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. 2013 Sep;87(17):9633-42.
doi: 10.1128/JVI.00714-13. Epub 2013 Jun 26.

Measles virus nonstructural C protein modulates viral RNA polymerase activity by interacting with host protein SHCBP1

Minako Ito  1 , Masaharu Iwasaki, Makoto Takeda, Takanori Nakamura, Yusuke Yanagi, Shinji Ohno
Affiliations

Measles virus nonstructural C protein modulates viral RNA polymerase activity by interacting with host protein SHCBP1

Minako Ito et al. J Virol. 2013 Sep.

Abstract

Most viruses possess strategies to circumvent host immune responses. The measles virus (MV) nonstructural C protein suppresses the interferon response, thereby allowing efficient viral growth, but its detailed mechanism has been unknown. We identified Shc Src homology 2 domain-binding protein 1 (SHCBP1) as one of the host proteins interacting with the C protein. Knockdown of SHCBP1 using a short-hairpin RNA greatly reduced MV growth. SHCBP1 was found to be required for viral RNA synthesis in the minigenome assay and to bind to the MV phosphoprotein, a subunit of the viral RNA polymerase. A stretch of 12 amino acid residues in the C protein were sufficient for SHCBP1 binding, and the peptide containing these 12 residues could suppress MV RNA synthesis, like the full-length C protein. The central region of SHCBP1 was found to bind to the C protein, as well as the phosphoprotein, but the two viral proteins did not compete for SHCBP1 binding. Our results indicate that the C protein modulates MV RNA polymerase activity by binding to the host protein SHCBP1. SHCBP1 may be exploited as a target of antiviral compounds.

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Figures

Fig 1
Fig 1
Effect of SHCBP1 knockdown on MV growth. (A) A549/hSLAM cells expressing shRNA against individual target genes or EGFP gene were infected with IC323-Luci. At 48 h postinfection (p.i.), intracellular luciferase activity was measured. The average value for cells expressing shEGFP was set to 100%, and relative values are indicated. (B) A549/hSLAM cells were infected with IC323-Luci (MV) at an MOI of 0.05 or stimulated with 2 μg of poly(I·C)/ml for 24 h (left panel) or treated with 1,000 IU of IFN-αA/D/ml for the indicated times (right panel). The expression of SHCBP1 was examined by Western blotting. (C) A549/hSLAM cells expressing shSHCBP1-1, shSHCBP1-2, or shEGFP were stimulated with poly(I·C) for 24 h, and the SHCBP1 expression was examined. (D) Growth of MV in SHCBP1- or EGFP-knockdown cells. A549/hSLAM cells expressing shSHCBP1-1, shSHCBP1-2, or shEGFP were infected with IC323-Luci at an MOI of 0.05. At various times p.i., the intracellular luciferase activity was measured. (E) Cells were infected with IC323-Luci as for panel D. At 48 h p.i., the cells were scraped into culture medium, and virus titers were determined on Vero/hSLAM cells. (F) Viability of shRNA-expressing A549/hSLAM cells. After puromycin selection, the cells were replated on 96-well plate, and the cell viability was determined at 6 and 48 h. The data were indicated as the value at 48 h relative to that at 6 h. The relative value in shEGFP-expressing cells was set to 100%. (G) shRNA-expressing A549/hSLAM cells were infected with EMCV and HSV-1, and the virus titers at 24 and 48 h p.i., respectively, were determined. The data indicate means ± the standard deviations (SD) (A, D, E, F, and G). *, P < 0.05; **, P < 0.01; ***, P < 0.001. All of the results in this and other figures are representative of at least two independent experiments.
Fig 2
Fig 2
Role of SHCBP1 in MV RNA synthesis. (A) Effect of SHCBP1 knockdown on MV RNA synthesis. A minigenome assay was performed in shRNA-expressing A549/hSLAM cells. The reporter activities were determined 48 h after transfection. The minigenome and support plasmids were supplemented with empty vector or the expression plasmid encoding the C protein. L(−), the plasmid encoding the L protein, was omitted. (B) Intracellular localization of proteins. The upper panel shows VV5-4 cells expressing Flag-tagged SHCBP1 (green). The middle panels show VV5-4 cells expressing the minigenome, the N, P (red), and L proteins, and Flag-tagged SHCBP1 (green). The lower panels show VV5-4 cells expressing the minigenome, the N, P, and L proteins, Flag-tagged SHCBP1 (green), and the C protein (red). (C) Interaction of SHCBP1 with the RNP complex. For the left panel, a coimmunoprecipitation (IP) assay was performed with lysates from HEK293T cells expressing the MV P protein with or without Flag-tagged SHCBP1. At 48 h after transfection, cell lysates were prepared, and proteins were precipitated with anti-Flag antibody and examined by Western blotting. For the right panel, a coimmunoprecipitation (IP) assay with lysates from cells expressing the MV N protein and HA-tagged-SHCBP1 or P protein was performed. Proteins were precipitated with anti-HA antibody. (D) A coimmunoprecipitation (IP) assay was performed with lysates from MV-infected A549/SLAM cells. Protein complexes were precipitated with anti-P/V antibody.
Fig 3
Fig 3
The interaction of the C protein with SHCBP1. (A) A coimmunoprecipitation (IP) assay was carried out with lysates from HEK293T cells expressing indicated combinations of Flag-tagged SHCBP1 and His-tagged wild type (wt) or mutant C protein. At 48 h after transfection, protein complexes were precipitated with anti-Flag antibody and examined by Western blotting. (B) Diagram of His-tagged wild-type (wt) and mutant C proteins. (C) Lysates from HEK 293T cells expressing indicated combinations of GFP-fused proteins and HA-tagged SHCBP1 were used for an immunoprecipitation assay. Proteins were precipitated with anti-HA antibody and examined by Western blotting. (D) Expression plasmids encoding the indicated proteins were included in the minigenome assay. Triangles below the lanes indicate increasing concentrations of the plasmids transfected (12.5, 50, and 200 ng). The average value in the sample expressing GFP was set to 100%. The data indicate means ± the SD. ***, P < 0.001. (E) Intracellular localization of CΔ127-138 and P proteins. A549/hSLAM cells were transfected with expression plasmids encoding P, N, and C proteins. At 48 h after transfection, the C (red) and P proteins (green) were immunostained.
Fig 4
Fig 4
Interaction of the central region of SHCBP1 with the P and C proteins. (A) Diagram of Flag-tagged SHCBP1 and its truncated mutants. (B) Cell lysates containing indicated Flag-tagged SHCBP1 constructs and the P (left panel) or C protein (right panel) were precipitated with anti-Flag antibody and examined by Western blotting. (C) Competition for SHCBP1 binding between the C and P proteins was examined. The indicated amounts (μg) of plasmids encoding Flag-tagged SHCBP1, HA-tagged P protein, and His-tagged C protein were introduced into HEK293T cells. The total amounts of transfected DNA were kept constant by using pCA7-EGFP. At 48 h after transfection, proteins were precipitated with anti-Flag antibody and examined by Western blotting. (D) SHCBP1 and its truncated mutants were included in the minigenome assay. The average value in the sample expressing GFP was set to 100%. The data indicate means ± the SD. ***, P < 0.001. (E) Multimer formation of SHCBP1. A coimmunoprecipitation assay was performed with lysates containing indicated HA- and Flag-tagged proteins. Proteins were precipitated with anti-HA antibody and examined by Western blotting. (F) A coimmunoprecipitation (IP) assay was performed with lysates containing indicated HA- and Flag-tagged SHCBP1 and MV C protein. Proteins were precipitated with anti-HA antibody and examined by Western blotting.
Fig 5
Fig 5
Model for the role of SHCBP1 in MV RNA synthesis and its modulation by the C protein.
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