Syncrip facilitates porcine parvovirus viral dna replication through the alternative splicing of ns1 mrna to promote ns2 mrna formation
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| Discussion
PPV is a major causative agent of stillbirth, mummifica- tion, and embryonic death in swine [ 1 ]. The PPV genome encodes four nonstructural proteins NS1, NS2, NS3 and late SAT protein [ 35 , 36 ]. NS1 protein has helicase and nickase activities [ 37 , 38 ], and can induce apoptosis and cell cycle arrest [ 39 ]. NS2 protein can block the expres- sion of IFN-β induced by ds-RNA [ 9 ], and associates with the nuclear egress of progeny virions efficiently [ 40 ]. A late non-structural protein SAT is expressed from the same mRNA as VP2, which can contribute to viral spreading by inducing irreversible endoplasmic reticu- lum stress in the progress of PPV infection [ 2 ]. Alternative splicing process of Parvovirus pre-mRNA plays a key role in regulating the expression of viral pro- teins [ 25 , 41 ]. Mature PPV NS2 mRNA is completely located in NS1 mRNA, which is formed by alternative splicing of NS1 mRNA. SYNCRIP has been reported as a splicing factor to be involved in post-transcriptional [ 42 , 43 ], miRNA exosomal sorting process [ 21 ], syn- aptic protein mRNA expression [ 11 ] and RNA virus replication [ 26 , 27 ]. SYNCRIP, a member of heteroge- neous nuclear ribonucleoproteins (hnRNP), is highly conserved among different species. SYNCRIP, which is a cytoplasm RNA-binding protein contains three typi- cal domains, RRM1 domain, RRM2 domain and RRM3 domain. Porcine SYNCRIP is encoded by the porcine SYNCRIP gene and possesses 99.84% identity to human SYNCRIP (GenBank: NM006372). In this study, we first reported that NS1 mRNA could directly interact with SYNCRIP, and found that SYNCRIP could be detected in most tissues of pigs, such as the spleen, lung, kidney, ovary and uterus. The expression of porcine SYNCRIP was upregulated with PPV infection levels. Impor- tantly, we found that SYNCRIP knockout significantly impaired PPV replication. This result agrees with the previous observations that SYNCRIP is involved in MHV and HCV RNA replication [ 26 , 27 ]. Previous studies have reported that some RNA bind- ing proteins are involved in alternative splicing either viral RNA or host cell RNA. HnRNP K can interact with influenza virulence factor NS1 binding protein (NS1-BP) to promote the splicing of the viral M1 RNA into M2 RNA [ 44 ], also modulate CD44 alternative splicing during the epithelial mesenchymal transition [ 45 ], and regulate the neuronal differentiation factor TRF2 alternative splicing. RBM38 is an essential host factor of B19V pre-mRNA splicing and it is important for the expression of the non-structural 11 kDa protein [ 24 ]. Because the hnRNP family has a highly conserved RNA recognition region, we speculated that SYNCRIP may directly interact with PPV virulence factor NS1 mRNA and regulate NS1 expression. When recombi- nant plasmids (pEGFP-NS1, pEGFP-NS2, pEGFP-VP1 and pEGFP-VP2) were transfected into PK-15 SYNCRIP , PK-15 and PK-15 V cells, the level of NS1 protein and mRNA were decreased in PK-15 SYNCRIP cells, compared to that in PK-15 or PK-15 V control cells, but the levels of VP1 and VP2 were not decreased in PK-15 SYNCRIP cells, compared to those in PK-15 or PK-15 V control cells. In addition, we noted that NS1 mRNA was signifi- cantly decreased in the PPV-infected PK-15 SYNCRIP cells relative to PK-15 and PK-15 V cells. Furthermore, NS2 mutant’s replication rate significantly decreased com- pared to parental PPV. These results suggest that |