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 and LGP2, 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.

FIG 1

Generation of a biologically active, V protein-insensitive LGP2. (A) Illustration of the key features of LGP2. LGP2 is shown as a box and positions of the helicase region, including conserved helicase motifs I to III of domain 1, motifs IV to VI of domain 2, and the C-terminal regulatory domain (CTD). Domain 2 coincides with the minimal V protein-binding region (MVBR) that is shared between MDA5 and LGP2 (7). Expanded sequence alignment illustrates relevant region of all three RLR proteins within the MVBR, and residues targeted are depicted with an asterisk. Arrow points to LGP2 R455, which is R806 of MDA5 and L714 of RIG-I; closed circle indicates RIG-I L714. (B) LGP2 interactions with V proteins. FLAG-tagged LGP2 or variants were coexpressed with HA-tagged V proteins from measles, PIV5, and Nipah virus in HEK293T cells. The cell lysates were subjected to FLAG immunoaffinity purification, and detection of coprecipitation was carried out by anti-HA immunoblotting. (C) LGP2 RGL-LEY is defective for interaction with PIV5 V protein in infected cells. HEK293T cells were transfected with FLAG-tagged LGP2 or mutant and infected with PIV5 at the indicated multiplicity of infection (MOI) for 24 h. The cell lysates were subjected to FLAG immunoaffinity purification, and detection of coprecipitation was carried out by immunoblotting with antiserum that recognizes P and V proteins. (D) LGP2 RGL-LEY retains biological activity. A total of 25 ng MDA5 was expressed with WT or mutant LGP2 titrated at 4, 20, 100, or 500 ng of vector. Cells were stimulated by transfection of high-molecular-weight (HMW) poly(I·C) for 6 h prior to luciferase assays. Student's t test indicated as follows: N.S. (not significant), P > 0.015; *, P < 0.015; **, P < 0.0015. LGP2 MI = motif I mutant (K30A); LGP2 MIII = motif III mutant (T167A S169A). Immunoblots below correspond to expression levels of representative lysates.

FIG 2

V proteins suppress MDA5 signaling enhancement by LGP2. (A) Similar to that described for Fig. 1D, WT MDA5 was expressed with various amounts of the indicated LGP2 vector (4, 20, 100, 500 ng) and stimulated with high-molecular-weight (HMW) poly(I·C) for 6 h prior to luciferase assays. Parallel experiments included measles virus V protein (MeV) or PIV5 V protein (PIV5-V) as indicated. (B) Similar to that described for panel A, but using WT MDA5 and V protein-insensitive LGP2 RGL-LEY mutant. (C) Similar to that described for panel A, but using the V protein-insensitive MDA5 R806L mutant and WT LGP2. (D) Similar to that described for panel A, but using the V protein-insensitive MDA5 R806L mutant and V protein-insensitive LGP2 RGL-LEY. For all panels, Student's t test indicated as follows: N.S. (not significant), P > 0.015; *, P < 0.015; **, P < 0.0015; ***, P < 0.00015.

FIG 3

RIG-I inhibition by LGP2 is independent of V proteins. (A) Similar to that described for Fig. 2, but using WT RIG-I coexpressed with various amounts of either WT LGP2 or LGP2 RGL-LEY in the absence or presence of MeV. (B) Similar to that described for panel A, but using PIV5 V. Student's t test indicated no significant differences for all conditions (P > 0.015).

FIG 4

Mutations to RIG-I enable direct V protein targeting. (A) RIG-I signaling assay similar to that described for Fig. 3A, but using a broader range of measles virus or PIV5 V protein expression (4, 20, 100, 500 ng). Immunoblots below demonstrate protein expression levels in representative lysates. (B) FLAG-tagged RIG-I or variants were coexpressed in HEK293T cells with HA-tagged V proteins from PIV5 and measles virus. Cell lysates were prepared and analyzed as described for Fig. 1B. (C) Combinatorial analysis of RIG-I mutations required for PIV5 V interaction. RIG-I mutants and PIV5 V were coexpressed and prepared as described above. (D) The RIG-I L714R E716G mutant is biologically active and suppressed by V proteins. Luciferase assays were carried out similar to those described for Fig. 4A, but using the RIG-I L714R E716G mutant in the presence and absence of measles virus or PIV5 V protein titration (4 ng, 20 ng, 100 ng, and 500 ng). Student's t test indicated as follows: N.S. (not significant), P > 0.05; *, P < 0.05.