Fig 1.
γb protein is associated with the surface of purified BSMV virions.
(A) Representative immunogold labeling images showing γb protein bound to purified BSMV virions (left three photos), the labeling without γb antibody was used as a negative control (right two photos). Representative data are shown and the experiments have done more than three biological replicates with similar results. Scale bar, 200 nm. (B) The average amount of γb proteins associated with purified BSMV virions as shown in Fig 1A. Different letters above the bars indicate statistically significant differences (p < 0.05) as determined by Duncan’s multiple range test (n = 10). (C) Western blot to detect the target protein expression in BSMV-infected N. benthamiana leaves with CP and γb antiserum.The BSMVmγb mutant has a UUG to AUG substitution to destroy the translation of γb. (D) Detection of the cross-reactivity between γb and CP antibodies. GST-CP and GST-γb were purified followed by Western blot using CP and γb antibodies.
Fig 2.
γb interacts with CP in vivo and in vitro.
(A) Interaction of γb with CP in the context of BSMV infection by using BiFC assay. CP-YFPn or CP-YFPc was co-expressed with BSMVγb-YFPn or BSMVγb-YFPc. Chloroplast autofluorescence is shown as false red color. The negative control of BiFC assay is shown in S3 Fig. Scale bars, 20 μm. (B) Yeast two-hybrid (Y2H) assay to analyze the interaction between CP and γb. γb was cloned as a translational fusion with BD, CP was cloned as a translational fusions with AD, and combinations containing empty AD or BD constructs were served as negative control. Serial 10-fold dilutions of liquid cultures were spotted on synthetic dextrose dropout medium, SD/-Trp-Leu or SD/-Trp-Leu-His-Ade. (C) GST pull-down assay for the interaction between CP and γb in vitro. Various γb mutants were fused with GST-tag and incubated with CP-His with or without the 10 μg RNaseA treatment. GST-GFP or GFP-His was used as a negative control. The immunoprecipitated proteins were analyzed by Western blot analysis using anti-GST or anti-His antibodies. (D) Validate the interaction between CP and γb by MST assay. The concentration of γb-GFP-His was held constantly at 10 μM and the concentration of CP-His was titrated from the 20 μM. MST measurements were performed at 25°C by using 20% LED-power and 40% MST-power. Data analyses were used the MO. Affinity Analysis (x86) software. The dissociation constant (Kd) was derived to be Kd = 0.46 μM.
Fig 3.
The interaction between γb and CP is specifically enhanced by Zn2+ in vivo and in vitro.
(A) MST assays showed that γb binds to the Zn2+. The γb-GFP-His protein was purified from E. coli. In this assay, the concentration of γb-GFP-His was consistent, while 5 μM Zn2+ or Mg2+ were used as ligands. The apparent dissociation constant between γb and Zn2+obtained was Kd = 1.01 μM. The obtained data were digitized using a Monolith NT.115 instrument. (B) MST assays showed that the interaction between γb and CP was enhanced by Zn2+. The γb protein alone or the γb protein containing 5 μM Zn2+ in the buffer was kept constant and the CP protein was added as a ligand. In the absence of Zn2+, γb was bound to the CP membrane with Kd = 0.63 μM. In the presence of Zn2+, the affinity increased 4-fold with Kd = 0.15 μM. The resulting data were digitized with Monolith NT.115 instrument. Error bars indicate the range of data points obtained from at least two measurements. (C) Zn2+ enhances CP-γb interaction by Co-IP assay. N. benthamiana leaf tissues co-agroinfiltrated with RNAα, RNAβ, and RNAγγb-3xFlag were harvested at 3 dpi. Various concentrations of Zn2+ were added to the crude protein extracts. Total proteins were immunoprecipitated with anti-Flag beads. Input and immunoprecipitated protein (IP) were analyzed by Western blot analysis with an anti-GFP or anti-Flag antibody. (D) Zn2+ can enhance the interaction between γb and CP by GST Pull-down assay. Purified GST-γb and CP-His were incubated with a concentration gradient of Zn2+ from 2 μM to 200 μM. Various concentrations of EDTA were used to destroy the zinc-binding activity. Western blot analysis of immunoprecipitated proteins was performed using anti-GST or anti-His antibodies. (E) Zn2+ specifically enhances the interaction between γb and CP. The concentration of divalent metal cations (Cu2+, Ni2+, Mn2+, Mg2+, Ca2+, and Zn2+) was 20 μM.
Fig 4.
γb physically associates with virions by interacting with CP in a Zn2+-dependent manner.
(A) Amino acid sequence alignment of CRP proteins in different genera of Virgaviridae. Sequence alignment was performed by using the Uniprot online server (https://www.uniprot.org/). The conserved CCCH motif (Cys-50, Cys-60, Cys-81 and His-85 for BSMV strain ND18) are highlighted in gray. (B) Amino acid sequence alignment of CPs in different genera of Virgaviridae. The N-terminal of CPs among Virgaviridae were analyzed by using the Uniprot online server. The conserved His 13 of BSMV strain ND18 is highlighted in gray. (C) Yeast two-hybrid (Y2H) assay to analyze the interaction between CP and γbH85A. γbH85A was cloned as a translational fusion with BD. The interaction between γb and CP was served as a positive control. Combinations containing the empty vector pGADT7 or pGBKT7 were used as negative controls. Yeast cells containing the indicated plasmids in the bottom panel were spotted on synthetic dextrose dropout medium, SD/-Trp-Leu or SD/-Trp-Leu-His-Ade. (D and E) His-85 of γb and His-13 of CP are required for zinc binding activity and interaction between CP and γb. γb and γbH85A were fused to GST-tag and incubated with CP-His or CPH13A-His. 200 μM EDTA was used to destroy zinc-binding activity in the GST pull-down assays. GFP-His was used as a negative control. Western blot analysis of immunoprecipitated proteins was performed using anti-GST or anti-His antibodies. (F) Immunogold labeling experiments showed that γb binds to BSMV virions by interacting with CP in a Zn2+-dependent manner. BSMV and its derivatives virions were purified from infected leaves of N. benthamiana at 5 dpi. Virions were adsorbed onto 200-mesh nickel grids and incubated with antibodies against the γb protein. The pictures were visualized by TEM at 80 kV. Scale bar, 100 nm. (G) Average amounts of γb protein that are associated with the surface of purified BSMV virions as shown in Fig 4F. Different letters above the bars denote statistically significant differences (p < 0.05) determined by the Duncan’s multiple range test (n = 13). (H) Quantification of the average number of virus particles. BSMV, BSMVmCP, and BSMVmγb were agroinfiltrated into N. benthamiana leaves. γb-3xFlag or γbH85A-3xFlag was expressed with BSMVmγb infectious cDNA clone. The virions of different derivatives were purified at 5 dpi. Different letters in the chart denote statistically significant differences among different groups according to the Duncan’s multiple range test (p < 0.05) (n = 10).
Fig 5.
γb enhances virion assembly by interacting with CP.
(A) Co-localization analyses of γb, CP, and plus-strand BSMV RNAs in RNAα + RNAγ- infected N. benthamiana epidermal cells at 3 dpi. Mixtures of A. tumefaciens containing CP-RFP, CFP-γb, and the split YFP-tagged Pumilio proteins were agroinfiltrated into RNAα and RNAγ(+)PUM-infected leaves [34]. Co-localization analyses were visualized by confocal microscopy at 3 dpi. Chloroplast autofluorescence was shown as false pink color. Figures on the right indicate the normalized fluorescence intensity of GFP, RFP, and CFP channels along the white dashed line in the merged confocal images. Scale bars, 30 μm. (B) Extracts of BSMV-, BSMVmγb-, and BSMVH85A- infected leaves were analyzed after sucrose gradient centrifugation. Ten fractions were collected from the bottom and the top eight fractions were subjected to CP-specific immunoblot analysis. The right panel indicates a schematic representation of VLPs separation by sucrose gradient centrifugation. The bottom panel indicates the fractions observed under electron microscope. Scale bars, 100 μm. (C) EMSA assay to analyze the effect of γb on the binding between CP and viral RNAs. The 300 bp RNA probe labeled with andy-fluor-647. Unlabeled RNA probe (cold probe) was used as a control. The fusion proteins and probes used for EMSA analyses are indicated on the right of each panel. (D) Immunogold labeling of γb and CP in BSMV-infected N. benthamiana cells show that γb protein binds to VLPs in BSMV-infected leaves. Chl, Chloroplast; CI, cytoplasmic invagination; VLP, virion-like particle. Scale bars, 0.2 μm.
Fig 6.
γb is required for the stability of BSMV virions.
(A) RNase sensitivity assay in BSMV and BSMVmγb virions extracts. 0.2 ng/μL RNaseA were added to the purified BSMV and BSMVmγb virions, and incubated at 37°C at 0 to 30 min. Viral RNA was extracted followed by Northern blot. (B) Quantification of viral RNA degradation rates in Fig 6A. The relative viral RNA level at 0 min was set to 1.0. Data are shown from three independent repeats; error bars indicate standard deviation (n = 3). (C) Endogenous RNase sensitivity assay in extracts from N. benthamiana infected with BSMV and its derivatives. Inoculated leaves were ground and incubated in PIPES buffer at 37°C at 0 to 30 min, to allow degradation of unprotected RNA by the endogenous RNase. The total RNAs were then extracted and analyzed by Northern blot. Methylene blue-stained rRNAs served as RNA loading controls. (D) Quantification of viral RNA degradation rates in Fig 6C. The relative viral RNA level at 0 min was set to 1.0. Data are shown from three independent repeats; error bars indicate standard deviation (n = 3). (E) Immunogold labeling experiments show that Zn2+ can enhance virion stability by promoting γb binding to virions. The purified BSMV virions were treated with 0.02 mM EDTA at 4°C for 12 h, followed by immunogold labeling assays. The first column (without EDTA treatment) serves as negative control. Average amounts of γb protein that associated with purified BSMV virions with or without EDTA treatment as shown in the right panel. Different letters above the bars denote statistically significant differences (p < 0.05) determined by the Duncan’s multiple range test (n = 8). Scale bars, 100 μm. (F) RNase sensitivity assay in extracts of BSMV virions under the EDTA treatment. The purified BSMV virions were treated with 0.02 mM EDTA or PBS buffer and incubated with 1 nM RNaseA at 37°C at 0 to 30 min. Viral RNA was extracted and detected by Northern blot.
Fig 7.
The physical binding of CRPs to the corresponding virions is a general feature among rod-shaped viruses.
The virions of LRSV, TRV, and BNYVV were purified from the infected N. benthamiana leaves, the virions of PSLV were purified from infected barley leaves (Yangfu 4056) at 10 dpi. The virions were adsorbed onto 200-mesh nickel grids, followed by incubated with antibodies against the corresponding CRPs or BL buffer (as control) at room temperature for 2 h. The pictures were visualized by TEM at 80 kV. Scale bars, 100 μm.