Meanwhile, a 3-terminal site mutant of NS2 infectious clone (Y-PPVpCmut) shows a lower replication rate compared to parental PPV, further demonstrating that the 3-terminal of NS1 mRNA as the splicing target of SYNCRIP is important for NS2 expression and PPV replication

Meanwhile, a 3-terminal site mutant of NS2 infectious clone (Y-PPVpCmut) shows a lower replication rate compared to parental PPV, further demonstrating that the 3-terminal of NS1 mRNA as the splicing target of SYNCRIP is important for NS2 expression and PPV replication. In summary, the data presented here demonstrate that a porcine RNA binding protein SYNCRIP can directly interact with PPV NS1 mRNA to modulate PPV replication by targeting the 3-termial site of NS1 mRNA to regulate NS2 expression. whereas deletion of SYNCRIP significantly reduced NS2 mRNA and protein levels and the ratio of NS2 to NS1, and further impaired replication of the PPV. Furthermore, we found that SYNCRIP was able to bind the 3-terminal site of NS1 mRNA to promote the cleavage of NS1 mRNA into NS2 mRNA. Taken together, the results presented here demonstrate that SYNCRIP OSI-420 is a critical molecule in the alternative splicing process of PPV mRNA, while revealing a novel function for this protein and providing a potential target of antiviral intervention for the control of porcine parvovirus disease. Supplementary Information The online version contains supplementary material available at 10.1186/s13567-021-00938-6. for 2?h. Virus titers in the culture supernatants were determined by the Reed-Muench method [28]. Plasmid constructions and reactions PPV open reading frame 1 (ORF1) coding NS1 protein and three deletion mutant fragments were constructed using the overlap PCR approach from PPV genome and cloned into the pCI-neo vector. SYNCRIP protein was constructed from PK-15 cDNA and sub-cloned into the pCDNA3.0 vector. All sequences were confirmed by sequencing analysis (Sangon Biotech, Shanghai, China). For the construction of NS2 knockout Y-PPV clone (Y-PPVNS2?), the three CTC in NS2 ORF OSI-420 of Y-PPV were mutated to TAG using the overlap PCR approach, as described previously [29]. CRISPR/Cas9 knockout cell: Lentiviral vector LentiCRISPR v2 with puro resistance gene was used to clone sgRNA sequences between I and I. The PCR primers used in this study are shown in Table ?Table11. Table 1 Primers used in this study for 15?min to obtain a pelleted nuclei. The nuclear pellet was resuspended in freshly prepared RIP buffer (150?mM KCl, 25?mM Tris pH 7.4, 5?mM EDTA, 0.5% NP40), 1?mM PMSF, 100 U/mL RNase inhibitor, as well as 1??protease OSI-420 inhibitor cocktail (Sigma). Centrifugation was then used to obtain a pellet containing the nuclear membrane and debris at 13?000?for 10?min. Then the specific antibody and control antibody IgG were added for 4?h at 4 , then 40 L protein APOD G beads were added for 2?h. After centrifugation to discard the supernatant, the beads were washed three times with the RIP buffer. The RNA co-precipitated with SYNCRIP was obtained by resuspending the beads in Trizol RNA extraction reagent and further extraction, and then was analyzed by PCR; GAPDH served as a negative control. RNA-pulldown assay RNA-pulldown assay was carried out as previously described [33]. Briefly, NS1 sequences were T7 transcription synthesized with biotinylation at 5 end. 20 L of streptavidin C1 was used for preclear nuclear extract in each sample for 30?min at 4 . Then they were centrifuged at 1000?for 5?min. The supernatant was collected and supplemented with yeast tRNA (0.1?g/L), 20?g biotinylated NS1 mRNA or LacZ mRNA for 60?min at 4 . 30 L of streptavidin C1 was added to isolate RNA bounding to protein for 60?min at 4 . Then the beads were washed five times with buffer A [150?mM KCl, 25?mM Tris pH 7.4, 5?mM EDTA, 0.5?mM DTT, 0.5% NP40, 1?mM PMSF, 100 U/mL SUPERAsin and 1??protease inhibitor cocktail (Sigma)]. After rotating at 1000?for 5?min, the precipitate was added with the protein loading buffer and OSI-420 loaded to precast 12% gradient BisCTris gel for further analysis by Coomassie brilliant blue staining. The different strips were then cut for mass spectrometry analysis. Electrophoretic mobility shift assay NS1 mRNA sequence were synthesized, using a T7 in vitro transcription synthesized with biotinylation at 5 end, following the manufacturers instruction. Glutathione (GST) and GST-SYNCRIP proteins were purified using the GST protein purification kit (P2262; Beyotime, China), and further incubated with biotinylation NS1 mRNA. Gel shift assays were performed using the Chemiluminescent EMSA Kit (GS009, Beyotime, China). Purification of GST-tagged SYNCRIP protein Bacterially OSI-420 optimized SYNCRIP ORF (1C1689?bp) was cloned in pGEX-4?T-1 vector between I and I restriction sites to express GST-SYNCRIP protein. The positive plasmids were transformed into bacteria strain Rosetta (DE3)plys. To induce fusion protein expression, isopropul -d-1-thiogalactopyranoside (IPTG) was added to bacteria culture medium at a final concentration of 1 1.0?mM for 6?h at 28 . GST-SYNCRIP protein was purified as previously described [34]. Statistics These data are shown.