Z proteins of New World arenaviruses bind RIG-I and interfere with type I interferon induction

Abstract

The retinoic acid-inducible gene I product (RIG-I) is a cellular sensor of RNA virus infection that regulates the cellular beta interferon (IFN-beta) response. The nucleoproteins (NP) of arenaviruses are reported to antagonize the IFN response by inhibiting interferon regulatory factor 3 (IRF-3). Here, we demonstrate that the Z proteins of four New World (NW) arenaviruses, Guanarito virus (GTOV), Junin virus (JUNV), Machupo virus (MAVC), and Sabia virus (SABV), bind to RIG-I, resulting in downregulation of the IFN-beta response. We show that expression of the four NW arenavirus Z proteins inhibits IFN-beta mRNA induction in A549 cells in response to RNA bearing 5' phosphates (5'pppRNA). NW arenavirus Z proteins interact with RIG-I in coimmunoprecipitation studies and colocalize with RIG-I. Furthermore, expression of Z proteins interferes with the interaction between RIG-I and MAVS. Z expression also impedes the nuclear factor kappa light chain enhancer of activated B cells (NF-kappaB) and IRF-3 activation. Our results indicate that NW arenavirus Z proteins, but not Z protein of the Old World (OW) arenavirus lymphocytic choriomeningitis virus (LCMV) or Lassa virus, bind to RIG-I and inhibit downstream activation of the RIG-I signaling pathway, preventing the transcriptional induction of IFN-beta.

FIG. 1.

New World arenavirus Z proteins decrease IFN-β induction in A549 cells stimulated with 5′pppRNA. A549 cells (5 × 10⁵) were transfected with 3 μg of pCAGGS-Z or empty vector by use of calcium phosphate. At 36 h posttransfection, cells were transfected with 1 μg of 5′pppRNA by use of Lipofectamine 2000. Six hours later, the cells were harvested and the IFN-β expression level was measured by qPCR, with normalization to the β-actin expression level. Data represent three biological replicates. *, significant differences (P < 0.05; t test).

FIG. 2.

New World arenavirus Z proteins interact with RIG-I in coimmunoprecipitation experiments. (A) 293A cells (10⁶) were cotransfected with 0.5 μg of pcDNA3.1-V5-RIG-I (N terminally V5 tagged [N-V5-RIG-I] or C terminally V5 tagged [C-V5-RIG-I]) and with 0.5 μg of pCAGGS-Z (ZG, GTOV Z; ZJ, JUNV Z; ZM, MACV Z; ZS, SABV Z), pCAGGS-HA-NS1, or pCAGGS-GFP. Thirty-six hours later, cells were lysed and clarified supernatants subjected to immunoprecipitation assays using anti-V5 antibody (IP:V5). Z, NS1, and GFP were detected after Western blotting using anti-Z (WB:Z), anti-hemagglutinin (anti-HA) (WB:HA), and anti-GFP (WB:GFP) antibodies, respectively. “Input” represents untreated lysate prior to precipitation subjected to Western blot analysis. (B) 293A cells (10⁶) were cotransfected with 0.5 μg of pCAGGS-Z (ZG, GTOV Z; ZJ, JUNV Z; ZM, MACV Z; ZS, SABV Z) and 0.5 μg of pcDNA3.1/nV5-RIG-I (N terminally V5 tagged [N-V5-RIG-I]) or 0.5 μg of pcDNA3.2/cV5-RIG-I (C terminally V5 tagged [C-V5-RIG-I]) and then immunoprecipitated with antisera to the respective Z protein (IP:Z) and analyzed by Western blotting with anti-V5 antibody (WB:V5).

FIG. 3.

New World arenavirus Z proteins bind RIG-I. (A) Purified recombinant Z and RIG-I were incubated in nondenaturing lysis buffer and subjected to immunoprecipitation assays using anti-Z antibody. RIG-I was detected by Western blotting using anti-RIG-I antibody. (B) 293A cells were transfected with pUNO1-RIG-I-GFP (I) or pCAGGS-ZJ (II) or cotransfected with both plasmids (III to V). Proteins were visualized by confocal immunofluorescence microscopy. (I, III) RIG-I-GFP (green). (II, IV) Staining with the ZJ antibody (anti-Z [α Junin Z]) (red). (V) Overlay (yellow).

FIG. 4.

JUNV Z protein blocks complex formation between RIG-I and MAVS. 293A cells (1.5 × 10⁶) were triple transfected with 0.5 μg of pUNO1-MAVS, 0.5 μg of pcDNA3.1nV5-RIG-I, and 0.5 μg of pCAGGS-ZJ or transfected with vector without Z instead of pCAGGS-ZJ. pCAGGS-GFP was used as a negative control. Amounts of transfected DNA were adjusted with empty pCAGGS plasmid. At 36 h posttransfection, cells were lysed and clarified supernatants subjected to immunoprecipitation assays using anti-MAVS antibody (IP:MAVS). RIG-I, Z, and GFP were detected by Western blotting using the respective antibodies (WB:MAVS, WB:V5, WB:Z, and WB:GFP).

FIG. 5.

JUNV Z protein interferes with NF-κB and IRF-3 activation. (A to C) 293A cells (10⁶) were transfected with 1 μg of pCAGGS-ZJ (Junin Z) or vector. At 24 h posttransfection, cells were mock infected or infected with SeV and protein exacts subjected to SDS-PAGE (A, B) or native PAGE (C) for subsequent immunoblot analysis with anti-NF-κB p100/p52 (A), anti-phospho-IRF-3 (Ser396) (B), or anti-IRF-3 (C) antibody. (D) 293A cells (2.5 × 10⁵) were transfected with 0.25 μg of vector (I, II, V, VI) or pCAGGS-ZJ (III, IV, VII, VIII) by use of Lipofectamine 2000. At 24 h posttransfection, cells were transfected with 0.5 μg of 5′pppRNA by use of Lipofectamine 2000, and 6 h later, cells were fixed for immunofluorescence analysis. Cells were stained with anti-IRF-3 antibody (I to IV) and DAPI (4′,6-diamidino-2-phenylindole) nuclear counterstain (V to VIII).