Creative BioMart to Present at
                        BIO-Europe Spring Creative BioMart to Present at AACR Annual Meeting|Apr. 5-10, 2024|Booth #2953

RNA Viruses Triggered Signal Pathway

Figure: RIG-I recognize RNA viruses by detecting short dsRNAs with 5’triphosphate ends. MDA5 recognize RNA viruses by detecting long dsRNAs. LGP2 positively regulates RIG-I and MDA5 mediated virus recognition. Ubiquitin ligases TRIM25 positively regulates the activation of RIG-I, RNF125 negatively regulates the activation of RIG-I. RIG-I and MDA5 interact with IPS-1 through CARD domains. IPS-1 then activates downstream signaling proteins TRAF3, TBK1/IKK-i, and IRF3/IRF7 to induce type I IFNs. Meanwhile, IPS-1 signaling induces nuclear translocation of NF-κB via TRADD and FADD and caspase-8/-10. A cleaved fragment of caspase-8/-10 is responsible for the activation of NF-κB.

Background

Viruses infect cells to activate innate immune signaling. Innate immunity induce type I interferons production and inflammatory response to clean up viruses from cells. Viruses are divided into DNA viruses and RNA viruses. DNA viruses are recognized by TLRs (Toll-like receptors) and cGAS (cyclic GMP-AMP synthase). On the other way, RNA viruses are recognized by RLRs (RIG-I-like receptors) like RIG-I, MDA5 and LGP2. TLRs and RLRs are both belong to PRRs (pattern recognition receptors). Innate immune signalings triggered by these PRRs lead to transcriptional expression of inflammatory mediators that coordinate the elimination of pathogens and infected cells.

Nucleic acid of RNA viruses is single-stranded RNA (ssRNA) or double-stranded RNA (dsRNA). Many human diseases are caused by RNA viruses include influenza, the common cold, hepatitis C, SARS, Ebola hemorrhoragic fever, West Nile fever and polio etc. It is very important for organism to virus killing and disease prevention by antiviral response. Innate immunity is the most critical process in antiviral response.

RIG-I, MDA5 (melanoma differentiation-associated gene 5), and LGP2 are belong to the RLR (RIG-I-like receptor) family. RLR family proteins are made up of a C-terminal regulatory domain, a central DEAD box helicase/ATPase domain, and two CARDs (N-terminal caspase recruitment domains). They recognize the genomic RNA like dsRNA viruses or dsRNA produced by the replication intermediate of ssRNA viruses. RLRs are localized in the cytoplasm. Virus infection or type I Interferons stimulation can greatly enhance the expression of RLRs.

RIG-I deficiency cells like cDCs and mouse fibroblasts cannot produce type I IFNs and inflammatory cytokines effectively in response to many RNA viruses. These RNA viruses include Sendai virus (SeV), vesicular stomatitis virus (VSV), Japanese encephalitis virus (JEV), Newcastle disease virus (NDV), influenza virus and so on.

To the contrary, MDA5 deficiency cells can respond normally to the RNA viruses above. However, MDA5 deficiency cells lack the IFN responses to several Picornaviridae, like Mengo virus, Theiler’s virus, and encephalomyocarditis virus (EMCV) while RIG-I-/- cells are not lacking.

Both RIG-I and MDA5 are not essential for recognizing Dengue virus and West Nile virus, whereas RIG-I is essential for recognizing hepatitis C virus (HCV). Reovirus is a double-stranded segmented RNA virus, it promotes IFN production mainly through MDA5. However, the lacking of RIG-I or MDA5 both completely reduces the IFN production, indicating that both RIG-I and MDA5 are essential for recognition of reovirus. RNA viruses infect RIG-I-/-MDA5-/-MEFs (mouse embryonic fibroblasts) cannot produce type I IFNs. It means that RIG-I and MDA5 are both essential and sufficient for inducing type I IFN production in response to RNA viruses.

Recognition of RNA viruses signal pathway

Short dsRNA (up to 1 kb) is recognized by RIG-I, and the type I IFN-inducing activity is greatly enhanced by the presence of its 5’triphosphate. It has been assumed that RIG-I ligand is 5’triphosphate ssRNA produced in vitro. However, RIG-I was not stimulated by chemically synthesized 5’triphosphate ssRNA, it means that the activation of RIG-I needs double-stranded RNA. As for the 19~21mer minimum length dsRNA with a 5’triphosphate end, it can strongly induce type I IFNs. Moreover, synthesized dsRNAs without a 5’phosphate end or with a 5’monophosphate end also activate RIG-I. It is indicated that a 5’triphosphate end is not always necessary for activating RIG-I, but the amount of production of type I IFNs is lower than the dsRNA with a 5’triphosphate end.

The presence of dsRNA is critical for RIG-I recognition. For example, VSV infects cells can produce dsRNA, IFN-b inducing activity will reduce when the produced dsRNA was disrupted. The dsRNA fragments produced by VSV infection are approximately 2~2.5 kb, the length is much shorter than the VSV genomic RNA. VSV-infected cells generate DI (defective interfering) particles, the size of DI particles are approximately 2.2 kb. Therefore, the dsRNA fragments produced by VSV infection maybe derive from DI particles. However, this hypothesis need further studies to affirm. We know that the DI particles strongly induced type I IFNs in VSV infection cells, RIG-I must play an important role in detecting the presence of dsRNA in DI particles. To the contrary, it is difficult to detect dsRNA in influenza virus infected cells. Influenza virus genomic RNA lost it’s an activity to induce IFN after a phosphatase treatment to remove it. Thus, we speculate that 5’triphosphate end of influenza virus is necessary for recognition by RIG-I.

Differ from RIG-I, MDA5 detects long dsRNA (more than 2 kb) like poly I:C. MDA5-/-mice show stongly reduced production of type I IFNs but not IL-12 in response to poly I:C stimulation in vivo. Poly I:C become a RIG-I ligand from an MDA5 ligand by cutting down the length of the poly I:C by dsRNA-specific nuclease. It is indicated that long, but not short, dsRNA is recognized by MDA5.

The other member of RLR family LGP2 lacks a CARD domain. In vitro studies suggested that LGP2 is a negative regulator of RIG-I and MDA5 signaling. LGP2 can isolate dsRNA and inhibit RIG-I conformational changes. However, the in vivo studies of LGP2-/-mice indicated that LGP2 positively regulates both RIG-I and MDA5 mediated production of type I IFNs. Nevertheless, LGP2 is dispensable for type I IFN production after stimulation by synthetic RNAs.

C-terminal regulatory domain (RD) of RLRs family is used for binding to dsRNAs. Recent studies have shown that RIG-I and LGP2’s RD have large basic surface to form RNA-binding loops. The termini of dsRNA bind to the RNA-binding loop. However, MDA5 C-terminal RD is different from RIG-I and LGP2. MDA5 RD also has a large basic surface, but it will not from RNA-binding loop. Therefore, the activity of RNA-binding of MDA5 is much weaker than RIG-I and LGP2. DExD/H helicase domain of RLRs catalyze ATP to induce type I IFN production.

RNA viruses triggered IPS-1 downstream signal pathway

The activation of RLR signaling requires RIG-I conformation modification. The RIG-I cofomation can be modulated by ubiquitination. K63-linked polyubiquitination of RIG-I is mediated by E3 ubiquitin ligases RNF135 and TRIM25.

RLRs adaptor IPS-1 (IFN-b-promoter stimulator 1), also known as MAVS or VISA, has N-terminal CARD-domain. The CARD-domain interacts with CARDs of RLRs for triggering signaling axis. IPS-1 is located on the mitochondrial membrane. When it was cut from mitochondria by HCV NS3/4A protease, RLR signaling activity would disappear. NOD9 is reported as an inhibitor of IPS-1 which belong to NLR family member and is located on mitochondria, but recent studies showed that NOD9 is responsible for reactive oxygen generation. Therefore, the relationship of NOD9 and RLR signaling needs further studies. IPS-1 activates downstream proteins TRAF3 and TRADD. TRAF3 activates IKK-i/TBK1 (TANK-binding kinase 1), then TBK1 dimerize and pass signaling to downstream protein IRF3 (Interferon regulatory Factor 3) and IRF7 (Interferon regulatory Factor 7). IRF3 and IRF7 are phosphorylated and enter nucleus to induce type I interferons production. Another signaling is activated by TRADD and FADD. TRADD and FADD transmit signal to result in the Cleavage of caspase-8/-10. This process activates NF-κB to induce cytokines production. Both TRIF and IPS-1 signaling pathways are regulating the IFN production and the expression of IFN-induced genes.

Expression of type I IFNs is regulated through both the activation of NF-κB, IRF3 and IRF7. OF these factors, IRF3 and IRF7 are activated in response to viral infection and mainly induced type I IFNs production. In the contrast, NF-κB are activated in response to stimulations to regulate the expression of inflammation, they also participate in inducing type I IFNs production cooperative interactions with IRF3 and IRF7.

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