Date Published: February 2, 2017
Publisher: Public Library of Science
Author(s): Danilo Dubrau, M. Alejandra Tortorici, Félix A. Rey, Norbert Tautz, Sara Cherry.
The viruses of the family Flaviviridae possess a positive-strand RNA genome and express a single polyprotein which is processed into functional proteins. Initially, the nonstructural (NS) proteins, which are not part of the virions, form complexes capable of genome replication. Later on, the NS proteins also play a critical role in virion formation. The molecular basis to understand how the same proteins form different complexes required in both processes is so far unknown. For pestiviruses, uncleaved NS2-3 is essential for virion morphogenesis while NS3 is required for RNA replication but is not functional in viral assembly. Recently, we identified two gain of function mutations, located in the C-terminal region of NS2 and in the serine protease domain of NS3 (NS3 residue 132), which allow NS2 and NS3 to substitute for uncleaved NS2-3 in particle assembly. We report here the crystal structure of pestivirus NS3-4A showing that the NS3 residue 132 maps to a surface patch interacting with the C-terminal region of NS4A (NS4A-kink region) suggesting a critical role of this contact in virion morphogenesis. We show that destabilization of this interaction, either by alanine exchanges at this NS3/4A-kink interface, led to a gain of function of the NS3/4A complex in particle formation. In contrast, RNA replication and thus replicase assembly requires a stable association between NS3 and the NS4A-kink region. Thus, we propose that two variants of NS3/4A complexes exist in pestivirus infected cells each representing a basic building block required for either RNA replication or virion morphogenesis. This could be further corroborated by trans-complementation studies with a replication-defective NS3/4A double mutant that was still functional in viral assembly. Our observations illustrate the presence of alternative overlapping surfaces providing different contacts between the same proteins, allowing the switch from RNA replication to virion formation.
The Flaviviridae family comprises positive-strand RNA viruses and consists of four genera, Flavivirus, Pegivirus, Hepacivirus, and Pestivirus, with the latter three genera showing a significantly higher degree of similarity [1–3]. Pestiviruses, like bovine viral diarrhea virus (BVDV-1 and -2) and classical swine fever virus (CSFV) are important animal pathogens which cause significant economic damage in livestock industries .
Due to their compact genome size viruses encode a severely limited number of gene products which therefore often exert multiple functions. Along these lines, the nonstructural proteins of the members of the Flaviviridae family have key functions during RNA replication and in the assembly of infectious particles . In addition, like most positive-strand RNA viruses, pestiviruses use regulated polyprotein processing by viral and cellular proteases as means to temporal and spatial regulate protein activity . An excellent example is the observation that in the pestiviral life cycle, RNA replication and virion morphogenesis are highly regulated processes both depending on different protein complexes which derive from the NS2-3-4A region of the polyprotein: while the NS3/4A complex is essential for RNA replication and cannot be functionally replaced by NS2-3/4A [6, 18], NS2-3/4A is indispensable for pestiviral particle formation but is not active in RNA replication [28, 29]. NS2-3 cleavage by the NS2 autoprotease is temporally restricted to the early time points of infection due to its dependency on a cellular cofactor available only in limiting amounts [6, 37]. This leads to a temporal gradient for the formation of NS3/4A vs. NS2-3/4A in pestivirus infected cells. In contrast to pestiviruses, the closely related HCV does not depend on uncleaved NS2-NS3 for infectious virion formation at least in cell culture [26, 41] and no major amounts of uncleaved NS2-NS3 can be detected in HCV-infected cells. Thus, HCV and pestiviruses show a major difference with regard to their dependency on uncleaved NS2-3 for the formation of infectious progeny.