Structural Analysis Of PRRSV Nsp9 And Its Specific Nanobodies

Porcine reproductive and respiratory syndrome(PRRS),which pathogen is PRRS virus(PRRSV),is a harmful viral disease and results in huge economic losses to the global pig industry.Non-structural protein(Nsp)9 of PRRSV possesses RNA-dependent RNA polymerase(Rd Rp)activity essential for virus replication.Furhtermore,Nsp9 is highly conserved among PRRSV Nsps,making it an ideal antiviral target.Our previous report demonstrated that a PRRSV Nsp9-specific nanobody(Nb),designated Nb6,inhibited PRRSV replication in vitro when expressed intracellularly in MARC-145 cells.Meanwhile,we have also demonstrated that Nb6 fused with cell-penetrating peptides(CPP)can enter the host cells and exhibit antiviral activities.However,the antiviral mechanism whereby Nb6 targets Nsp9 to disrupt PRRSV function remains elusive.In addition,the three-dimensional structure of Nsp9 is still unknown.In this study,small-angle X-ray scattering(SAXS)was used to obtain the molecular envelopes of Nsp9 and Nsp9-Nb6 complexes,and further identified the interaction sites of Nsp9-Nb6 complexes.Then,we generated recombinant Nsp9-mutant viruses using a reverse genetics-based approach and then assessed mutant virus replication efficiencies in vitro to determine key residues involved in viral replication.In addition,the crystal structure of Nb7 recombinant protein,another Nsp9-specific nanobody,was resolved at a high resolution(1.8 ?).This study support clues for fighting against PRRSV infection and the development of structure-based antiviral agents against the virus.The main works and results of this study are as follows:1.Recombinant Nsp9 was purified by Q column,Ni-NTA column and gel filtration chromatography column and crystallized.The structure of the full-length Nsp9 was predicted through the I-TASSER online server.The overall structure was divided into two parts and showed barrel-shape with narrow top and wide bottom.According to the predicted structure and several studies,Nsp9 was truncated and expressed.Dynamic light scattering(DLS)analysis was performed to detect the particle size of the full-length Nsp9 in solution,showing that Nsp9 was monodisperse.SAXS measurement of Nsp9 was performed at the Shanghai Synchrotron Radiation Facility(SSRF).Different concentrations of Nsp9 were used for measurements.The molecular envelope of Nsp9 was asymmetric and showed barrel shape.2.Nb6/Nb7 gene was amplified and cloned into p MECS vector to construct the p MECS-Nb6/Nb7 recombinant expression vector.Soluble Nb6/Nb7 was expressed in BL21 cells and analyzed by SDS-PAGE.Nb6/Nb7 was purified using Ni-NTA column and gel chromatography column.Next,Nb6/Nb7 gene was cloned into p PICZ?A vector to construct the p PICZ?A-Nb6/Nb7 recombinant expression vector.The recombinant expression vector was linearized and transformed into Pichia pastoris strain X-33 competent cells.After methanol induction,SDS-PAGE analysis showed that Nb6/Nb7 was successfully expressed in the cell supernatant.Nb6/Nb7 was purified by Ni-NTA column and gel filtration chromatography column.We obtained the crystals of Nb6/Nb7,and successfully resolved the structure of Nb7.There are two Nb7 in each asymmetric unit.The shape of the recombinant protein is generally compact with short CDR3,which is different from other nanobodies.3.The purified Nsp9 and the excess purified Nb6 were mixed.The mixture was purified by gel filtration chromatography column to obtain the Nsp9-Nb6 complex.Bio-layer interferometry(BLI)were carried out to study the interaction between recombinant Nsp9 and Nb6.Nb6 bound to Nsp9 with high affinity.DLS analysis suggested that the complex was homogeneous.SAXS measurement of Nsp9-Nb6 complex was carried out at SSRF.Different concentrations of Nsp9-Nb6 complex were used for measurements.The molecular envelope was asymmetric and showed Z-shape.The final model of Nsp9-Nb6 complex was compared to that of Nsp9 monomer.In our model,the complex formed through the residues Ile588,Asp590,Leu643 and Asp646 in Nsp9 and Trp105,Pro107,Tyr62 and Asp64 in Nb6.BLI and ELISAs were carried out to verify the residues responsible for the interaction.The results confirmed that the residues at positions 588,590,643 of Nsp9 and the ones at positions 105,107,62 of Nb6 were involved in their interaction.Using the infectious clone of highly pathogenic PRRSV(HP-PRRSV)SD16 strain as the backbone,single-site-mutated viruses at positions 588,590 and 643 of Nsp9 were constructed.The replication kinetics on MARC-145 cells showed that the mutant viruses with substitutions at positions 588 and 643 had significantly lower replication efficiency than that of r SD16.There were also significant differences of the negative strand genome RNA levels between r SD16-I588 A,r SD16-L643 A and r SD16.In summary,in the current research,the molecular envelopes of Nsp9 and Nsp9-Nb6 complex were analyzed by SAXS for the first time and two novel residues of Nsp9 crucial to PRRSV replication were reported.These findings enrich the knowledge related to PRRSV Nsp9 and the replication and pathogenicity of the virus,and contribute to the development of drugs and vaccines against PRRSV infection.