Analysis Of The Solution Structure Of Nonstructural Protein 7α(NSP7α) From Porcine Reproductive And Respiratory Syndrome Virus (PRRSV) And Preliminary Study For Its Functions

Porcine reproductive and respiratory syndrome virus（PRRSV）has a single strand positive-sense RNA genome and it is the causative agent of porcine reproductive and respiratory syndrome（PRRS）,one of the most destructive disease of swine in the global swine industry.Previous studies have shown that NSP7 participates in humoral immune reaction and strongly reacts with PRRSV infected pig serum.After an internal cleavage,NSP7can yield NSP7αand NSP7β.In this research,antisera were used to detect the expression of NSP7αand NSP7βin the infected cells;the solution structure of NSP7αwas determined by NMR then the structures of PRRSV NSP7αand EAV NSP7αwere compared and analyzed;the interaction of NSP7αand RNA-dependent RNA polymerase（NSP9）was detected,and the key amino acids on NSP7αfor the NSP7α-NSP9 interaction were predicted and further identified.The results obtained in this study may provide some new insights into the function of NSP7αand the molecular mechanism of PRRSV infectious cycle.The main results obtained in the research were as followed:1.Anti-NSP7αand anti-NSP7βantibodies were generated in rabbits by immunization with the proteins purified from prokaryotic expression system.The expression of NSP7α,NSP7βand their precursors in the PRRSV infected MARC-145 cells and the primary host cell porcine alveolar macrophages（PAMs）at different time points were examined by Western blot.The distribution of NSP7α,NSP7βand their precursors in PRRSV infected MARC-145 cells at different time points were observed by immunofluorescence.The result of Western blot showed that anti-NSP7αantiserum detected a band with the predicted size of NSP7αand some cleavage precursor and intermediate forms of NSP7α.However,NSP7 was not detected.Anti-NSP7βantiserum detected some precursor proteins,but no mature NSP7βand NSP7.In immunofluorescence analysis,anti-NSP7αantiserum detected the fluorescence of NSP7αand the precursors in cytoplasm.This result indicated that they are only involved in the physiological functions in the cytoplasm.2.The structure of NSP7αwas solved by solution NMR.Isotope ¹³C and ¹⁵N labeled NSP7αwas purified using prokaryotic expression system and NMR spectrums of NSP7αwere collected.After the assignment of the spectrums,the protein structure was determined.Comparison of the solution structure of PRRSV and EAV NSP7αproteins showed their secondary structures have the similar spatial distribution and the region of helixα2 to helixα3is the best superimposed region in the two proteins.3.The interactions between NSP7αand other PRRSV nonstructural proteins were detected by yeast two hybrid.The results showed that NSP7αonly interacted with NSP9.To verify the interaction between NSP7αand NSP9,Pull-down and bimolecular fluorescence complementation（BiFC）assays were conducted.NSP9 is the viral RNA dependent RNA polymerase and a critical component of the replication complex.The interaction between NSP7αand NSP9 indicates that NSP7αmight play a role in the viral replication and transcription complex（RTC）and/or assist NSP9 to carry out its function in PRRSV RNA synthesis.Based on the solved structure of NSP7α,the key amino acids of NSP7αinvolved in the NSP7α-NSP9 interaction were predicted by structure docking,and the predicted amino acids were subsequently analyzed by using mutagenesis analysis and Pull-down.L69 and F72on NSP7αwere identified as the key amino acids for NSP7α-NSP9 interaction.