Molecular mechanisms of NS2^pro activation in hepaciviruses and pestiviruses: regulation, intrinsic activity, and structural determinants

Replication of all positive strand-RNA viruses depends on translation of a viral polyprotein, which is cleaved by viral and/or cellular proteases. The nonstructural protein 2 (NS2) of hepaciviruses and pestiviruses is responsible for NS2-NS3 cleavage, thus playing a highly specific and crucial role in viral polyprotein processing. Cleavage at the NS2/NS3 junction is essential for viral replication complex assembly and thus strictly regulated. Activation of hepaciviral and pestiviral NS2 cysteine proteases relies on different cofactors for optimal activity: while hepaciviral NS2 is reliant on the NS3 protease domain, classical pestiviral NS2 protease activation depends on the cellular chaperon DNAJC14. Despite its importance for the viral life cycle, how hepaci- and pestiviral NS2 protease activation is achieved remains ill-defined. Structural and functional data of hepaciviral NS2 protease revealed a dimeric composition with two composite active sites. Hepaciviral NS2 proteases harbor an intrinsic proteolytic activity that can be activated through specific surface residues. These surface residues are proposed to be pivotal for NS3-stimulated conformational changes in NS2. The activation mechanism for pestiviral NS2 protease is structurally less defined, but a cofactor-dependent, regulated NS2^pro activation is conserved for a large number of pestivirus species. This review will focus on recent insights into NS2 protease activation, with a focus on structural findings, host factor dependencies, and the conservation of proteolytic mechanisms among different members of the Flaviviridae family. Additionally, it will discuss perspectives on viral protein maturation, precursor function, and assembly of multiprotein complexes pivotal for the life cycle of these different viruses.