Rhesus macaque dendritic cells efficiently transmit primate lentiviruses independently of DC-SIGN

Abstract

Here, we describe the isolation and characterization of the rhesus macaque homolog for human DC-SIGN, a dendritic cell-specific C-type lectin. mac-DC-SIGN is 92% identical to hu-DC-SIGN. mac-DC-SIGN preserves the virus transmission function of hu-DC-SIGN, capturing and efficiently transducing simian and human immunodeficiency virus to target CD4(+) T cells. Surprisingly, however, mac-DC-SIGN plays no discernable role in the ability of rhesus macaque dendritic cells to capture and transmit primate lentiviruses. Expression and neutralization analyses suggest that this process is DC-SIGN independent in macaque, although the participation of other lectin molecules cannot be ruled out. The ability of primate lentiviruses to effectively use human and rhesus dendritic cells in virus transmission without the cells becoming directly infected suggests that these viruses have taken advantage of a conserved dendritic cell mechanism in which DC-SIGN family molecules are significant contributors but not the only participants.

Figure 1

Alignment of the presumptive amino acid sequence of hu-DC-SIGN and mac-DC-SIGN. Predicted N-linked glycosylation sites (NXT/S) (34) and cytoplasmic functional motifs (LL, dileucine motif; YXXL, potential internalization motif) (–38) are annotated (italicized and underlined). The beginning of each of the following is marked: TM, transmembrane domain (underlined); NR, neck repeats (underlined); CRD, carbohydrate recognition domain (bold type). Nonconserved amino acids are boxed. Amino acids that precede the transmembrane domain represent cytoplasmic residues, and those that follow the transmembrane domain jut into the extracellular matrix.

Figure 2

FACS analysis of reactivity of mac-DC-SIGN with anti-hu-DC-SIGN mAbs. Stable THP-1/mac-DC-SIGN and THP-1/hu-DC-SIGN cells were stained separately with six mAbs recognizing hu-DC-SIGN; three of the six mAbs (507, 516, and 526) are mac-DC-SIGN cross-reactive. On all histograms, the gray curve represents staining with an isotype control antibody, whereas the black curve represents DC-SIGN mAb staining. The mean fluorescence is shown in the top right corner of the histograms. One of three representative experiments is shown.

Figure 3

Cross-reactive DC-SIGN mAbs 507, 516, and 526 (10 μg/ml) block adhesion of ICAM-3-coated fluorescent beads to THP-1/hu-DC-SIGN and THP-1/mac-DC-SIGN cells. ICAM-3 bead binding to THP-1 cells was uniformly less than 5% in repeat experiments. Mouse IgG was used as a background antibody control. One of four representative experiments is shown.

Figure 4

Transmission of HIV-1 pseudotypes mediated by hu-DC-SIGN or mac-DC-SIGN. THP-1, THP-1/hu-DC-SIGN, or THP-1/mac-DC-SIGN donor cells were preincubated with DC-SIGN mAb 526 (10 μg/ml). Next, HIV-luc pseudotyped with Env from R5-tropic HIV-1_JRFL, X4-tropic HIV-1_HXB2, SIV_MAC1A11, SIV_MAC239, SIV_MAC239MER, or SIV_AGM was incubated with cells for 3 h at 37°C. Cells were washed and cocultured with Hut/CCR5 target cells. HIV-1 infection was determined after 2 days by measuring the transduction of luciferase activity. Each data set represents the mean of three separate wells of infected cells.

Figure 5

Transmission of SIV to Hut/CCR5 cells by macaque DC is not blocked with anti-DC-SIGN. The virus capture and transmission assay was performed as described in Fig. 4. DC-SIGN mAb 507 (10 μg/ml) was preincubated with the DC before pulsing with HIV-luc pseudotyped with SIV_MAC1A11 Env. DC isolated from two individual macaques (L655 and CN72) were use as donor cells, respectively. Each data set represents the mean of three separate wells of infected cells. CPS, counts per second.

Figure 6

Expression of mac-DC-SIGN in vivo. (A) FACS analysis of mac-DC-SIGN expression on macaque DC stained with the cross-reactive DC-SIGN mAbs 507, 516, and 526. On all histograms, the gray curve represents staining with an isotype control antibody, whereas the black curve indicates DC-SIGN staining. The mean fluorescence is shown in the top right corner of the histograms. (B) Expression of mac-DC-SIGN mRNA. RNA isolated from hu-DC, mac-DC, or mac-PBMC was hybridized with a 1.2-kb mac-DC-SIGN cDNA probe. The human β-actin probe was used as a control for RNA loading.