HIV-1 Transmission by Dendritic Cell-specific ICAM-3-grabbing Nonintegrin (DC-SIGN) Is Regulated by Determinants in the Carbohydrate Recognition Domain That Are Absent in Liver/Lymph Node-SIGN (L-SIGN)

Nancy P.Y Chung,Sabine K.J Breun,Arman Bashirova,Joerg G Baumann,Thomas D Martin,Jaideep M Karamchandani,Jason W Rausch,Stuart F.J Le Grice,Li Wu,Mary Carrington,Vineet N Kewalramani

doi:10.1074/jbc.m109.030619

Abstract

In this study, we identify determinants in dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) necessary for human immunodeficiency virus, type 1 (HIV-1), transmission. Although human B cell lines expressing DC-SIGN efficiently capture and transmit HIV-1 to susceptible target cells, cells expressing the related molecule liver/lymph node-specific ICAM-3-grabbing nonintegrin (L-SIGN) do not. To understand the differences between DC-SIGN and L-SIGN that affect HIV-1 interactions, we developed Raji B cell lines expressing different DC-SIGN/L-SIGN chimeras. Testing of the chimeras demonstrated that replacement of the DC-SIGN carbohydrate-recognition domain (CRD) with that of L-SIGN was sufficient to impair virus binding and prevent transmission. Conversely, the ability to bind and transmit HIV-1 was conferred to L-SIGN chimeras containing the DC-SIGN CRD. We identified Trp-258 in the DC-SIGN CRD to be essential for HIV-1 transmission. Although introduction of a K270W mutation at the same position in L-SIGN was insufficient for HIV-1 binding, an L-SIGN mutant molecule with K270W and a C-terminal DC-SIGN CRD subdomain transmitted HIV-1. These data suggest that DC-SIGN structural elements distinct from the oligosaccharide-binding site are required for HIV-1 glycoprotein selectivity.

Highlights

In this study, we identify determinants in dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) necessary for human immunodeficiency virus, type 1 (HIV-1), transmission
Because we previously observed that DC-SIGN carbohydrate-recognition domain (CRD) segments 1 and 2 as well as segments 1 and 3 could enable HIV-1 transmission by lymph node-specific ICAM-3-grabbing nonintegrin (L-SIGN) chimeras (Fig. 5C), we investigated whether K270W required residues from DC-SIGN CRD segment 2 or 3 to acquire the HIV-1 transmission phenotype
In contrast to DC-SIGN, we show that L-SIGN does not support HIV-1 transmission when expressed on human B cell lines

Summary

EXPERIMENTAL PROCEDURES

Plasmids—Human DC-SIGN and L-SIGN with five, six, or seven complete repeats in the repeat domain were cloned into pcDNA3.1/V5-His-TOPO vector (Invitrogen). DC-SIGN, L-SIGN, or chimeras were incubated with 300 ␮l of VLPs for 1 h at 37 °C and washed extensively with RPMI 1640 medium containing 2% FBS to remove unbound VLPs. For blocking experiments, cells were preincubated with either cross-reactive mAb 612. The inclusion bodies were washed with 20 mM Tris-HCl, expressing DC-SIGN or L-SIGN were incubated in the absence pH 7.4, and solubilized in 6 M guanidine HCl, pH 8, at 4 °C for or presence of single-round infectious HIV-luciferase vectors. Consistent with HIV-1 transmission data, Western Blot Analysis—Recombinant proteins and cleared DC-SIGN interacted with VLPs in an Env-dependent manner, cell lysates from Raji lines and 293T cells transfected with chi- whereas L-SIGN did not (Fig. 1D).

RESULTS

Other Human B Cell Lines

DDDDL ϩ

DISCUSSION