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. 2002 Nov;76(22):11476-83.
doi: 10.1128/jvi.76.22.11476-11483.2002.

Nipah virus V protein evades alpha and gamma interferons by preventing STAT1 and STAT2 activation and nuclear accumulation

Jason J Rodriguez  1 , Jean-Patrick Parisien, Curt M Horvath
Affiliations

Nipah virus V protein evades alpha and gamma interferons by preventing STAT1 and STAT2 activation and nuclear accumulation

Jason J Rodriguez et al. J Virol. 2002 Nov.

Abstract

Characterization of recent outbreaks of fatal encephalitis in southeast Asia identified the causative agent to be a previously unrecognized enveloped negative-strand RNA virus of the Paramyxoviridae family, Nipah virus. One feature linking Nipah virus to this family is a conserved cysteine-rich domain that is the hallmark of paramyxovirus V proteins. The V proteins of other paramyxovirus species have been linked with evasion of host cell interferon (IFN) signal transduction and subsequent antiviral responses by inducing proteasomal degradation of the IFN-responsive transcription factors, STAT1 or STAT2. Here we demonstrate that Nipah virus V protein escapes IFN by a distinct mechanism involving direct inhibition of STAT protein function. Nipah virus V protein differs from other paramyxovirus V proteins in its subcellular distribution but not in its ability to inhibit cellular IFN responses. Nipah virus V protein does not induce STAT degradation but instead inhibits IFN responses by forming high-molecular-weight complexes with both STAT1 and STAT2. We demonstrate that Nipah virus V protein accumulates in the cytoplasm by a Crm1-dependent mechanism, alters the STAT protein subcellular distribution in the steady state, and prevents IFN-stimulated STAT redistribution. Consistent with the formation of complexes, STAT protein tyrosine phosphorylation is inhibited in cells expressing the Nipah virus V protein. As a result, Nipah virus V protein efficiently prevents STAT1 and STAT2 nuclear translocation in response to IFN, inhibiting cellular responses to both IFN-alpha and IFN-gamma.

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Figures

FIG. 1.
FIG. 1.
Expression of the Nipah virus V protein inhibits IFN-α and IFN-γ signaling. (A) Human fibrosarcoma 2FTGH cells were transfected with empty vector (lane C) or expression vectors for HA-tagged SV5 V (lane SV), HPIV2 V (lane HV), or Nipah V (lane NV), and lysates were separated by SDS-PAGE (12% polyacrylamide). V proteins were visualized by immunoblotting with antiserum to the HA epitope tag. (B) 2FTGH cells were transfected with an ISRE-luciferase reporter gene and either empty vector, SV5 V (SV), or Nipah V (NV) expression vectors as indicated. The cells were treated with 1,000 U of IFN-α per ml 8 h prior to lysis and luciferase assays (+) or left untreated (−). (C) Same as in panel B but using an IFN-γ-responsive GAS-luciferase reporter gene and treatment with 5 ng/ml of IFN-γ (or no treatment). All bars represent average values from triplicate samples, normalized to cotransfected CMV-LacZ and expressed as percentages of IFN stimulated controls; error bars indicate standard deviations.
FIG. 2.
FIG. 2.
Nipah virus V binds to STAT1 and STAT2 without inducing STAT degradation. (A) 293T cells were transfected with empty vector (lane C), FLAG-tagged SV5 V (lane SV), or FLAG-tagged Nipah virus V (lane NV) expression vectors. Whole-cell extracts were prepared 36 h later, and 20 μg of protein was separated by SDS-PAGE (12% polyacrylamide) and processed for immunoblotting with antisera to STAT1, STAT2, or FLAG. Positions of prestained molecular weight standards (in thousands) and STAT1, STAT2, Nipah V protein (NV), and SV5 V protein (SV) are indicated. (B) Extracts as in panel A (700 μg of total protein) were immunoprecipitated with FLAG affinity gel (Sigma) and analyzed by immunoblotting. (C and D) Gel filtration analysis. Whole-cell extracts from control or NV-expressing cells were separated by chromatography on a Superdex-200 column. Positions of STAT1 (C) and STAT2 (D) were determined in control cells (+GFP) or in the presence of Nipah V protein (+NV) by immunoblotting of every third fraction. (E) Gel filtration data plotted in comparison to a standard curve. Elution positions are indicated for STAT1 and STAT2 in the presence and absence of Nipah V protein (NV) with respect to calibration standards (open circles).
FIG. 3.
FIG. 3.
Nipah virus V protein prevents nuclear import of STAT1 and STAT2. (A) 2FTGH cells were transfected with empty vector or HA-tagged Nipah V expression plasmid (+NV) and were either unstimulated (UNT) or treated for 30 min with 1,000 U of IFN-α per ml (+IFNα). Cells were fixed, permeabilized, and stained sequentially for HA and then for STAT1 or STAT2. (B) Independent block on STAT1 translocation. Same as in panel A, but the cells were treated for 30 min with 5 ng of IFN-γ per ml prior to processing for NV (HA), STAT1, and STAT2 fluorescence. Left panels indicate the parental cell line 2FTGH (WT). Right panels indicate the STAT2-deficient daughter cell line, U6A (STAT2−). Arrows indicate Nipah V protein-expressing cells.
FIG. 4.
FIG. 4.
Nipah virus V-STAT complexes accumulate in the cytoplasm by a Crm1 nuclear export system. (A) Localization of V proteins. 2FTGH cells were transfected with the HA-tagged V expression vectors and treated 24 h later with LMB (10 ng/ml) for 3 (+) or left untreated (−), fixed, permeabilized, and stained as described in the legend to Fig. 3. (B) Localization of STAT proteins and Nipah virus V in the presence of LMB. Cells were processed as described above, but double staining was used to visualize effects on STAT1 and STAT2 proteins. (C) Nipah virus V blocks IFN-α signaling in the presence of LMB. 2FTGH cells were transfected with an ISRE-luciferase reporter gene, empty vector, SV5 V (SV), or Nipah virus V (NV) expression vectors. The cells were treated with 1,000 U of IFN-α per ml and/or 10 ng of LMB per ml (+) or left untreated (−) prior to lysis and luciferase assays, as in Fig. 1.
FIG. 5.
FIG. 5.
Nipah virus V protein inhibits activating tyrosine phosphorylation of STAT1. Individual 60-mm plates of 293T cells were transfected with 15 μg of empty vector or FLAG-tagged Nipah virus V expression vector (NV). Cells were treated 24 h later with 1,000 U of IFN-α per ml or 5 ng of IFN-γ per ml for 30 min prior to lysis. Lysates (50 μg of total protein) were separated by SDS-PAGE (7% polyacrylamide), transferred to nitrocellulose, and visualized by immunoblotting with antisera to STAT1, phosphotyrosine 701-modified STAT1 (STAT1-P), and FLAG epitope tag.
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