Quantitative Micro-Spectroscopic Imaging Reveals Viral and Cellular RNA Helicase Interactions in Live Cells [Signal Transduction]

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 sometimes are 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 micro-spectroscopic imaging (Q-MSI), a technique that determines Forster Resonance Energy Transfer (FRET) efficiency and sub-cellular donor and acceptor concentrations. HEK293T cells were transfected with various vector combinations to express cyan fluorescent protein (CFP) or yellow fluorescent protein (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 LGP2 oligomers disperse, so LGP2 can bind MDA5. Data support a new model where a LGP2-MDA5 oligomer shuttles NS3 to the mitochondria to block antiviral signaling.

from #AlexandrosSfakianakis via Alexandros G.Sfakianakis on Inoreader http://ift.tt/2pdtcJy
via IFTTT