Article Figures & Data
Figures
- FIG. 1.
Purification of M2-1 from E. coli and analysis of the presence of nucleic acids. (A and B) Agarose gel electrophoresis analysis of nucleic acids present in the GST-M2-1 complexes (A) and SDS-PAGE analysis of GST-M2-1 complexes from the same samples (B). GST-M2-1 fusion proteins from standard bacterial lysates containing 150 mM NaCl were purified by using glutathione-Sepharose beads, resuspended in an equal volume of PBS, and then treated with RNase A, RQ1 DNase, or both or washed with 1 M NaCl, as indicated. Lane M, molecular mass markers. (C) SDS-PAGE analysis of M2-1 purified from standard bacterial lysates (lanes 2, 5, and 7) or high-salt lysates (lanes 3, 6, and 8). Proteins were cleaved by thrombin, and the pellets (lanes 5 and 6) or soluble fractions (lanes 7 and 8) were analyzed in parallel with uncleaved GST-M2-1 adsorbed on glutathione-Sepharose beads (lanes 2 and 3; 1 μl of beads per lane). Lanes 1 and 4, molecular mass markers. (D) UV spectra of tRNA and M2-1 purified from standard (150 mM NaCl) or high-salt (1 M NaCl) bacterial lysates, as indicated.
- FIG. 2.
Far-UV CD spectra of wild-type (wt) M2-1 and the deletion mutants M2-1Δ31N, M2-1Δ58N, and M2-1Δ35-58. The results are expressed as degrees per square centimeter per decamole (deg.cm2.dmol−1).
- FIG. 3.
M2-1 forms oligomers in solution. (A) DLS analysis. Purified M2-1 (solid line) and M2-1Δ35-58 (dashed line) in filtered PBS at 20°C were subjected to DLS with a Malverne Zetasizer Nano series instrument. The results are presented as volume-based particle size distributions. M2-1 is shown to exist as a monomodal, monodisperse protein with a DH of approximately 7.25 nm. M2-1Δ35-58 is also monodisperse, with a DH of ∼5 nm. d. nm, diameter in nanometers. (B) M2-1 cross-linking. Samples of 2 μg of purified recombinant M2-1 free of RNA were subjected to cross-linking with increasing concentrations of EGS as indicated below each lane. The reaction products were resolved on an SDS-4 to 12% gradient PAGE gel and stained with Coomassie blue. The molecular mass markers are indicated. 1X, monomers; 2X, dimers; 3X, trimers; 4X, tetramers.
- FIG. 4.
Analytical ultracentrifugation of M2-1. (A) Sedimentation velocity analysis of RNA-free M2-1 in PBS. The positions of the moving boundaries shown were recorded at intervals of 5 min by spectrometric scanning at 280 nm. The continuous lines are best fits of the experimental data (points) analyzed by the Lamm equation using SEDFIT. The rotor speed was 45,000 rpm, and the temperature was 15°C. OD, optical density. (B) The calculated c(s) distribution was plotted as a function of the sedimentation coefficient and reveals the presence of a major species with an s value of 4.6 (at 15°C in 1× PBS-1 mM DTT) that corresponds to an s20,w value of 5.4 (at 20°C in H2O).
- FIG. 5.
Electron microscopy image of M2-1. Original magnification, ×35,000. Bar, 50 nm.
- FIG. 6.
Analysis of M2-1 oligomerization and mapping of the M2-1 oligomerization domain. (A) Schematic representation of the deletion mutants of the GST-M2-1 fusion protein that were used to map the M2-1 oligomerization domain by coexpression with M2-1 in bacteria and sequence of the putative M2-1 ZnF motif. The column to the right summarizes the interactions between GST-M2-1 mutants and His-M2-1 analyzed as depicted in panel B. (B) Coomassie blue-stained SDS-12% polyacrylamide gel with purified GST-M2-1 deletion mutants expressed alone (−) or coexpressed with M2-1 (+) in E. coli. The arrowhead points to the His-M2-1 band. Lane M, molecular mass markers. (C) M2-1Δ35-58 cross-linking. Two micrograms of purified M2-1Δ35-58 free of RNA was subjected to cross-linking and analyzed as described in the legend to Fig. 3. The upper band corresponds to M2-1Δ35-58, and the faster-migrating band indicated by a star results from an N-terminal cleavage of the M2-1Δ35-58 protein.
- FIG. 7.
The domain of residues 59 to 177 is sufficient for binding to P and RNA in a competitive manner. (A) Binding of different RNAs to GST-M2-1. GST-M2-1 complexes bound to glutathione-Sepharose beads (100 μg) were incubated with 100 μg of RNA transcribed from pCDNA3 (lane 1), pET-P (lane 2), or a leader-NS1 sequence (lane 3) or 100 μg of yeast tRNA (lane 4) for 1 h at room temperature. After extensive washing, GST-M2-1-RNA complexes were analyzed by agarose gel electrophoresis by double staining with EtBr (top) and amido black (bottom) to reveal RNAs and proteins, respectively. (B) Schematic representation of the deletion mutants of the GST-M2-1 fusion protein that were used in the pulldown assays. (C and D) Mapping of the RNA (C)- and P (D)-binding domains on M2-1 by in vitro pulldown assays using deletion mutants of the GST-M2-1 fusion protein, tRNA, and purified recombinant RSV P protein. After extensive washing, the sample of GST-M2-1-RNA complexes was separated into two equal parts and analyzed by agarose gel electrophoresis and EtBr staining (C, bottom) and SDS-PAGE and Coomassie blue staining (C, top) to reveal RNA and proteins, respectively. Lane M, molecular size markers; +, M2-1 was inoculated with P in vitro. (E) Evidence for competition between RNA and P for binding to M2-1. Glutathione-Sepharose beads containing RNA-free GST-M2-1 (lane 1) were incubated with tRNA alone (lane 2), P alone (lane 3), or P in the presence of yeast tRNA (lane 4), an 81-nucleotide RNA transcribed from pBluescript plasmid (lane 5), or an 80-nucleotide RNA containing the HRSV leader sequence in the plus (lane 6) or minus (lane 7) polarity. After further incubation for 1 h at 20°C, the reaction products were washed extensively and separated into two parts for analysis to detect the presence of both P (top) and RNA (bottom) bound to M2-1, as described above. The GST-M2-1 and P protein bands are indicated by arrowheads.
- FIG. 8.
In vitro M2-1 phosphorylation has no visible effect on binding to P or RNA or on oligomerization. (A) In vitro phosphorylation of M2-1. GST-M2-1 was in vitro phosphorylated (lane 1) or not (lane 2) by CKI in the presence of [γ-32P]ATP32, cleaved by thrombin, resolved by SDS-PAGE, stained with Coomassie blue (lanes 1 and 2), and exposed by autoradiography (lanes 3 and 4). +, with. (B) Unphosphorylated GST-M2-1 (lanes 1 and 3) and phosphorylated GST-M2-1 (lanes 2 and 4) were incubated with P (lane 5) and washed, and the presence of P pulled down by GST-M2-1 was revealed by SDS-PAGE after thrombin cleavage. (C and D) Unphosphorylated GST-M2-1 (lanes 3 and 4) and phosphorylated GST-M2-1 (lanes 5 and 6) were incubated with tRNA and resolved by agarose gel electrophoresis. The agarose gel was stained with EtBr (C) and amido black (D). Lanes: 1, GST; 2, GST and tRNA; 3, GST-M2-1; 4, GST-M2-1 and tRNA; 5, phosphorylated GST-M2-1; 6, phosphorylated GST-M2-1 and tRNA; and 7, tRNA. (E) Phosphorylated M2-1 cross-linking. In vitro-phosphorylated M2-1 was cross-linked with EGS as indicated in the legend to Fig. 6.
- FIG. 9.
Organization of HRSV M2-1. (A) Sequence alignment of HRSV, BRSV, mouse pneumovirus (MPV), human metapneumovirus (HMPV), and avian metapneumovirus (AMPV) M2-1 proteins by ClustalW. Acidic residues are in red, basic residues are in blue, hydrophobic residues are in green, and glycines are in boldface. Asterisks and colons indicate identities and homologies, respectively. (B) Hydrophobic cluster analysis plot for HRSV M2-1. Structured regions are characterized by a high number of hydrophobic clusters, while unstructured regions have low numbers or are devoid of hydrophobic clusters. Secondary structure predictions were performed with PSI-PRED (21) and are shown below the plot.
Tables
- TABLE 1.
Estimation of proportions of secondary structuresa present in M2-1 and deletion mutants
Protein % Comprising: α-Helix structure β-Sheet structure Turns Random structure M2-1 wild type 38 12 10 40 M2-1Δ31N 49 7 8 35 M2-1Δ58N 45 6 13 37 M2-1Δ35-58 28 17 12 43 ↵ a The proportions of secondary structures were determined using the Dicroprot program.
- TABLE 2.
Effect of deletion of the M2-1 oligomerization domain on luciferase minigenome activity
System content Luc/β-Gal ratioa M2-1 0.425 ± 0.066 M2-1Δ35-58 0.040 ± 0.015 No M2-1 0.065 ± 0.060 No L 0.037 ± 0.015 ↵ a Each luciferase minigenome activity value was normalized based on β-Gal expression and is the average ± the standard deviation of results from three separate experiments performed in duplicate.
