Identification of an NTPase motif in classical swine fever virus NS4B protein

Classical swine fever (CSF) is a highly contagious and often fatal disease of swine caused by CSF virus (CSFV), a positive-sense single-stranded RNA virus within the Pestivirus genus of the Flaviviridae family. Here, we have identified conserved sequence elements observed in nucleotide-binding motifs (NBM) that hydrolyze NTPs within the CSFV non-structural (NS) protein NS4B. Expressed NS4B protein hydrolyzes both ATP and GTP. Substitutions of critical residues within the identified NS4B NBM Walker A and B motifs significantly impair the ATPase and GTPase activities of expressed proteins. Similar mutations introduced into the genetic backbone of a full-length cDNA copy of CSFV strain Brescia rendered no infectious viruses or viruses with impaired replication capabilities, suggesting that this NTPase activity is critical for the CSFV cycle. Recovered mutant viruses retained a virulent phenotype, as parental strain Brescia, in infected swine. These results have important implications for developing novel antiviral strategies against CSFV infection.