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. 2003 Jul;77(13):7635-44.
doi: 10.1128/jvi.77.13.7635-7644.2003.

STAT protein interference and suppression of cytokine signal transduction by measles virus V protein

Heidi Palosaari  1 , Jean-Patrick Parisien, Jason J Rodriguez, Christina M Ulane, Curt M Horvath
Affiliations

STAT protein interference and suppression of cytokine signal transduction by measles virus V protein

Heidi Palosaari et al. J Virol. 2003 Jul.

Abstract

Measles virus, a paramyxovirus of the Morbillivirus genus, is responsible for an acute childhood illness that infects over 40 million people and leads to the deaths of more than 1 million people annually (C. J. Murray and A. D. Lopez, Lancet 349:1269-1276, 1997). Measles virus infection is characterized by virus-induced immune suppression that creates susceptibility to opportunistic infections. Here we demonstrate that measles virus can inhibit cytokine responses by direct interference with host STAT protein-dependent signaling systems. Expression of the measles V protein prevents alpha, beta, and gamma interferon-induced transcriptional responses. Furthermore, it can interfere with signaling by interleukin-6 and the non-receptor tyrosine kinase, v-Src. Affinity purification demonstrates that the measles V protein associates with cellular STAT1, STAT2, STAT3, and IRF9, as well as several unidentified partners. Mechanistic studies indicate that while the measles V protein does not interfere with STAT1 or STAT2 tyrosine phosphorylation, it causes a defect in IFN-induced STAT nuclear accumulation. The defective STAT nuclear redistribution is also observed in measles virus-infected cells, where some of the STAT protein is detected in cytoplasmic bodies that contain viral nucleocapsid protein and nucleic acids. Interference with STAT-inducible transcription may provide a novel intracellular mechanism for measles virus-induced cytokine inhibition that links innate immune evasion to adaptive immune suppression.

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Figures

FIG. 1.
FIG. 1.
Measles virus V protein blocks IFN signal transduction. (A) 293T cells were transfected with an ISRE-luciferase reporter gene and either empty vector or measles virus V protein expression vector as indicated. Cells were treated with 1,000 U of IFN-α per ml for 14 h prior to lysis and luciferase assays (+) or left untreated (−). (B) The same experiment was carried out using a GAS-luciferase reporter gene and treatment with 5 ng of IFN-γ per ml. (C) Murine NIH3T3 fibroblasts were subject to ISRE-luciferase assays as above, but using 1,000 U of murine IFN-β per ml.
FIG. 2.
FIG. 2.
Affinity purification of a measles virus V protein-dependent complex. (A) Silver stain of the eluate from FLAG affinity purification. Migration positions of prestained molecular mass markers and FLAG-GFP, FLAG-SV5 V (SV), or FLAG-measles virus V (MeV) are indicated on the left in kilodaltons. SV5 VIP subunits are indicated on the right, as are prominent MeVIPs (indicated by asterisks). (B) Identification of MeVIP polypeptides. Parallel samples from the experiment in panel A were processed for immunoblotting with antisera for STAT1, STAT2, STAT3, IRF9, DDB1, or Cul4A.
FIG. 3.
FIG. 3.
Measles virus V protein STAT3 interference inhibits IL-6 and v-Src transcriptional responses. (A) Measles virus V inhibits IL-6 signaling. GAS-luciferase reporter gene assays were carried out with 2fTGH cells as in Fig. 1, but the cells were transfected with empty vector or measles virus V expression vector as indicated and then treated with IL-6 (400 ng/ml) plus soluble IL-6 receptor (500 ng/ml) or left untreated, as described previously (14, 57). (B) Measles virus V inhibits v-Src signaling. The GAS-luciferase assay was carried out as in panel A, but with cotransfected v-Src expression vector as the STAT3 activator.
FIG. 4.
FIG. 4.
Effect of measles virus V protein on STAT phosphorylation and dimerization. (A) IFN-α-induced STAT1 and STAT2 activating tyrosine phosphorylation was tested by immunoblotting with STAT phosphopeptide-specific antisera in 293T and 2fTGH cell lines transfected with empty vector (−) or measles virus V cDNA (MeV). Total STAT1 and STAT2 levels and V protein expression were analyzed in parallel. The results are shown in the left panels. A similar analysis was performed with a stable 293 Tet-On cell line harboring a doxycycline (Dox)-inducible measles virus V cDNA. The results are shown in the right panels. (B) IFN-γ-induced STAT1 tyrosine phosphorylation was tested as in panel A by immunoblotting with STAT1 phosphopeptide-specific antisera. (C) Measles virus V induces constitutive STAT association. Cells transfected with empty vector (−) or measles virus V cDNA (MeV) were treated with 1,000 U of IFN-α per ml for 30 min (+) or left untreated and whole-cell lysates were immunoprecipitated (IP) with STAT2-specific antiserum (left) or directly applied to the gel (right) and then processed for immunoblotting with STAT1 antiserum or STAT1 phosphopeptide-specific antiserum.
FIG. 5.
FIG. 5.
Measles virus V protein prevents IFN-induced STAT nuclear accumulation. (A) Human 2fTGH cells were transfected with HA-tagged measles virus V cDNA and were untreated (UNT) or treated with IFN for 30 min 36 h later. The cells were stimulated with IFN-α (1,000 U/ml) to visualize STAT2 nuclear redistribution and IFN-γ (5 ng/ml) to visualize STAT1 nuclear redistribution. They were fixed, permeabilized, and stained with antisera to HA tag (red), and STAT1 or STAT2 (green). Merged images illustrate overlap in yellow; arrows point to V-expressing cells for clarity. (B) Same as in panel A, except that the cells were also treated with LMB (10 ng/ml) for 5 h.
FIG. 6.
FIG. 6.
Measles virus infection alters STAT protein distribution. (A) Human 2fTGH cells were infected with 0.1 PFU measles virus per cell. The cells were processed as for the experiment in Fig. 5 but stained for measles virus nucleocapsid protein (N; red) or STAT1 or STAT2 (green), TOTO3 was used to stain nucleic acids (blue). Arrows point to infected cells for clarity. The cells were visualized with a 40× objective; the dashedbox indicates a cell viewed at higher magnification in panel B. (B) Infected cells from panel A visualized with a 100× objective. Arrows point to regions where N and STAT1 colocalize in cytoplasmic bodies. (C) Same as in panel A, but measles virus-induced syncytia were visualized. For all panels. STAT+N indicates merged images for protein colocalization (yellow) and TOTO3+N indicates merged images for nucleic acid and N-protein colocalization (purple).
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