Macrophage-tropic simian/human immunodeficiency virus chimeras use CXCR4, not CCR5, for infections of rhesus macaque peripheral blood mononuclear cells and alveolar macrophages

Abstract

After the nearly complete and irreversible depletion of CD4(+) T lymphocytes induced by highly pathogenic simian/human immunodeficiency virus chimeric viruses (SHIVs) during infections of rhesus monkeys, tissue macrophages are able to sustain high levels (>10(6) viral RNA copies/ml) of plasma viremia for several months. We recently reported that the virus present in the plasma during the late macrophage phase of infection had acquired changes that specifically targeted the V2 region of gp120 (H. Imamichi et al., Proc. Natl. Acad. Sci. USA 99:13813-13818, 2002); some of these SHIV variants were macrophage-tropic (M-tropic). Those findings have been extended by examining the tropic properties, coreceptor usage, and gp120 structure of five independent SHIVs recovered directly from lymph nodes of late-stage animals. All of these tissue-derived SHIV isolates were able to infect alveolar macrophages. These M-tropic SHIVs used CXCR4, not CCR5, for infections of rhesus monkey PBMC and primary alveolar macrophages. Because the starting highly pathogenic T-tropic SHIV inoculum also utilized CXCR4, these results indicate that the acquisition of M-tropism in the SHIV-macaque system is not accompanied by a change in coreceptor usage. Compared to the initial T-tropic SHIV inoculum, tissue-derived M-tropic SHIVs from individual infected animals carry gp120s containing similar changes (specific amino acid deletions, substitutions, and loss of N-linked glycosylation sites), primarily within the V1 and/or V2 regions of gp120.

FIG. 1.

Viral RNA loads in plasma and peripheral blood CD4⁺-T-cell profiles of SHIV-infected rhesus monkeys. Each animal was inoculated with the indicated amounts of SHIV_DH12R, SHIV_DH12R-PS1, or SHIV_DH12R-CL-7 intravenously. Viral RNA levels in plasma and the peripheral blood CD4⁺-T-cell numbers were measured at the indicated times. Daggers indicate the time of euthanasia.

FIG. 2.

SHIV and SIV replication in rhesus monkey PBMC. The replication kinetics of virus controls (a) or SHIVs recovered from lymph nodes of animals during the macrophage phase of SHIV infections (b) are shown. Culture supernatants were collected at the indicated time points, and the RT activity was determined. The data shown in the two panels were obtained from the same experiment.

FIG. 3.

SHIV and SIV replication in primary monkey AM. The replication kinetics of virus controls (a) or SHIVs recovered from lymph nodes of animals during the macrophage phase of SHIV infections (b) are shown. Culture supernatants were collected at the indicated time points, and the RT activity was measured. The data shown the two panels were obtained from the same experiment.

FIG. 4.

gp120 V1, V2, and V3 sequence alignments of M-tropic SHIVs recovered from lymph nodes of macrophage-phase animals. env gene segments of 3 kbp were RT-PCR amplified from the five indicated lymph node-derived virus stocks. Six to eight independent PCR clones were sequenced from animals originally inoculated with SHIV_DH12R (top), SHIV_DH12R-PS1 (middle), and SHIV_DH12R-CL7 (bottom). The previously described (19) gp120 V1, V2 and V3 sequences, associated with SHIVs present in the plasma of late-stage monkeys infected with SHIV_DH12R or SHIV_DH12R-PS1, are also shown. Identical residues are indicated by a dash, deleted residues are indicated by a period, and potential N-linked glycosylation sites are shaded.

FIG. 5.

V1 and V2 loop length polymorphism analyses of SHIVs recovered from lymph nodes during the macrophage phase of infection. Fluorescently labeled V1 or V2 PCR products, amplified from the five indicated SHIV stocks, were separated on a DNA sequencing gel, and their sizes were determined as described in Materials and Methods. The molecularly cloned SHIV_DH12R-CL-7 inoculum, which contains full-length V1 (28 residues) and V2 (40 residues) regions, was used as a reference (top). The percentage of each deleted species in the virus stock was calculated from the fluorescence intensity of each isoform divided by the total intensity.

FIG. 6.

Coreceptor inhibitor sensitivity of the three SHIV inocula and SIV controls. The inoculum viruses, SHIV_DH12R, SHIV_DH12R-PS1, or SHIV_DH12R-CL-7 were spinoculated onto rhesus PBMC in the presence of the indicated small molecule coreceptor inhibitors. The inhibitor concentrations used were 0.05, 0.1, 0.5, 1.0, 5.0, and 10 μM. The RT activity released into the medium on day 5 postinfection was determined in the absence (dashed line) or presence of inhibitor. SIV_mac239 and SIV_mac316 were also analyzed as representative of R5 viruses that can infect macaque PBMC.

FIG. 7.

Coreceptor usage of five M-tropic SHIVs for entry into macaque PBMC. The indicated SHIVs, all isolated from lymph nodes of late-stage animals, were spinoculated onto rhesus PBMC in the presence of the indicated small molecule coreceptor inhibitors. Progeny virus production was monitored as described in the legend to Fig. 6.

FIG. 8.

M-tropic SHIVs use CXCR4 for infections of rhesus monkey AM. SHIVs, recovered from lymph nodes of animals 891631 or WBJ and SIV_mac316 were used to infect macaque AM in the presence of AD101 or AMD3100 coreceptor inhibitors. Progeny virus production was monitored on day 10 for M-tropic SHIVs and on day 12 for SIV_mac316 by the RT activity released into the culture fluid. Symbols: □, AD101 (CCR5 inhibitor); ○, AMD3100 (CXCR4 inhibitor); - -, no inhibitor.