The N-terminus of classical swine fever virus (CSFV) nonstructural protein 2 modulates viral genome RNA replication

•A novel function for CSFV NS2, located in the N-terminus, in the regulation of viral RNA replication and infectious virus production was found, and the regulatory roles of NS2 for infectious virus production were identified to occur at the viral RNA replication level.•The single amino acid within the NS2 N-terminus modulated viral RNA replication independently of NS2-3 cleavage efficiency.•Two aspartic acids at positions D60, D78 and an arginine at position R100 in the N-terminus of NS2 are key amino acids for regulating viral replication.•The mutation of arginine for alanine at position 100 in the N-terminus of NS2 (NS2/R100A) caused a significant decrease in virus titers, and the down-regulation of virus production due to R100A was reversed by an NS2/I90L compensatory mutation.

Pestivirus nonstructural protein 2 (NS2) is a multifunctional, hydrophobic protein with an important but poorly understood role in viral RNA replication and infectious virus production. In the present study, based on sequence analysis, we mutated several representative conserved residues within the N-terminus of NS2 of classical swine fever virus (CSFV) and investigated how these mutations affected viral RNA replication and infectious virus production. Our results demonstrated that the mutation of two aspartic acids, NS2/D60A or NS2/D60K and NS2/D78K, in the N-terminus of NS2 abolished infectious virus production and that the substitution of arginine for alanine at position 100 (NS2/R100A) resulted in significantly decreased viral titer. The serial passage of cells containing viral genomic RNA molecules generated the revertants NS2/A60D, NS2/K60D and NS2/K78D, leading to the recovery of infectious virus. In the context of the NS2/R100A mutant, the NS2/I90L mutation compensated for infectious virus production. The regulatory roles of the indicated amino acid residues were identified to occur at the viral RNA replication level. These results revealed a novel function for the NS2 N-terminus of CSFV in modulating viral RNA replication.