Structural Characterization of Non-structural Protein 9 Complexed With Specific Nanobody Pinpoints Two Important Residues Involved in Porcine Reproductive and Respiratory Syndrome Virus Replication

FIGURE 1. Expression and analysis of recombinant Nsp9 and Nb6. (A) Size-exclusion chromatography of recombinant Nsp9 on calibrated Superdex 200 increase 10/300 GL column. The target protein was eluted at a retention volume of 13.5 ml, corresponding to approximately 72 kDa as monomer. Characterization of recombinant Nsp9 after two-step purification was analyzed by SDS–PAGE (Inset), M: protein ladder (10–180 kDa, Sorlarbio, Beijing, China), Gel: 12% precast polyacrylamide gel (Item No. NP0341BOX, Thermo Fisher Scientific). (B) Size-exclusion chromatography of recombinant Nb6 on calibrated Superdex 75 10/300 GL column. The target protein was eluted at a retention volume of 13 ml, corresponding to approximately 15 kDa as monomer. Characterization of recombinant Nb6 after two-step purification was analyzed by SDS–PAGE (Inset), M: protein ladder (10–180 kDa, Sorlarbio, Beijing, China), Gel: 12% precast polyacrylamide gel (Item No. NP0341BOX, Thermo Fisher Scientific). (C) Analysis of purified Nb6 binding to Nsp9. Gradient concentrations (62.5 nM–1 μM) of Nb6 in PBS buffer (pH7.4), containing 0.02% tween-20 and 0.1% BSA were passing through the streptavidin biosensor loaded with biotinylated…

FIGURE 2. The Nsp9-Nb6 complex exists as a heterodimer. (A) The Nsp9-Nb6 complex was purified with high purity by calibrated Superdex 75 10/300 GL column. Two peaks were observed, with the retention volume of the Peak1 was 9.5 ml, corresponding to the heterodimer of the Nsp9-Nb6 complex, and the retention volume of the Peak 2 was about 13 ml, corresponding to the monomer of Nb6. Characterization of the complex after purification was analyzed by SDS–PAGE (Inset) M: protein ladder (10–180 kDa, Sorlarbio, Beijing, China), Gel: 12% precast polyacrylamide gel (Item No. NP0341BOX, Thermo Fisher Scientific). (B) DLS of Nsp9-Nb6 complex at 1.5 μM. The hydrodynamic molecular radius of the complex is 8.414 nm.

FIGURE 3. Structural characterization of Nsp9 and Nsp9-Nb6 complex by SAXS. (A) Experimental SAXS curve (open circles) of Nsp9 in solution. The Guinier region and the corresponding linear fitting were shown in the inset. (B) Pair-distance distribution function derived from the experimental curve. (C) Kratky plot calculated from the experimental data. Bell shaped curves indicate compact structures. (D) Superimposition of the experimental envelope (as shown in surface) onto the model of Nsp9 represented in cartoon. (E) Experimental SAXS curve (open circles) of Nsp9-Nb6 complex in solution. The Guinier region and the corresponding linear fitting were shown in the inset. (F) Pair-distance distribution function derived from the experimental curve. (G) Kratky plot calculated from the experimental data. Bell shaped curves indicate compact structures. (H) Superimposition of the experimental envelope (as shown in surface) onto the heterogeneous model of Nsp9 and Nb6 heterodimer represented in cartoon.

FIGURE 4. Predicted model of Nsp9-Nb6 complex. The surface representations of Nsp9-Nb6 complex are shown in (A–D). Nsp9 is colored in green, Nb6 is in blue, Ile588 (A), Asp590 (B), Leu643 (C) and Asp646 (D) in Nsp9 are highlighted in red, Trp105 (A), Pro107 (B), Tyr62 (C) and Asp64 (D) in Nb6 are highlighted in magentas. (E,F) The predicted complex of Nsp9 and Nb6 are shown in cartoon diagrams.

FIGURE 5. Identification of residues involved in the interaction between Nsp9 and Nb6 by ELISAs. (A) Binding efficiency of the WT and mutant Nsp9 to Nb6. 96-well microtiter plates were coated with the WT and mutant Nsp9 (200 ng/well). After incubation with biotinylated Nb6, bound proteins were detected by HRP-conjugated streptavidin. (B) Binding efficiency of the WT and mutant Nb6 to Nsp9. 96-well microtiter plates were coated with the WT and mutant Nb6 (200 ng/well). After incubation with biotinylated Nsp9, bound proteins were detected by HRP-conjugated streptavidin. The binding efficiency was calculated on the basis of optical density (OD450). Assays were performed in triplicate and data are presented as means ± SD.

FIGURE 6. Rescue of recombinant PRRSV bearing Nsp9 mutation. (A) MARC-145 cells transfected with DNA-launched infectious clones of wild type PRRSV-SD16 strain (rSD16) or infectious clones bearing indicated mutations in the putative interaction sites (I588A, D590A, L643A) between Nsp9 and Nb6 on rSD16 backbone. IFA were performed to detect PRRSV-N protein expression and visualized by FITC-conjugated goat anti-mouse IgG. Overlay images were visualized for cell nuclei (blue) and N protein (green) with mock-transfected cells were included as control. The scale bars are 100 μm. (B) WB analysis of PRRSV-N protein expression in MARC-145 cells transfected with DNA-launched infectious clones of indicated mutants or wild type virus. Mock-transfected cells were used as control.

FIGURE 7. The effect of Nsp9 mutants on PRRSV replication. (A) The growth kinetics of the rescued recombinant viruses bearing different mutations in Nsp9 were analyzed. (B) Relative levels of PRRSV RNA of recombinant viruses were further analyzed for early time points. Data represent means ± SD from three independent experiments. Asterisk (*) indicates a significant difference between rSD16 and rSD16-I588A (*p < 0.05; **p < 0.01; ***p < 0.001). Pound (#) shows a significant difference between rSD16 and rSD16-L643A (#p < 0.05; ##p < 0.01; ###p < 0.001); ns, not significant. (C) Total cellular RNAs were extracted from recombinant virus-transfected MARC-145 cells at 24 hpi, and then subjected to reverse transcribed-PCR (RT-PCR) and agarose gel electrophoresis. The mRNA level of β-actin was included as reference for total RNA input. (D) The ratio of the gray value for PCR products of (−) gRNA/β-actin calculated using Image J software. Asterisk (*) indicates a significant difference between rSD16 and rSD16-I588A (***p < 0.001). Pound (#) shows a significant difference between rSD16 and rSD16-L643A (###p < 0.001).

FIGURE 8. Recombinant PRRSV viruses bearing mutations of residues 588 and 643 of Nsp9 impaired PRRSV replication in vitro. MARC-145 Cells (A) and CRL-2843-CD163 cells (C) were infected with mutated viruses or wild type rSD16 (0.1 MOI). IFA was performed at 48 hpi with the monoclonal antibody recognized PRRSV-N protein (Clone No. 6D10) and visualized by FITC-conjugated goat anti-mouse IgG. Overlay images were visualized for cell nuclei (blue) and N protein (green). The scale bars are 100 μm. (B,D) The ratio of virus-infected cells/total cells calculated using Image J software. Asterisk (*) indicates a significant difference between rSD16 and rSD16-I588A (***p < 0.001). Pound (#) shows a significant difference between rSD16 and rSD16-L643A (###p < 0.001).

FIGURE 9. The sequence alignment of aa residues 561-689 in PRRSV Nsp9. The sequences of Nsp9 from the 32 PRRSV strains were pulled from Genbank and then analyzed using Lasergene. The residues at 588 and 643 of Nsp9 are shown in red frame with dots indicating identical amino acid residues.