Natural resistance-associated macrophage protein is a cellular receptor for sindbis virus in both insect and mammalian hosts

Abstract

Alphaviruses, including several emerging human pathogens, are a large family of mosquito-borne viruses with Sindbis virus being a prototypical member of the genus. The host factor requirements and receptors for entry of this class of viruses remain obscure. Using a Drosophila system, we identified the divalent metal ion transporter natural resistance-associated macrophage protein (NRAMP) as a host cell surface molecule required for Sindbis virus binding and entry into Drosophila cells. Consequently, flies mutant for dNRAMP were protected from virus infection. NRAMP2, the ubiquitously expressed vertebrate homolog, mediated binding and infection of Sindbis virus into mammalian cells, and murine cells deficient for NRAMP2 were nonpermissive to infection. Alphavirus glycoprotein chimeras demonstrated that the requirement for NRAMP2 is at the level of Sindbis virus entry. Given the conserved structure of alphavirus glycoproteins, and the widespread use of transporters for viral entry, other alphaviruses may use conserved multipass membrane proteins for infection.

Figure 1

Drosophila NRAMP is required for Sindbis virus infection. dsRNA against dNRAMP, the vATPase component vha26, the SINV reporter GFP and the negative control luciferase were challenged with a. Sindbis virus-GFP (HRsp) in DL1 cells b. Sindbis virus-GFP (HRsp) in Kc167 cells c. Sindbis virus-GFP (dsTE12H) in DL1 cells. d. Sindbis-GFP (S.A.AR86) in DL1 cells. e. Wild type WNV detected using anti-E in DL1 cells. F. VSV GFP in DL1 cells. a–f Number on bottom left is robust Z-score and bottom right percent infection (virus, green; nuclei, blue).

Figure 2

dNRAMP is required for binding and entry in Drosophila cells. a. Cells pre-treated with the indicated dsRNA were either infected (gray) or transfected with vRNA (black) and triplicate Mean± S.D. shown; *p<0.05. b. Cells were pre-treated with dsRNA against dNRAMP or control dsRNA and bound with biotinylated Sindbis virus (virus, red; nuclei, blue). c. The percentage of cells that had at least one viral particle bound is graphed with Mean± S.D. for triplicate experiments; *p<0.05.

Figure 3

dNRAMP is required for Sindbis infection of adult flies. a. Survival curve of SINV challenged flies compared to control. b. Viral titers of flies challenged with the indicated SINV concentrations or control at 6 days post infection. Mean± S.D. of three experiments. c. Sindbis virus titers from flies of the indicated genotypes at either 3 (gray) or 6 (black) days post infection. Mean± S.D. of three experiments; *p<0.05. d. VSV virus titers from flies of the indicated genotypes at either 3 (gray) or 6 (black) days post infection; Mean± S.D. of three experiments.

Figure 4

Sindbis virus infection is sensitive to iron treatment in both Drosophila and mosquito cells. a–c. Iron treatment of Drosophila DL1 cells (black) or Aedes aegypti Aag-2 cells (gray) challenged with (a) Sindbis virus (HRsp), (b) Sindbis virus (S.A.AR86), or (c) VSV. Mean± S.D. of three experiments; * p<0.05.

Figure 5

NRAMP2 facilitates binding and infection of Sindbis virus in mammalian cells. a. U2OS cells challenged with the indicated virus in the presence or absence of iron. Mean± S.D. of three experiments; * p<0.05. b. U2OS cells transfected with hNRAMP2 or the control vector (mock) were challenged with the indicated virus. Mean± S.D. of three experiments; *p<0.05. c. The percentage of cells that had at least one viral particle bound is graphed for the indicated virus with Mean± S.D. for triplicate experiments shown; *p<0.05. d. Mock or hNRAMP2-transfected U2OS cells were either untreated or iron treated and bound with biotinylated Sindbis virus, precipitated with steptavidin beads and visualized by immunoblot. Left panel shows that cells that were uninfected do not have a band while input labeled virus is visualized as a 60kD band. Representative of 3 experiments. e. U2OS cells were either untreated or iron treated and bound with biotinylated Sindbis virus, biotinylated VSV or biotinylated IgG, precipitated with streptavidin beads and visualized by immunoblot. GAPDH and NRAMP2 are observed in the input, whereas only Sindbis virus precipitates with NRAMP2; and only in the absence of iron treatment which reduces NRAMP2 levels. Representative of 5 experiments.

Figure 6

NRAMP2 is required for Sindbis virus infection and entry. a. NRAMP2^fl/fl cells were transduced with MSCV-Cre-ires-GFP or MSCV-ires-GFP retroviruses and challenged with the indicated virus. The percent infection of retrovirally transduced cells (GFP⁺) is shown for triplicate experiments Mean± S.D.; *p<0.05. b. Sindbis and Ross River chimeric viruses were used to challenge NRAMP2^fl/fl cells transduced with MSCV-Cre-ires-GFP or MSCV-ires-GFP retroviruses as indicated. Mean± S.D. of three experiments; * p<0.05. (gp, glycoproteins; nsp, nonstructural proteins)