Structure of Orthoreovirus RNA Chaperone σNS, a Component of Viral Replication Factories

Abstract

The reovirus {sigma}NS RNA-binding protein is required for formation of intracellular compartments during viral infection that support viral genome replication and capsid assembly. Despite its functional importance, a mechanistic understanding of {sigma}NS is lacking. We conducted structural and biochemical analyses of an R6A mutant of {sigma}NS that forms dimers instead of the higher-order oligomers formed by wildtype (WT) {sigma}NS. The crystal structure of selenomethionine-substituted {sigma}NS-R6A reveals that the mutant protein forms a stable antiparallel dimer, with each subunit having a well-folded central core and a projecting N-terminal arm. The dimers interact with each other by inserting the N-terminal arms into a hydrophobic pocket of the neighboring dimers on either side to form a helical assembly that resembles filaments of WT {sigma}NS in complex with RNA observed using cryo-EM. The interior of the crystallographic helical assembly is positively charged and of appropriate diameter to bind RNA. The helical assembly is disrupted by bile acids, which bind to the same hydrophobic pocket as the N-terminal arm, as demonstrated in the crystal structure of {sigma}NS-R6A in complex with bile acid. This finding suggests that the N-terminal arm functions in conferring context-dependent oligomeric states of {sigma}NS, which is supported by the structure of {sigma}NS lacking the N-terminal arm. We discovered that {sigma}NS displays RNA helix destabilizing and annealing activities, likely essential for presenting mRNA to the viral RNA-dependent RNA polymerase for genome replication. The RNA chaperone activity is reduced by bile acids and abolished by N-terminal arm deletion, suggesting that the activity requires formation of {sigma}NS oligomers. Our studies provide structural and mechanistic insights into the function of {sigma}NS in reovirus replication.