Syncrip coordinates with hnRNPK to regulate PPV NS1 mRNA splicing and facilitate viral replication.

Abstract

Porcine parvovirus (PPV) relies on alternative splicing of the nonstructural protein 1 (NS1) transcript to generate nonstructural protein 2 (NS2), but the host factors coordinating this process remain incompletely defined. Here, we identified heterogeneous nuclear ribonucleoprotein K (hnRNPK) as a regulator of PPV NS1-to-NS2 mRNA splicing in PK-15 cells. hnRNPK specifically associated with NS1 mRNA, and PPV infection increased hnRNPK expression in a time- and multiplicity-of-infection-dependent manner. hnRNPK overexpression decreased the NS1/NS2 mRNA and protein ratios and increased viral DNA accumulation, whereas hnRNPK knockout produced the opposite effects. hnRNPK and the previously identified splicing regulator synaptotagmin-binding, cytoplasmic RNA-interacting protein (Syncrip) additively promoted NS1-to-NS2 splicing and PPV replication. Double-knockout and reconstitution experiments further showed that co-expression of hnRNPK and Syncrip restored NS2 production and viral replication more effectively than either factor alone. Domain-mapping analyses identified the K-protein interaction region (KI) of hnRNPK and the RNA recognition motif 2 (RRM2) region of Syncrip as the principal regions supporting this activity. hnRNPK associated directly with Syncrip, and Syncrip was required for efficient recruitment of hnRNPK to NS1 mRNA. In addition, hnRNPK associated with serine- and arginine-rich splicing factor 1 (SRSF1) in an RNA-independent manner during PPV infection. These findings support a model in which a Syncrip-hnRNPK complex promotes PPV NS1-to-NS2 alternative splicing and thereby facilitates viral replication, while identifying SRSF1 as a candidate component of the associated host splicing machinery.IMPORTANCEPorcine parvovirus (PPV) causes reproductive failure in swine and relies extensively on host RNA-processing machinery because of its limited coding capacity. Alternative splicing of the PPV nonstructural protein 1 (NS1) transcript generates nonstructural protein 2 (NS2), but the host factors controlling this process remain poorly defined. Here, we identify heterogeneous nuclear ribonucleoprotein K (hnRNPK) as a host regulator of PPV mRNA splicing and replication. hnRNPK acts additively with synaptotagmin-binding, cytoplasmic RNA-interacting protein (Syncrip) to promote NS1-to-NS2 mRNA splicing, increase NS2 production, and support viral DNA replication. Syncrip facilitates the association of hnRNPK with NS1 mRNA, and defined regions within both proteins contribute to this activity. hnRNPK also associates with the cellular splicing factor serine- and arginine-rich splicing factor 1 (SRSF1) during PPV infection. These findings reveal a host RNA-binding protein network that PPV exploits to expand its coding capacity and promote replication.