Paramyxovirus V Protein Interaction with the Antiviral Sensor LGP2 Disrupts MDA5 Signaling Enhancement but Is Not Relevant to LGP2-Mediated RLR Signaling Inhibition

Abstract

The interferon antiviral system is a primary barrier to virus replication triggered upon recognition of nonself RNAs by the cytoplasmic sensors encoded by retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology gene 2 (LGP2). Paramyxovirus V proteins are interferon antagonists that can selectively interact with MDA5 and LGP2 through contact with a discrete helicase domain region. Interaction with MDA5, an activator of antiviral signaling, disrupts interferon gene expression and antiviral responses. LGP2 has more diverse reported roles as both a coactivator of MDA5 and a negative regulator of both RIG-I and MDA5. This functional dichotomy, along with the concurrent interference with both cellular targets, has made it difficult to assess the unique consequences of V protein interaction with LGP2. To directly evaluate the impact of LGP2 interference, MDA5 and LGP2 variants unable to be recognized by measles virus and parainfluenza virus 5 (PIV5) V proteins were tested in signaling assays. Results indicate that interaction with LGP2 specifically prevents coactivation of MDA5 signaling and that LGP2's negative regulatory capacity was not affected. V proteins only partially antagonize RIG-I at high concentrations, and their expression had no additive effects on LGP2-mediated negative regulation. However, conversion of RIG-I to a direct V protein target was accomplished by only two amino acid substitutions that allowed both V protein interaction and efficient interference. These results clarify the unique consequences of MDA5 and LGP2 interference by paramyxovirus V proteins and help resolve the distinct roles of LGP2 in both activation and inhibition of antiviral signal transduction. IMPORTANCE Paramyxovirus V proteins interact with two innate immune receptors, MDA5 andLGP2, but not RIG-I. V proteins prevent MDA5 from signaling to the beta interferon promoter, but the consequences of LGP2 targeting are poorly understood. As the V protein targets MDA5 and LGP2 simultaneously, and LGP2 is both a positive and negative regulator of both MDA5 and RIG-I, it has been difficult to evaluate the specific advantages conferred by LGP2 targeting. Experiments with V-insensitive proteins revealed that the primary outcome of LGP2 interference is suppression of its ability to synergize with MDA5. LGP2's negative regulation of MDA5 and RIG-I remains intact irrespective of V protein interaction. Complementary experiments demonstrate that RIG-I can be converted to V protein sensitivity by two amino acid substitutions. These findings clarify the functions of LGP2 as a positive regulator of MDA5 signaling, demonstrate the basis for V-mediated LGP2 targeting, and broaden our understanding of paramyxovirus-host interactions.

Antiviral RNA recognition and assembly by RLR family innate immune sensors

“…The negative regulatory activity of LGP2 is independent of enzymatic activity and remains intact despite mutations that target key helicase domain residues [8, 35, 47, 79]. In contrast, helicase domain mutations disrupt LGP2’s ability to co-activate MDA5 signaling, which requires both ATP hydrolysis and RNA binding [35, 47, 79]. Unlike the RNA-induced activity of other RLRs, LGP2 has a high basal ATP hydrolysis activity that is independent of RNA binding [8, 35].…”

“…For example, we have proposed a working model in which LGP2 switches between MDA5-specific enhancement and a more general RLR negative regulation [47]. Titrating LGP2 expression into MDA5-dependent signaling demonstrates that low levels of LGP2 are synergistic with MDA5 [35, 47, 79, 82], a process that requires LGP2 ATP hydrolysis and RNA binding activities. The MDA5-stimulating activity of LGP2 is revealed in a narrow stoichiometric range, and further titration of LGP2 expression ultimately achieves an inhibitory concentration that disrupts RLR signaling activity [35, 76] [11, 66, 67].…”

MDA5 and LGP2: Accomplices and Antagonists of Antiviral Signal Transduction

“…This model would support the concept that viral evasion mechanisms could evolve to inactivate positive signaling effects of LGP2 in the MDA5 axis, as has been accomplished by paramyxovirus V proteins that antagonize both MDA5 and LGP2. By disrupting LGP2 ATP hydrolysis, V proteins prevent LGP2 RNA recognition, but negative regulatory effects remain intact (77), providing the virus with a double benefit in overcoming host immunity. Although this hypothetical model is currently based on the idea that the concentration of LGP2 determines its differential activity, it is possible and likely that additional research will reveal posttranslational modifications to LGP2 or MDA5 that regulate the switch between positive and negative actions or control more nuanced signaling activities.…”

Resurrecting ancestral structural dynamics of an antiviral immune receptor: adaptive binding pocket reorganization repeatedly shifts RNA preference

“…Viral RNA sequences can change extremely rapidly, and although structural analyses and early functional studies have suggested that RLR-RNA interactions are not strongly affected by sequence variation [52, 84], changes in viral RNA sequences might impact RLR immune signaling under some circumstances [105–107]. Many viruses biochemically ‘hide’ their RNAs to avoid host detection [108–110], and viruses are known to antagonize various aspects of the RLR system [111–113]. Unfortunately, we know almost nothing about the ecologically important viruses that may have plagued ancient animals, so determining the precise native ligands that may have driven the evolution of RLRs is not possible.…”