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

Yan Wang,Rui Li,Lei Yang,Maoyang Weng,Gaiping Zhang,Julian A Hiscox,Songlin Qiao,Hongliang Liu,En-Min Zhou,Hongfang Ma,Haiyu Ruan,Jiaxi Wang,Yuchen Nan,James P Stewart,Zhijun Li

doi:10.3389/fmicb.2020.581856

Abstract

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), is a widespread viral disease that has led to huge economic losses for the global swine industry. Non-structural protein 9 (Nsp9) of PRRSV possesses essential RNA-dependent RNA polymerase (RdRp) activity for viral RNA replication. Our previous report showed that Nsp9-specific nanobody, Nb6, was able to inhibit PRRSV replication. In this study, recombinant Nsp9 and Nsp9-Nb6 complex were prepared then characterized using bio-layer interferometry (BLI) and dynamic light scattering (DLS) analyses that demonstrated high-affinity binding of Nb6 to Nsp9 to form a homogeneous complex. Small-angle X-ray scattering (SAXS) characterization analyses revealed that spatial interactions differed between Nsp9 and Nsp9-Nb6 complex molecular envelopes. Enzyme-linked immunosorbent assays (ELISAs) revealed key involvement of Nsp9 residues Ile588, Asp590, and Leu643 and Nb6 residues Tyr62, Trp105, and Pro107 in the Nsp9-Nb6 interaction. After reverse genetics-based techniques were employed to generate recombinant Nsp9 mutant viruses, virus replication efficiencies were assessed in MARC-145 cells. The results revealed impaired viral replication of recombinant viruses bearing I588A and L643A mutations as compared with replication of wild type virus, as evidenced by reduced negative-strand genomic RNA [(−) gRNA] synthesis and attenuated viral infection. Moreover, the isoleucine at position 588 of Nsp9 was conserved across PRRSV genotypes. In conclusion, structural analysis of the Nsp9-Nb6 complex revealed novel amino acid interactions involved in viral RNA replication that will be useful for guiding development of structure-based anti-PRRSV agents.

Highlights

Porcine reproductive and respiratory syndrome (PRRS), first reported in 1987 in the United States, is one of the most widespread and severe viral disease threats to the global swine industry (Wensvoort et al, 1991; Nan et al, 2017)
To discover the molecular structure of PRRS virus (PRRSV)-Non-structural protein 9 (Nsp9), soluble Nsp9 was recombinantly expressed in a prokaryotic expression system and Nb6 was produced using a Pichia pastoris expression system
To generate the Nsp9-Nb6 complex, Nsp9 was mixed with an excess of Nb6 and the mixture was incubated at 4◦C overnight

Summary

Introduction

Porcine reproductive and respiratory syndrome (PRRS), first reported in 1987 in the United States, is one of the most widespread and severe viral disease threats to the global swine industry (Wensvoort et al, 1991; Nan et al, 2017). This disease is characterized by reproductive failures in sows and respiratory diseases in pigs of all ages (Lunney et al, 2010). In 2006, an atypical PRRS outbreak caused by a highly pathogenic PRRSV strain (HPPRRSV) occurred in China, leading to a country-wide epidemic (Tian et al, 2007; Tong et al, 2007)

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Results

Conclusion