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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 (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.

SUBMITTER: Corby MJ

PROVIDER: S-EPMC5500786 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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

Corby M J MJ Stoneman Michael R MR Biener Gabriel G Paprocki Joel D JD Kolli Rajesh R Raicu Valerica V Frick David N DN

The Journal of biological chemistry 20170508 27

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 ...[more]

PMID: 28483922

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Project description:Immunomodulators of pathogens frequently affect multiple cellular targets, thus preventing recognition by different immune cells. For instance, the K5 modulator of immune recognition (MIR2) from Kaposi sarcoma-associated herpesvirus prevents activation of cytotoxic T cells, natural killer cells, and natural killer T cells by downregulating major histocompatibility complex (MHC) class I molecules, the MHC-like molecule CD1, the cell adhesion molecules ICAM-1 and PECAM, and the co-stimulatory molecule B7.2. K5 belongs to a family of viral- and cellular-membrane-spanning RING ubiquitin ligases. While a limited number of transmembrane proteins have been shown to be targeted for degradation by this family, it is unknown whether additional targets exist. We now describe a quantitative proteomics approach to identify novel targets of this protein family. Using stable isotope labeling by amino acids, we compared the proteome of plasma, Golgi, and endoplasmic reticulum membranes in the presence and absence of K5. Mass spectrometric protein identification revealed four proteins that were consistently underrepresented in the plasma membrane of K5 expression cells: MHC I (as expected), bone marrow stromal antigen 2 (BST-2, CD316), activated leukocyte cell adhesion molecule (ALCAM, CD166) and Syntaxin-4. Downregulation of each of these proteins was independently confirmed by immunoblotting with specific antibodies. We further demonstrate that ALCAM is a bona fide target of both K5 and the myxomavirus homolog M153R. Upon exiting the endoplasmic reticulum, ALCAM is ubiquitinated in the presence of wild-type, but not RING-deficient or acidic motif-deficient, K5, and is targeted for lysosomal degradation via the multivesicular body pathway. Since ALCAM is the ligand for CD6, a member of the immunological synapse of T cells, its removal by viral immune modulators implies a role for CD6 in the recognition of pathogens by T cells. The unbiased global proteome analysis therefore revealed novel immunomodulatory functions of pathogen proteins.

Dataset Information

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

Publications

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

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