Phosphorylation of bluetongue virus nonstructural protein 2 is essential for formation of viral inclusion bodies

Abstract

In bluetongue virus (BTV)-infected cells, large cytoplasmic aggregates are formed, termed viral inclusion bodies (VIBs), which are believed to be the sites of viral replication and morphogenesis. The BTV nonstructural protein NS2 is the major component of VIBs. NS2 undergoes intracellular phosphorylation and possesses a strong single-stranded RNA binding activity. By changing phosphorylated amino acids to alanines and aspartates, we have mapped the phosphorylated sites of NS2 to two serine residues at positions 249 and 259. Since both of these serines are within the context of protein kinase CK2 recognition signals, we have further examined if CK2 is involved in NS2 phosphorylation by both intracellular colocalization and an in vitro phosphorylation assay. In addition, we have utilized the NS2 mutants to determine the role of phosphorylation on NS2 activities. The data obtained demonstrate that NS2 phosphorylation is not necessary either for its RNA binding properties or for its ability to interact with the viral polymerase VP1. However, phosphorylated NS2 exhibited VIB formation while unmodified NS2 failed to assemble as VIBs although smaller oligomeric forms of NS2 were readily formed. Our data reveal that NS2 phosphorylation controls VIBs formation consistent with a model in which NS2 provides the matrix for viral assembly.

FIG. 1.

Phosphorylation state analysis of NS2 and NS2 mutants expressed in mammalian cells and insect cells. (A) 293T cells were transfected with plasmids encoding NS2 and NS2 mutants in which serine residue 249 or 259 was substituted by alanine residues as indicated. The mutant NS2_2A had both serines exchanged for alanines. At 24 h (lanes 1 through 4) or 40 h (lanes 5 through 9) posttransfection, proteins were metabolically labeled with ³²P_i and immunoprecipitated from the cellular lysate using an anti-NS2 antiserum. Precipitated proteins were visualized by Western blotting (wb) and radioactive signals detected by exposure to a phosphorimaging screen. (B) Quantification of the phosphorylation state of NS2. The ³²P signals of NS2 or NS2 mutants from expression in 293T cells were quantified using ImageQuant 5.0 software and normalized to the expression level of the respective protein (Western blot signals). Each mutant was tested in three independent experiments. (C) Sf9 cells were infected with a recombinant baculovirus carrying the genes for NS2 or NS2 mutants. NS_2A contains serine residues 249 and 259 exchanged for alanines and NS2_2D, both serines substituted by aspartates. At 28 h postinfection, proteins were labeled, immunoprecipitated, and visualized as described above.

FIG. 2.

Phosphorylation of NS2 by protein kinase CK2. (A) Amino acid sequence of positions 246 through 262 of NS2 aligned to the consensus recognition sequence of protein kinase CK2. Serine residues 249 and 259 are in bold. (B) Colocalization of NS2 and CK2 in BHK-21 cells 40 h posttransfection. NS2 was visualized using an FITC-conjugated antibody and protein kinase CK2 with a TRITC-conjugated antibody. (C) S-tagged NS2 (lanes 1 and 2) or S-tagged NS2 mutants (lanes 3 through 5) synthesized by in vitro transcription/translation were incubated with purified CK2 and [γ-³²P]ATP, pulled down using S-agarose protein beads, separated on an SDS-PAGE gel, and visualized by autoradiography. Myricetin was added to inhibit CK2 activity when indicated at 1 μM.

FIG. 3.

Influence of NS2 phosphorylation on its RNA binding properties. (A) Coomassie blue-stained SDS-10% PAGE comparison of NS2 and NS2 mutants expressed in Sf9 cells and purified by column chromatography. (B through D) Electrophoretic mobility shift assays of NS2 or NS2 mutants bound to ³²P-labeled transcripts (8 nM) of BTV segment S8 (1,125 nt) and in the presence of increasing amounts (80 pM to 800 nM) of unlabeled competitor, either BTV S8 (panel B, lanes 3 through 7 and 9 through 13, and panel D, lanes 3 through 7) or pGEM3zf (+)-derived RNA (1,165 nt) (panel C, lanes 3 through 7 and 9 through 13, and panel D, lanes 8 through 12). Competitors were used in 10-fold increments (left to right). (B, C, and D) Lane 1, ³²P-labeled transcript only; all other lanes, ³²P-labeled transcript and NS2 or NS2 mutants. NS2 was used in panels B and C, lanes 2 through 7; NS2_2A was used in panels B and C, lanes 8 through 13; and NS2_2D was used in panel D, lanes 2 through 12.

FIG. 4.

Interaction of NS2 with BTV core proteins. (A) Colocalization of NS2 with VP1, VP4, and VP6 in transfected HeLa cells at 40 h posttransfection. VP1, VP4, and VP6 were visualized by using a TRITC-conjugated antibody and NS2 by using an FITC-conjugated antibody. (B) Coimmunoprecipitation of VP1_his with NS2 or NS2 mutants. Proteins were subjected to SDS-10% PAGE and detected by Coomassie blue staining (left panel) or by Western blotting (right panel) using an antipolyhistidine antibody. In a control reaction, no NS2 (−) or NS2 mutant was added in the assay. Numbers on the right are in kilodaltons.

FIG. 5.

Immunoprecipitation of NS2 complexes. (A) 293T cells were transfected with plasmids encoding NS2 and NS2f and labeled with [³⁵S]methionine. Proteins were immunoprecipitated with an anti-NS2 serum (lanes 1 through 4) or an anti-FLAG antibody (lanes 5 through 12), separated by SDS-PAGE, and visualized by phosphorimager analysis. RNase A was omitted during immunoprecipitation in lanes 1 through 8 and used in lanes 9 through 12. Transfections of plasmids encoding NS2 (lanes 1, 5, and 9), NS2 plus NS2f (lanes 2, 6, and 10), or NS2_2A plus NS2_2Af (lanes 3, 7, and 11) or without any insertion (lanes 4, 8, and 12) are shown. (B) Quantification of ³⁵S signals of NS2 and NS2 mutants from lanes 6, 7, 10, and 11 of panel A. Signals were quantified using ImageQuant 5.0 software.

FIG. 6.

The formation of inclusion bodies by NS2 is impaired when NS2 is not phosphorylated. (A) BHK-21 cells were transfected with plasmids encoding NS2 or NS2 mutants as indicated or infected with BTV-10. Expressed recombinant NS2 and NS2 mutants were visualized using an FITC-conjugated antibody (green). For BTV-10, cells were infected with BTV-10 and NS2 was visualized using a TRITC-conjugated antibody (red). BHK-21 cells were cotransfected with plasmids encoding Flag-tagged NS2 and untagged NS2 (B) or Flag-tagged NS2_2A and untagged NS2 (C). BHK-21 cells were transfected with Flag-tagged NS2 (D) or Flag-tagged NS2_2A (E). (D and E) At 24 h posttransfection, cells were infected with BTV-10. (B through E) Flag-tagged NS2 was visualized using an FITC-conjugated antibody (green). NS2 or NS2 mutants were visualized using a TRITC-conjugated antibody (red).