N-terminal phosphorylation of phosphoprotein of vesicular stomatitis virus is required for preventing nucleoprotein from binding to cellular RNAs and for functional template formation

Abstract

The phosphoprotein (P) of vesicular stomatitis virus (VSV) plays essential roles in viral RNA synthesis. It associates with nascent nucleoprotein (N) to form N(0)-P (free of RNAs), thereby preventing the N from binding to cellular RNAs and maintaining the N in a viral genomic RNA encapsidation-competent form for transcription and replication. The contributions of phosphorylation of P to transcription and replication have been studied intensively, but a concrete mechanism of action still remains unclear. In this study, using a VSV minigenome system, we demonstrated that a mutant of P lacking N-terminal phosphorylation (P3A), in which the N-terminal phosphate acceptor sites are replaced with alanines (S60/A, T62/A, and S64/A), does not support transcription and replication. However, results from protein interaction assays showed that P3A self-associates and interacts with N and the large protein (L) as efficiently as P does. Furthermore, purified recombinant P3A from Sf21 cells supported transcription in an in vitro transcription reconstitution assay. We also proved that P3A is not distributed intranuclearly in vivo. CsCl gradient centrifugation showed that P3A is incapable of preventing N from binding to cellular RNAs and therefore prevents functional template formation. Taken together, our results demonstrate that N-terminal phosphorylation is indispensable for P to prevent N from binding to nonviral RNAs and to maintain the N-specific encapsidation of viral genomic RNA for functional template formation.

Fig 1

N-terminal phosphorylation of P is essential for VSV minigenome transcription in vivo. (A) Schematic diagram of minigenome and replication-defective minigenome (ΔTr) systems. The negative-strand minigenome or ΔTr minigenome could be generated from transcription driven by T7 RNA polymerase in cells. Of the two, the former was encapsidated for transcription into the reporter gene mRNA, resulting in expression of CAT and its use as a template for replication by a complex of N, P, and L, but the latter was encapsidated and transcribed for reporter gene expression only. (B) BHK cells expressing T7 polymerase were transfected with plasmids encoding N, L, minigenome RNA, and P or P3A. Relative CAT expression levels in lysates were measured via CAT ELISA. The relative CAT expression level of cells transfected with a plasmid encoding P, as a positive control, was defined as 100%. (C) BHK cells expressing T7 polymerase were transfected with increasing quantities of plasmids encoding P or P3A together with the indicated plasmids in the minigenome system. Relative CAT expression levels were measured via CAT ELISA, and expression of P and P3A was detected via Western blot analysis (WB) with anti-HA antibody. (D and E) Transfection was performed as described for panel B. Relative CAT antigenomic RNA levels and mRNA levels were analyzed via RT-PCR, using β-actin as a reference gene. (F) BHK cells expressing T7 polymerase were transfected with plasmids encoding N, P, L, and the ΔTr minigenome, and relative CAT expression levels were measured as described for panel C. The CAT expression level of cells transfected with the minigenome, as a positive control, was defined as 100%. (G) The ΔTr minigenome is supported by P3A. The assay described for panel B was performed, but with the minigenome replaced with the ΔTr minigenome.

Fig 2

Self-association of P3A and interaction of P3A with N or L. (A) Domain structures of P and P3A. P structures with three functionally defined domains (I, II, and III) and a hinge region are shown. The phosphate acceptor sites S60, T62, and S64 and their respective mutations to alanine (A60, A62, and A64) are indicated with solid circles. (B) Interaction of P3A with P and itself in a yeast two-hybrid assay. A yeast mating assay was performed between yeast AH109 strains (type a) expressing BD and BD-baits and Y187 strains (type α) expressing AD and AD-preys, as indicated. Experiments were repeated at least three times for accuracy. (C) Myc-tagged P or P3A and HA-tagged P or P3A were coexpressed in HeLa cells as indicated. (Left) Protein expression was detected via Western blot analysis using anti-Myc and anti-HA monoclonal Abs. (Right) Immunoprecipitation (IP) was performed using anti-Myc polyclonal Ab, and immune complexes were detected with anti-Myc and anti-HA monoclonal Abs. (D) Cell lysates of E. coli BL21 expressing GST, GST-P, or GST-P3A were mixed with His-P purified from Sf21 cells to allow their direct interaction. GST pulldown assays were performed. Input (left) and bound (right) proteins were assayed via Western blotting using anti-GST and anti-His Abs. (E) Myc-tagged N and HA-tagged P or P3A were coexpressed in HeLa cells as indicated. Protein expression (left) and immunoprecipitation (right) were detected as described for panel C. (F) Flag-tagged L and Myc-tagged P or P3A were coexpressed in HeLa cells as indicated. (Left) Protein expression was detected via Western blot analysis using anti-Flag and anti-Myc monoclonal Abs. (Right) Immunoprecipitation was performed using anti-L polyclonal Ab, and immune complexes were detected using anti-Flag and anti-Myc monoclonal Abs.

Fig 3

Transcription activity of P3A in vitro. (A) Recombinant P or P3A and L were subjected to in vitro transcription with the N-RNA template as described in Materials and Methods. [α-³²P]GTP-labeled transcripts were analyzed via urea–5% PAGE followed by autoradiography. The positions of the viral mRNAs are indicated on the right. (B) Relative transcription activities (percentages) of P and P3A in transcription reactions. The relative transcription activity of P was defined as 100%. All assays were repeated at least three times for accuracy.

Fig 4

Localization of P3A within cells. (A) HA-tagged P and P3A were expressed in BHK cells. Nuclear and cytoplasmic extracts were analyzed via Western blotting using anti-HA antibody. GAPDH was used as a reference for cytoplasmic extracts. (B) RFP-fused P or P3A was expressed in HeLa cells as described in Materials and Methods. Localization of P and P3A within the cells was detected via immunofluorescence microscopy. The nucleus was stained with DAPI.

Fig 5

Ability of N binding of cellular RNAs and viral genomic RNA in the presence of P and P3A. (A and B) P3A is incapable of preventing N from binding to cellular RNAs. BHK cells were transfected with plasmids encoding N alone or N plus P or P3A. (A) Lysates from transfected cells were centrifuged, and an aliquot of 10 μl of supernatant was analyzed via Western blotting to detect N, P, and P3A expression. The remaining supernatant was analyzed via CsCl gradient centrifugation as described in Materials and Methods. After centrifugation, the pellet was used for N detection (A) and further purification of RNAs (B). The RNAs or RNAs treated with RNase A or DNase I were analyzed in a 1% agarose gel. (C and D) Assay performed as described for panels A and B, but with the addition of minigenome expression for all transfection combinations. CAT antigenomic RNA and GAPDH and β-actin RNAs were amplified via RT-PCR, using RNAs in the pellet as a template. (E and F) The assay described for panels C and D was performed, but the minigenome was replaced with the ΔLe-Tr minigenome.

Fig 6

Proposed model of the role of N-terminal phosphorylation of P in facilitating viral RNA-specific encapsidation by N and functional template formation. P prevents N not only from aggregating but also from binding to cellular RNAs. Instead, the N⁰-P complex forms and maintains N-specific encapsidation of viral genomic RNA. P3A prevents N from aggregating but is unable to prevent it from binding to cellular RNAs. Alternatively, N binds to any RNAs in the presence of P3A, irrespective of sequence specificity (see the text for details).