Identification and characterization of the 480-kilodalton template-specific RNA-dependent RNA polymerase complex of red clover necrotic mosaic virus

Abstract

Replication of positive-strand RNA viruses occurs through the assembly of membrane-associated viral RNA replication complexes that include viral replicase proteins, viral RNA templates, and host proteins. Red clover necrotic mosaic virus (RCNMV) is a positive-strand RNA plant virus with a genome consisting of RNA1 and RNA2. The two proteins encoded by RNA1, a 27-kDa protein (p27) and an 88-kDa protein containing an RNA-dependent RNA polymerase (RdRP) motif (p88), are essential for RCNMV RNA replication. To analyze RCNMV RNA replication complexes, we used blue-native polyacrylamide gel electrophoresis (BN/PAGE), which enabled us to analyze detergent-solubilized large membrane protein complexes. p27 and p88 formed a complex of 480 kDa in RCNMV-infected plants. As a result of sucrose gradient sedimentation, the 480-kDa complex cofractionated with both endogenous template-bound and exogenous template-dependent RdRP activities. The amount of the 480-kDa complex corresponded to the activity of exogenous template-dependent RdRP, which produced RNA fragments by specifically recognizing the 3'-terminal core promoter sequences of RCNMV RNAs, but did not correspond to the activity of endogenous template-bound RdRP, which produced genome-sized RNAs without the addition of RNA templates. These results suggest that the 480-kDa complex contributes to template-dependent RdRP activities. We subjected those RdRP complexes to affinity purification and analyzed their components using two-dimensional BN/sodium dodecyl sulfate-PAGE (BN/SDS-PAGE) and mass spectrometry. The 480-kDa complex contained p27, p88, and possible host proteins, and the original affinity-purified RdRP preparation contained HSP70, HSP90, and several ribosomal proteins that were not detected in the 480-kDa complex. A model for the formation of RCNMV RNA replication complexes is proposed.

FIG. 1.

Interaction of p27 with both p27 and p88. (A) Coimmunoprecipitation analysis of the interaction between p27-HA and p27-FLAG. Protein extracts from Agrobacterium-infiltrated leaves expressing viral proteins were subjected to immunoprecipitation with anti-HA antibody (IP:αHA) or anti-FLAG antibody (IP:αFLAG), followed by Western blotting using anti-HA antibody (WB:αHA) and anti-FLAG antibody (WB:αFLAG). (B) Coimmunoprecipitation analysis of the interaction between p27-FLAG and p88-T7. Protein extracts from Agrobacterium-infiltrated leaves expressing viral proteins and RNA2 were subjected to immunoprecipitation with anti-FLAG antibody (IP:αFLAG) or anti-T7 antibody (IP:αT7), followed by Western blotting using anti-FLAG antibody (WB:αFLAG) and anti-T7 antibody (WB:αT7).

FIG. 2.

RCNMV replicase proteins form two different patterns of high-molecular-mass complexes. Membrane fractions were prepared from Agrobacterium-infiltrated leaves expressing RCNMV RNAs (A), from Agrobacterium-infiltrated leaves expressing various combinations of p27-FLAG, p88-T7, and RNA2 (B), from BYL incubated with RNA1 derivatives (C), or from BYL incubated with various combinations of p27 mRNA, p88 mRNA, RNA2, RAN2-dSLF, or uncapped or capped Luc and RLuc mRNAs (D). All membrane fractions were solubilized with Triton X-100 and subjected to BN/PAGE (upper panel) and SDS-PAGE (lower panel). Western blots for BN/PAGE and SDS-PAGE were performed using appropriate antibodies. Coomassie brilliant blue (CBB)-stained cellular proteins on SDS-PAGE gels are shown below the Western blots as a loading control (Prot).

FIG. 3.

Effects of membrane washes on association of p27 and p88 and their complexes with the ER. (A) Membrane fractions prepared from Agrobacterium-infiltrated leaves expressing RNA1 plus RNA2 were incubated on ice for 30 min with 1 M KCl, 0.1 M Na₂CO₃ (pH 10.5), or 4 M urea. After incubation, samples were centrifuged to obtain the supernatant (lanes S) and the pellet (lanes P) fractions. Both fractions were subjected to SDS-PAGE, followed by Western blotting with anti-p27 antisera and anti-BiP antibody. CBB-stained cellular proteins are shown below the Western blots as a loading control. (B) The pellet fractions were solubilized with 0.5% Triton X-100 and subjected to BN/PAGE, followed by Western blotting with anti-p27 antisera.

FIG. 4.

RdRP activity of the solubilized membrane fractions prepared from Agrobacterium-infiltrated leaves expressing RNA1 plus RNA2. The RdRP assays were performed without RNA templates (lanes 1 and 2) and with RCNMV RNA1 and -2 (lanes 3 to 6). The RdRP products represented in lanes 5 and 6 were treated with S1 nuclease.

FIG. 5.

Sucrose gradient sedimentation analysis of RCNMV replicase proteins, the 380-kDa and the 480-kDa complexes, and the assay for their RdRP activity. (A and B) The solubilized membrane fractions prepared from Agrobacterium-infiltrated leaves expressing RNA1 plus RNA2 were loaded onto 5 to 40% continuous sucrose gradients and centrifuged at 170,000 × g for 16 h at 4°C. Twelve fractions (numbered 1 to 12 from the top to the bottom of the mixture) were collected and subjected to BN/PAGE (A) and SDS-PAGE (B), followed by Western blotting with anti-p27 antisera. (C and D) The RdRP activity of each fraction was assayed with or without RCNMV RNA templates.

FIG. 6.

The RdRP activity of the 480-kDa complex exhibits high template specificity. The RdRP activity of fraction 6 of the sucrose gradients was assayed without templates (lanes 1 and 3), with RCNMV RNA1 and -2 (lanes 2 and 4), with RCNMV RNA1-dSLF and RNA2-dSLF (lane 5), with BMV RNA1, -2, and -3 (lane 6), and with ToMV RNA (lane 7). The RdRP products represented in lanes 3 to 7 were treated with S1 nuclease before electrophoresis.

FIG. 7.

Identification of the components of the 480-kDa complex. The solubilized membrane fractions prepared from Agrobacterium-infiltrated leaves expressing p27 plus p88-T7 plus RNA2 or p27-FLAG plus p88-T7 plus RNA2 were subjected to affinity purification with anti-FLAG antibody-conjugated agarose beads. (A) The affinity-purified fractions were subjected to BN/PAGE and SDS-PAGE, followed by Western blotting with anti-p27 antisera. (B) The RdRP activity of the affinity-purified fractions was assayed in the absence or presence of RCNMV RNA templates. (C) The affinity-purified fraction prepared from Agrobacterium-infiltrated leaves expressing p27-FLAG plus p88-T7 plus RNA2 was subjected to two-dimensional BN/SDS-PAGE, followed by Western blotting with anti-T7 and anti-FLAG antibodies.

FIG. 8.

Identification of proteins copurified with RCNMV RdRP complexes. The solubilized membrane fractions prepared from Agrobacterium-infiltrated leaves expressing p27 plus RNA1-p88 plus RNA2 or p27-FLAG plus RNA1-p88 plus RNA2 were subjected to affinity purification with anti-FLAG antibody-conjugated agarose beads. (A) The affinity-purified fractions were subjected to BN/PAGE and stained with MS-compatible silver staining. A protein band corresponding to the 480-kDa complex was excised, subjected to in-gel digestion, and analyzed by tandem mass spectrometry. Proteins that showed Mascot search scores above 30 and that were absent in control protein bands are indicated on the right side of the panel. (B) The affinity-purified fractions were subjected to SDS-PAGE and stained using MS-compatible silver staining. Protein bands of interests were excised, subjected to in-gel digestion, and analyzed by tandem mass spectrometry. Proteins that showed Mascot search scores above 50 and that were absent in control protein bands, and proteins that showed significantly higher scores than control proteins, are indicated on the right sides of the panels. The NCBI accession numbers of the identified proteins are also indicated. Rep, RCNMV replicase proteins.