Abstract
Human cells detect RNA viruses through a set of helicases called RIG-I-like receptors (RLRs) that initiate the interferon response via a mitochondrial signaling complex. Many RNA viruses also encode helicases, which are sometimes covalently linked to proteases that cleave signaling proteins. One unresolved question is how RLRs interact with each other and with viral proteins in cells. This study examined the interactions among the hepatitis C virus (HCV) helicase and RLR helicases in live cells with quantitative microspectroscopic imaging (Q-MSI), a technique that determines FRET efficiency and subcellular donor and acceptor concentrations. HEK293T cells were transfected with various vector combinations to express cyan fluorescent protein (CFP) or YFP fused to either biologically active HCV helicase or one RLR (i.e. RIG-I, MDA5, or LGP2), expressed in the presence or absence of polyinosinic-polycytidylic acid (poly(I:C)), which elicits RLR accumulation at mitochondria. Q-MSI confirmed previously reported RLR interactions and revealed an interaction between HCV helicase and LGP2. Mitochondria in CFP-RIG-I:YFP-RIG-I cells, CFP-MDA5:YFP-MDA5 cells, and CFP-MDA5:YFP-LGP2 cells had higher FRET efficiencies in the presence of poly(I:C), indicating that RNA causes these proteins to accumulate at mitochondria in higher-order complexes than those formed in the absence of poly(I:C). However, mitochondria in CFP-LGP2:YFP-LGP2 cells had lower FRET signal in the presence of poly(I:C), suggesting that LGP2 oligomers disperse so that LGP2 can bind MDA5. Data support a new model where an LGP2-MDA5 oligomer shuttles NS3 to the mitochondria to block antiviral signaling.
Highlights
Human cells detect RNA viruses through a set of helicases called RIG-I-like receptors (RLRs) that initiate the interferon response via a mitochondrial signaling complex
This project was initially designed to test the notion that homologous protein/protein interaction motifs shared between nonstructural protein 3 (NS3) and RLRs enable the NS3 helicase domain to position the NS3 protease where it can cleave key cellular targets needed for the interferon response
Seven plasmids were constructed to encode cyan fluorescent protein (CFP)-NS3, CFPRIG-I, CFP-MDA5, CFP-LGP2, YFP-RIG-I, YFP-MDA5, and YFP-LGP2
Summary
Human cells detect RNA viruses through a set of helicases called RIG-I-like receptors (RLRs) that initiate the interferon response via a mitochondrial signaling complex. A direct interaction between NS3 and an RLR has not yet been demonstrated; nor has there been convincing evidence that RLR oligomerization occurs in living cells [30] This project was initially designed to test the notion that homologous protein/protein interaction motifs shared between NS3 and RLRs enable the NS3 helicase domain to position the NS3 protease where it can cleave key cellular targets needed for the interferon response. NS3 might locate MAVS by forming hetero-oligomers with RIG-I, MDA5, and/or LGP2 To test this hypothesis, we designed pairs of fluorescent fusion proteins [31, 32] to be used in cell-based FRET assays.
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