Quantitative microspectroscopic imaging reveals viral and cellular RNA helicase interactions in live cells

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 ( 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.

Alternative Splicing Transcripts of Zebrafish LGP2 Gene Differentially Contribute to IFN Antiviral Response

“…The unique structure of LGP2 proteins, which lack N-terminal CARD domains, makes it difficult to understand how they trigger IFN antiviral response as RIG-I and MDA5 do. In mammals, whereas initial experiments do not reveal an ability of LGP2 to activate IFNb promoter and inhibit virus replication (7,8), subsequent studies have obtained the opposite results (12,46). In these cases, LGP2directed ISRE stimulation in 293T cells might be attributable to its association with endogenous RLRs that contain CARDs (46).…”

Ab Initio Derivation of the FRET Equations Resolves Old Puzzles and Suggests Measurement Strategies

“…Förster resonance energy transfer (FRET) (1-3) is, without doubt, a very useful physical phenomenon that enjoys broad popularity among researchers in various science areas (4)(5)(6)(7)(8). Defined as the transfer of energy from an excited fluorescent tag to an unexcited one, FRET has become an indispensable tool in a gamut of applications ranging from estimation of intramolecular distances within a protein or DNA molecule (2,9), through probing the structure of molecular complexes (10)(11)(12)(13), to the determination of the proportion of complexes with different size and their dissociation constants in living cells (11,14). Fully quantitative analysis has been facilitated by the use of the kinetic theory of FRET (15,16) as well as the availability of multiphoton microscopy with spatial and spectral resolution (7,10,12,17,18).…”

“…Alternatively, one avoids use of corrections or additional measurements altogether by using spectral resolution to quantify a reduction in the donor emission as well as acceptor-sensitized emission simultaneously, thereby separating the donor and acceptor signals upon a single sample scan (10,(23)(24)(25). This latter approach has led to the introduction of different variants of FRET-based imaging, including FRET spectrometry (12,14,26), simultaneous unmixing of excitation and emission spectra (27), fully quantitative spectral imaging (11,13) and, more recently and only theoretically for now, FRET-induced color contrast shift spectrometry (28).…”

Ab initio derivation of the FRET equations resolves old puzzles and suggests measurement strategies

“…Defined as the transfer of energy from an excited fluorescent tag to an unexcited one, both of which are attached to macromolecules of interest in vivo or in vitro, FRET has become an indispensable tool in a gamut of applications ranging from estimation of intra-molecular distances within a protein or DNA molecules (2,9), through probing the structure of oligomeric complexes (10)(11)(12)(13) and to the determination of the proportion of various oligomeric species and their dissociation constants in living cells (11,14). Fully quantitative analysis has been facilitated by the use of the kinetic theory of FRET (15,16) as well as the availability of multiphoton microscopy with spatial and spectral resolution (7,10,12,17,18).…”

“…Alternatively, one avoids use of corrections or additional measurements altogether by using spectral resolution to quantify a reduction in the donor emission as well as acceptor sensitized emission simultaneously, thereby separating the donor and acceptor signals upon a single sample scan (10,(23)(24)(25). This latter approach has led to the introduction of different variants of FRET-based imaging, including FRET spectrometry (12,14,26), fully quantitative spectral imaging (FSI) (11,13) and, more recently and only theoretically for now, FRET-induced color contrast shift (FiCoS) spectrometry (27).…”