Identification Of A Linear B-cell Epitope On PRRSV Nsp10 And The Molecular Basis Of The Production Of Nsp10a

Porcine reproductive and respiratory syndrome virus (PRRSV) is a pathogen that severely affects the swine industry. The non-structural protein 10 (Nsp10) of PRRSV has helicase activity and is closely related with viral replication. The previous study in our laboratory showed that there are two forms of Nsp10 in HP-PRRSV JXwn06 infected cells, a full length one (Nsp10) and a truncation (Nsp10a). To explain this phenomenon, point-mutant viruses were constructed by using site-directed mutagenesis and reverse genetic technique on the backbone of HP-PRRSV JXwn06 and LP-PRRSV HB-1/3.9 to uncover the molecular basis of the production of Nsp10a and its meaning, which would provide theoretical basis for elucidating the biological functions of different forms of Nsp10 during the replication of PRRSV,At the beginning, a monoclonal antibody (mAb) against Nsp10 was generated by immunizing mouse with prokaryotic expressed Nsp10. The B-cell epitope recognized by the mAb was 286AIQPDYRDKL295, which was highly conserved among genotype 2 PRRSV but varied in genotype 1 PRRSV. The mAb could differentiate genotype 2 PRRSV from genotype 1 PRRSV. IFA and Western Blot analysis were conducted in PRRSV infected cells using the anti-Nsp10 mAb and the results showed that the Nsp10 products were first detected at 12 h post-infection and distributed in the perinuclear area. JXwn06 infection produced both Nsp10 and Nsp10a, while HB-1/3.9 infection only generated the full-length Nsp10. The results of mass spectrometry and expression of a series of truncated Nsp10 demonstrated that the amino acid (aa) sequences of Nsp10a contained at least 78-Thr and 423-Arg and Nsp10a may lack of about 70 amino acids at its N terminal compared to Nsp10.To explore the molecule basis of the production of Nsp10a, four chimeric viruses were constructed by swapping the Nsp10 gene or both the Nsp9 and Nsp10 genes together on the backbone of JXwn06 and HB-1/3.9. Unlike their parent viruses, the infection of RvHJn10 and RvHJn9n10 but not RvJHn10 and RvHJn9n10 produced Nsp10a, which means that Nsp10 gene is the determinant of the production of NsplOa. The 69-Glu in Nsp10 is proved to be the key aa that affects the production of Nsp10a by using a series of rescued point mutant viruses. To verify whether the viral protease Nsp2 or Nsp4 could cleave Nsp10, Nsp10 was cotransfected with Nsp2 or Nsp4 in vitro. The results showed that Nsp10 could not be cut by Nsp2 or Nsp4, although Nsp10 could interact with Nsp2. The production of Nsp10a was unrelated with the ubiquitin-proteasome pathway by adding the inhibitor MG132. The production of Nsp10a had little influence on the replication of PRRSV through analyzing the propagation dynamics of RvJX-E69G and RvHB-G69E and their parent viruses on MARC-145 cells and PAMs.In summary, a mAb that could differentiate genotype 2 from genotype 1 PRRSV was generated,and the mAb specific B-cell epitope was identified. The 69-Glu was proved to be the key aa affecting the production of NsplOa. The production of Nsp10a was not related with ubiquitin-proteasome pathway and had little influence on the replication of PRRSV. The findings will provide powerful tools and scientific data for elucidating the increased pathogenicity of HP-PRRSV.