The RIG-I ATPase core has evolved a functional requirement for allosteric stabilization by the Pincer domain.

Retinoic acid-inducible gene I (RIG-I) is a pattern recognition receptor expressed in metazoan cells that is responsible for eliciting the production of type I interferons and pro-inflammatory cytokines upon detection of intracellular, non-self RNA. Structural studies of RIG-I have identified a novel Pincer domain composed of two alpha helices that physically tethers the C-terminal domain to the SF2 helicase core. We find that the Pincer plays an important role in mediating the enzymatic and signaling activities of RIG-I.

The linker region of NS3 plays a critical role in the replication and infectivity of hepatitis C virus.

Hepatitis C virus (HCV) NS3-4A is required for viral replication and assembly. We establish that virus assembly is sensitive to mutations in the linker region between the helicase and protease domains of NS3-4A. However, we find that the protease cleavage, RNA binding, and unwinding rates of NS3 are minimally affected in vitro.

Defining the functional determinants for RNA surveillance by RIG-I.

Retinoic acid-inducible gene-I (RIG-I) is an intracellular RNA sensor that activates the innate immune machinery in response to infection by RNA viruses. Here, we report the crystal structure of distinct conformations of a RIG-I:dsRNA complex, which shows that HEL2i-mediated scanning allows RIG-I to sense the length of RNA targets. To understand the implications of HEL2i scanning for catalytic activity and signalling by RIG-I, we examined its ATPase activity when stimulated by duplex RNAs of varying lengths and 5' composition.

A short hairpin RNA screen of interferon-stimulated genes identifies a novel negative regulator of the cellular antiviral response.

The type I interferon (IFN) signaling pathway restricts infection of many divergent families of RNA and DNA viruses by inducing hundreds of IFN-stimulated genes (ISGs), some of which have direct antiviral activity. We screened 813 short hairpin RNA (shRNA) constructs targeting 245 human ISGs using a flow cytometry approach to identify genes that modulated infection of West Nile virus (WNV) in IFN-β-treated human cells. Thirty ISGs with inhibitory effects against WNV were identified, including several novel genes that had antiviral activity against related and unrelated positive-strand RNA viruses.

The thermodynamic basis for viral RNA detection by the RIG-I innate immune sensor.

RIG-I is a cytoplasmic surveillance protein that contributes to the earliest stages of the vertebrate innate immune response. The protein specifically recognizes 5'-triphosphorylated RNA structures that are released into the cell by viruses, such as influenza and hepatitis C. To understand the energetic basis for viral RNA recognition by RIG-I, we studied the binding of RIG-I domain variants to a family of dsRNA ligands.

Molecular mechanics of RNA translocases.

Historically, research on RNA helicase and translocation enzymes has seemed like a footnote to the extraordinary progress in studies on DNA-remodeling enzymes. However, during the past decade, the rising wave of activity in RNA science has engendered intense interest in the behaviors of specialized motor enzymes that remodel RNA molecules. Functional, mechanistic, and structural investigations of these RNA enzymes have begun to reveal the molecular basis for their key roles in RNA metabolism and signaling.

Structural insights into RNA recognition by RIG-I.

Intracellular RIG-I-like receptors (RLRs, including RIG-I, MDA-5, and LGP2) recognize viral RNAs as pathogen-associated molecular patterns (PAMPs) and initiate an antiviral immune response. To understand the molecular basis of this process, we determined the crystal structure of RIG-I in complex with double-stranded RNA (dsRNA). The dsRNA is sheathed within a network of protein domains that include a conserved "helicase" domain (regions HEL1 and HEL2), a specialized insertion domain (HEL2i), and a C-terminal regulatory domain (CTD).

Mechanism of Mss116 ATPase reveals functional diversity of DEAD-Box proteins.

Mss116 is a Saccharomyces cerevisiae mitochondrial DEAD-box RNA helicase protein that is essential for efficient in vivo splicing of all group I and group II introns and for activation of mRNA translation. Catalysis of intron splicing by Mss116 is coupled to its ATPase activity. Knowledge of the kinetic pathway(s) and biochemical intermediates populated during RNA-stimulated Mss116 ATPase is fundamental for defining how Mss116 ATP utilization is linked to in vivo function.

Unmasking the active helicase conformation of nonstructural protein 3 from hepatitis C virus.

The nonstructural protein 3 (NS3) helicase/protease is an important component of the hepatitis C virus (HCV) replication complex. We hypothesized that a specific β-strand tethers the C terminus of the helicase domain to the protease domain, thereby maintaining HCV NS3 in a compact conformation that differs from the extended conformations observed for other Flaviviridae NS3 enzymes. To test this hypothesis, we removed the β-strand and explored the structural and functional attributes of the truncated NS3 protein (NS3ΔC7).