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PACT is required for MDA5-mediated immunoresponses triggered by Cardiovirus infection via interaction with LGP2
Masahiko Miyamoto1, Akihiko Komuro1
1Department of Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata 956-8603, Japan.
Abstract:
Laboratory of genetics and physiology 2 (LGP2) and melanoma differentiation-associated gene 5 (MDA5) cooperatively detect viral RNA in the cytoplasm of Cardiovirus-infected cells and activate innate immune responses. Here, we evaluated whether the double-stranded RNA-binding protein PACT plays a role in this anti-viral response to further elucidate the mechanism. Immunoprecipitation experiments demonstrated that PACT interacts with LGP2 and that this interaction is enhanced by encephalomyocarditis virus (EMCV) infection. In vitro interaction analyses using purified recombinant proteins confirmed that the single-stranded Theiler's murine encephalitis virus genome enhanced the interaction between LGP2 and PACT. Small interfering RNA knockdown experiments further indicated that PACT is required for Cardiovirus-triggered interferon responses. To support this functional interaction with LGP2, overexpressed PACT was shown to enhance EMCV-triggered interferon promoter activity only when LGP2 and MDA5 were co-expressed but not when MDA5 is expressed alone. Together, our findings indicate a possible role of PACT in regulating the Cardiovirus-triggered immune responses mediated by MDA5 and LGP2, which opens the door to novel therapeutic strategies in interferon-related autoimmune diseases and cancer.
Insights
The protein PACT interacts with LGP2 and MDA5 to enhance antiviral responses against Cardiovirus. This discovery may lead to new therapies for autoimmune diseases and cancer.
Area of Science:
- Immunology
- Molecular Biology
- Virology
Background:
- Laboratory of genetics and physiology 2 (LGP2) and melanoma differentiation-associated gene 5 (MDA5) detect viral RNA and trigger innate immunity.
- The role of the double-stranded RNA-binding protein PACT in this antiviral pathway was unclear.
Purpose of the Study:
- To investigate the role of PACT in the antiviral response mediated by LGP2 and MDA5.
- To elucidate the mechanism of PACT's involvement in Cardiovirus detection and immune activation.
Main Methods:
- Immunoprecipitation assays to detect protein interactions.
- In vitro interaction analyses with recombinant proteins.
- Small interfering RNA (siRNA) knockdown experiments.
- Interferon promoter activity assays.
Main Results:
- PACT directly interacts with LGP2, and this interaction is strengthened by encephalomyocarditis virus (EMCV) infection.
- The single-stranded Theiler's murine encephalitis virus genome enhances the LGP2-PACT interaction.
- PACT is essential for Cardiovirus-induced interferon responses, as shown by siRNA knockdown.
- PACT enhances EMCV-triggered interferon promoter activity only when LGP2 and MDA5 are co-expressed.
Conclusions:
- PACT plays a significant role in regulating Cardiovirus-triggered innate immune responses via the LGP2-MDA5 pathway.
- These findings suggest potential therapeutic strategies targeting PACT for interferon-related autoimmune diseases and cancer.