ and estimating the number of infected cells as described previously (27). Macrophage infectivity is plotted as a percentage of that recorded on HeLa TZM-BL cells. Light symbols represent envelopes that failed to confer any macrophage infectivity, i.e., macrophage infectivity was less than the value shown.

FIG. 3.

Macrophage-tropic envelopes require less CD4 for infection. HeLa cell clones expressing different CD4 and CCR5 levels (29) were infected with Env⁺ pseudoviruses as described in Materials and Methods. After 72 h, cells were fixed and immunostained for p24 antigen, and foci of infection were counted. Infectivities for Env⁺ pseudotypes prepared with envelopes amplified from adult blood and semen (A) and pediatric plasma (B) are shown. Envelopes that conferred infection of macrophages (SQ43 380.1, C98-15, C98-18, J92-14, and S94-CSF-11) also conferred infection of HeLa cells expressing low levels of CD4. (C) Infectivities of an extended panel of brain and lymph node envelopes that were previously reported (27) are shown for comparison. Envelopes from pediatric patients P-1206, P-1114, and P-1031 are designated by the letters S, C, and J, respectively.

FIG. 4.

Infectivities of replication-competent viruses carrying highly macrophage-tropic and non-macrophage-tropic envelopes evaluated on HeLa TZM-BL cells, primary macrophages, PBMCs, and CD4⁺ T cells. A. Infectivity titers of replication-competent viruses carrying macrophage-tropic (YU2, NA20 B59, and NA420 B33) or non-macrophage-tropic (JR-CSF, NA420 LN85, and NA118 LN33) envelopes. Infectivities for HeLa TZM-BL cells and macrophages were estimated by FFU assays, and the results shown represent averaged FFU counts for duplicate wells from one of several experiments with similar results. Infectivities for PBMCs and CD4⁺ T cells were estimated by TCID₅₀ assay, using replicates of six wells, and are representative of at least two experiments. B. Ratios of infectivity for macrophages compared to HeLa TZM-BL cells, PBMCs, and CD4⁺ T cells. C. Ratios of infectivity for macrophages, PBMCs, and CD4⁺ T cells compared to HeLa TZM-BL cells.