Biological analysis of human immunodeficiency virus type 1 R5 envelopes amplified from brain and lymph node tissues of AIDS patients with neuropathology reveals two distinct tropism phenotypes and identifies envelopes in the brain that confer an enhanced tropism and fusigenicity for macrophages

Abstract

Complete envelope genes were amplified from autopsy brain tissue of five individuals who had died of AIDS and had neurological complications. Lymph node samples were included for two of the patients. Nineteen different envelope clones from the five patients had distinct V1V2 sequences. Thirteen of the envelopes were functional and conferred fusigenicity and infectivity for CD4(+) CCR5(+) cells. Infectivity and cell-cell fusion assays showed that most envelopes used both CCR5 and CCR3. One brain-derived envelope used a broad range of coreceptors, while three other brain envelopes from one individual were restricted to CCR5. However, there was no correlation between tissue of origin and coreceptor use. Envelopes showed two very distinct phenotypes depending on their capacity to infect macrophages and to exploit low levels of CD4 and/or CCR5 for infection. Envelopes that were highly fusigenic and tropic for macrophages were identified in brain tissue from four of the five patients. The enhanced macrophage tropism correlated with reduced sensitivity to inhibition by Q4120, a CD4-specific antibody, but not with sensitivity to the CCR5 inhibitor, TAK779. The highly macrophage-tropic envelopes were able to infect cells expressing low levels of CD4 and/or CCR5. Comparison with several well-characterized macrophage-tropic envelopes showed that the four identified patient envelopes were at the top limit of macrophage tropism. In contrast, all four lymph node-derived envelopes exhibited a non-macrophage-tropic phenotype and required high levels of CD4 for infection. Our data support the presence of envelopes that are highly fusigenic and tropic for macrophages in the brains of patients with neurological complications. These envelopes are able to infect cells that express low levels of CD4 and/or CCR5 and may have adapted for replication in brain macrophages and microglia, which are known to express limited amounts of CD4.

FIG. 1.

Coreceptor use of NA20 B59 envelope evaluated by cell-cell fusion assays. NA20 B59-induced syncytia in GHOST and NP2/CD4 cells expressing a range of different coreceptors but not in the parental cell lines lacking coreceptors.

FIG. 2.

Infection of primary macrophages by patient LN and brain envelopes. (A) Infectivity titers for patient envelope-positive pseudotype viruses titrated on primary macrophages from different donors and on GHOST/CCR5 cells. Arrows indicate that infection is below the level of detection. Data are derived from duplicate infections on two donor macrophage batches. GHOST/CCR5 titers were derived from infectivity titrations carried out with the same batch of virus as in macrophage infections. (B) Macrophage-GHOST/CCR5 infectivity ratios indicate that patient envelopes NA20 B59, NA353 B27, NA420 B33, and NA176 B93 are highly macrophage tropic.

FIG. 3.

Syncytium induction in primary macrophages by patient brain-derived envelopes. Large spreading syncytia induced by patient brain envelopes NA176 B93 and NA353 B27 are shown. Note that the syncytia shown contain hundreds of nuclei (top panels). No syncytia were induced by NA176 B72 or the nonfunctional NA353 B13 (bottom panels).

FIG. 4.

Infection of cells expressing low levels of CD4 and/or CCR5 by patient envelopes. Infection of HeLa/HiCD4 cells expressing high levels of CD4 and three different amounts of CCR5 (top panels). Infection of HeLa/LowCD4 expressing low levels of CD4 and three different amounts of CCR5 (bottom panels). Infectivity was assessed as FFU per milliliter by immunostaining for p24 as described in Materials and Methods. Values plotted are the average FFU counts for duplicate wells in a single experiment. Similar data were obtained from at least one repeat experiment. Results for different envelopes are grouped into separate graphs to avoid congestion.

FIG. 5.

Cell-to-cell fusion induced by patient envelopes in MOLT 4 cells that express very low levels of CCR5. 293T cells expressing different patient envelopes were cocultivated with MOLT 4 cells. The presence of syncytia was assessed by light microscopy after overnight incubation. Arrows mark examples of syncytia. The control panel shows cocultivation of MOLT 4 with 293T cells transfected with a pSVIIIenv carrying a defective envelope.

FIG. 6.

Sensitivity of patient envelopes to CCR5 and CD4 ligands. (A) TAK779 (CCR5 inhibitor) IC0₉₀ 0 inhibitory doses. (B) Q4120 (anti-CD4 domain 1) IC0₉₀ 0 inhibitory doses. Black symbols represent macrophage-tropic patient envelopes. Gray symbols are other patient envelopes. Striped symbols are control, well-characterized HIV-1 envelopes. Data shown are derived from a single experiment and are representative of data from several experiments.