Interaction of vesicular stomatitis virus P and N proteins: identification of two overlapping domains at the N terminus of P that are involved in N0-P complex formation and encapsidation of viral genome RNA

Abstract

The nucleocapsid (N) protein of nonsegmented negative-strand (NNS) RNA viruses, when expressed in eukaryotic cells, aggregates and forms nucleocapsid-like complexes with cellular RNAs. The phosphoprotein (P) has been shown to prevent such aggregation by forming a soluble complex with the N protein free from cellular RNAs (designated N(0)). The N(0)-P complex presumably mediates specific encapsidation of the viral genome RNA. The precise mechanism by which the P protein carries out this function remains unclear. Here, by using a series of deleted and truncated mutant forms of the P protein of vesicular stomatitis virus (VSV), Indiana serotype, we present evidence that the N-terminal 11 to 30 amino acids (aa) of the P protein are essential in keeping the N protein soluble. Furthermore, glutathione S-transferase fused to the N-terminal 40 aa by itself is able to form the N(0)-P complex. Interestingly, the N-terminal 40-aa stretch failed to interact with the viral genome N-RNA template whereas the C-terminal 72 aa of the P protein interacted specifically with the latter. With an in vivo VSV minigenome transcription system, we further show that a deletion mutant form of P (PDelta1-10) lacking the N-terminal 10 aa which is capable of forming the N(0)-P complex was unable to support VSV minigenome transcription, although it efficiently supported transcription in vitro in a transcription-reconstitution reaction when used as purified protein. However, the same mutant protein complemented minigenome transcription when expressed together with a transcription-defective P deletion mutant protein containing N-terminal aa 1 to 210 (PDeltaII+III). Since the minigenome RNA needs to be encapsidated before transcription ensues, it seems that the entire N-terminal 210 aa are required for efficient genome RNA encapsidation. Taking these results together, we conclude that the N-terminal 11 to 30 aa are required for N(0)-P complex formation but the N-terminal 210 aa are required for genome RNA encapsidation.

FIG. 1.

Schematic representation of the wild-type P protein and the mutant P proteins used in this study. The wild-type P protein is divided into four regions designated I (aa 1 to 137), Hinge (aa 138 to 210), II (aa 211 to 244), and III (aa 245 to 265). The letters S at position 60, T at position 62, S at position 64, S at position 226, and S at position 227 represent the phosphorylation sites of the P protein. Wild-type P and the mutant P proteins were fused with an HA tag. The numbers represent amino acid positions.

FIG. 2.

Amino acids 11 to 30 of the P protein are essential to keep N protein soluble. Myc-tagged N protein and HA-tagged P protein or mutant P proteins were coexpressed in HeLa cells as indicated. Supernatants (sup) from centrifugation at 13,000 rpm (A, B, C, and D) were detected by WB with anti-Myc, anti-HA, and anti-GAPDH monoclonal Abs. GAPDH was used as a loading control. (A) The pellet (ppt) was also detected by WB with an anti-Myc monoclonal Ab.

FIG. 3.

The N-terminal 40 aa of the P protein are sufficient to keep N protein soluble. (A) Schematic representation of the N-terminal 40 and 60 aa and the C-terminal 72 aa fused with GST and an HA tag. Numbers represent amino acid positions. (B) Myc-tagged N and HA-tagged GST, GST-1-40 (PN40), GST-1-60 (PN60), or GST-192-265 (PC72) were coexpressed in HeLa cells as indicated. Supernatants (sup) from centrifugation at 13,000 rpm were detected by WB with anti-Myc, anti-HA, and anti-GAPDH monoclonal Abs. GAPDH was used as a loading control. (C) PN40 binds to N⁰, and PC72 binds to the N-RNA complex. Myc-tagged N protein was expressed alone or coexpressed with increasing concentrations of PN40 or PC72. Supernatants from centrifugation at 13,000 rpm were further centrifuged at 35,000 rpm (see Materials and Methods). N⁰ and the N-RNA complex were detected in U-sup or U-ppt by WB with anti-Myc Ab.

FIG. 4.

The N-terminal 40-aa region is unable to bind to the N-RNA template in vitro. Purified RNP, N-RNA, and L-P complexes were analyzed by SDS-PAGE, followed by Coomassie brilliant blue (CBB) staining (A), and detected by WB with an anti-N polyclonal Ab (B). (C) PC72 interacted with the N-RNA template in vitro, but PN40 did not. Equal amounts of HeLa cell lysates expressing GST, PN40, or PC72 were detected by WB with anti-HA Ab (left side), and the same lysates were mixed with 200 ng of the purified N-RNA template in GST pull-down assays. Eluted GST beads were assayed by WB with anti-GST and anti-N Abs.

FIG. 5.

PΔ1-10, PΔ11-20, PΔ21-30, and PΔ31-40 all interact with L protein in vivo. HA-tagged wild-type P protein or mutant P proteins were coexpressed with Flag-tagged L protein (Flag-L) in HeLa cells as indicated. Protein expression was detected in lysates by WB with anti-HA and anti-Flag Abs (B). Immunoprecipitation (IP) was performed with anti-L polyclonal Ab, and immune complexes were detected by WB with anti-HA and anti-Flag Abs (A).

FIG. 6.

The N-terminal 40-aa region is required for encapsidation of viral genome RNA. (A) Purified, recombinant, His-tagged wild-type P protein and mutant P proteins were analyzed by SDS-PAGE, followed by Coomassie brilliant blue (CBB) staining, and by WB with anti-His and anti-P Abs. (B) In vitro transcription reconstituted with an N-RNA template and recombinant P protein or mutant P proteins and L protein. The recombinant P protein or mutant P proteins and L protein were subjected to in vitro transcription with the N-RNA template. ³²P-labeled transcripts were analyzed by urea-5% PAGE, followed by autoradiography. The positions of the viral mRNAs are indicated on the right. (C) The relative transcription activities (percent) of wild-type P protein and mutant P proteins in transcription reactions are indicated. The relative transcription activity of wild-type P protein was defined as 100%. (D) Roles of wild-type P protein and mutant P proteins in VSV minigenome transcription in vivo. BHK cells expressing T7 polymerase were transfected with pBS-N, pBS-L, pVSV-CAT2, and a plasmid encoding HA-tagged wild-type P protein or mutant P proteins as indicated. A CAT ELISA was performed with lysates of the transfected cells to measure relative CAT expression levels as viral minigenome transcription activities. The relative CAT expression level of the wild-type P protein was defined as 100%. The expression levels of the wild-type P protein and mutant P proteins were detected by WB with an anti-HA monoclonal Ab.

FIG. 7.

Complementation assay of the ability of mutant proteins to support the transcription of the VSV minigenome in vivo. BHK cells expressing T7 polymerase were transfected with pBS-N, pBS-L, pVSV-CAT2, and a plasmid encoding the wild-type P protein or mutant P proteins as indicated. (A) A CAT ELISA was performed with lysates of transfected cells to measure relative CAT expression levels as viral minigenome transcription activities. The relative CAT expression level of the wild-type P protein was defined as 100%. (B) Expression levels of HA-tagged mutant proteins and the Myc-tagged P protein and PΔ1-10 were detected by WB with anti-HA and anti-Myc monoclonal Abs, respectively.