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

Abstract

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.

Selection of Classical Swine Fever Virus with Enhanced Pathogenicity Reveals Synergistic Virulence Determinants in E2 and NS4B

“…NS4B also harbors a nucleoside triphosphatase (NTPase) motif. The NTPase activity is an absolute requirement for CSFV replication, but there is no evidence yet that modulating the NTPase activity may affect virulence (10). The 2 amino acids of NS4B identified in the present study are not located within the NTPase motif but clearly determine the replication efficiency of the virus in vivo and in vitro.…”

“…This is reflected in numerous reports in which mutations of different functional domains in the structural proteins C, E rns , E1, and E2 and in the nonstructural proteins p7 and NS4B were shown to reduce the pathogenicity in pigs, indicating that these genes may be involved in determining CSFV virulence (7,8,9,10,11,12,13,32,36,37,38,39,40,46,54).…”

“…Comparison of the genomes of the GPE Ϫ vaccine virus and the parent ALD virus revealed 225 nucleotide substitutions and 46 amino acid differences (22), which did not provide any conclusive information on the molecular basis of the attenuation. Numerous reports suggest that the viral proteins N pro , C, E rns , E1, E2, p7, and NS4B may be involved in determining the virulence of CSFV in pigs (7,8,9,10,11,12,13,32,36,37,38,39,40,43,46,54). So far, however, all conclusions on CSFV virulence determinants rely on mutations that result in downregulation of pathogenicity.…”

Identification and characterization of the biochemical function of Agrobacterium T‐complex‐recruiting protein Atu5117

“…To further confirm the substrate preference of Atu5117, the kinetic parameters of Atu5117 for eight (d)NTPs were measured. We compared the kinetic parameters of Atu5117 (d)NTPase with those of other (d)NTPases [25][26][27][28][29]. The kinetic parameters of (d)NTPase for different nucleotide substrates were determined by analyzing the effects of substrate concentrations on hydrolysis rates.…”

Complex Virus–Host Interactions Involved in the Regulation of Classical Swine Fever Virus Replication: A Minireview

“…NS4B contains two conserved domains, Walkers A (aa 209–216) and B (aa 335–342). Walker A exhibits NTPase activity and is essential for RNA replication [ 46 ]. Analysis of simple modular architecture research tool (SMART) has revealed that NS4B contains a Toll/interleukin-1 receptor (TIR)-like domain, and mutations in the TIR-like domain of NS4B significantly attenuate the virulence of CSFV in pigs [ 47 ].…”