Wang, Yan, Li, Rui, Qiao, Songlin, Wang, Jiaxi, Liu, Hongliang, Li, Zhijun, Ma, Hongfang, Yang, Lei, Ruan, Haiyu, Weng, Maoyang et al (show 5 more authors)
(2020)
Structural Characterization of Non-structural Protein 9 Complexed With Specific Nanobody Pinpoints Two Important Residues Involved in Porcine Reproductive and Respiratory Syndrome Virus Replication.
FRONTIERS IN MICROBIOLOGY, 11.
581856-.
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Structural Characterization of Non-structural Protein 9 Complexed With Specific Nanobody Pinpoints Two Important Residues In.pdf - Published Version Download (5MB) | Preview |
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.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | PRRSV, Nsp9, RdRp, nanobody, SAXS |
| Depositing User: | Symplectic Admin |
| Date Deposited: | 15 Jan 2021 09:20 |
| Last Modified: | 18 Jan 2023 23:03 |
| DOI: | 10.3389/fmicb.2020.581856 |
| Open Access URL: | http://doi.org/10.3389/fmicb.2020.581856 |
| Related URLs: | |
| URI: | https://livrepository.liverpool.ac.uk/id/eprint/3113642 |
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