Structural and Biochemical Characterization of N-Terminal Protease of Classical Swine Fever Virus

Pestiviruses are single-strand positive sense RNA viruses that belong to the flaviviridae family. They express their genome as a single polypeptide that is subsequently cleaved into individual proteins by host- and virus-encoded proteases. N-terminal protease Npro is the first translated protein of the pestivirus genome. Npro is a cysteine auto-protease that cleaves between its own C-terminus (Cys168) and the N-terminus of the core protein (Ser169). Npro is essential for initiation of viral polyprotein processing and generation of a viable core protein for viral assembly. The predicted catalytic triad of Npro is Glu22, His49 and Cys69, which differs from the known catalytic triads in either serine or cysteine proteases. Due to its unique sequence and catalytic site, Npro forms its own cysteine protease family C53. After the initial self-cleavage, Npro is no longer active as a protease and no trans activity has ever been observed. Npro also plays a critical role in subverting the host’s innate immune response by targeting interferon regulatory factor-3 (IRF3) for proteasomal degradation, and thus preventing the transcriptional activation of interferon-α/β genes. We determined the crystal structure of wild type Npro and that of a cleavage site mutant (Npro-C168A) to 1.6 Å resolution. The structures show that Npro is structurally distinct from other known cysteine proteases and has a novel “clam shell” fold consisting of two domains, the protease domain carrying the active site of Npro and the zinc-binding domain that harbors a conserved metal binding sequence motif viz., the TRASH motif formed by Cys112-Cys134-Asp136-Cys138. The structure conclusively shows that the catalytic site of Npro is a dyad formed by His49 and Cys69 and does not include Glu22. Rather, Glu22 has a key role in maintaining the structural integrity of the protein. The C-terminus is not only bound in the active site, but it also contributes a strand to the beta-sheet that makes up the active site. Thus, this structure explains the autocatalytic, and subsequent auto-inhibition mechanism of Npro (i.e., why there is no trans activity), and furthermore provides insight into the interaction of Npro with IRF3 and its role in subversion of host immune response.