RNA‐virus proteases counteracting host innate immunity

2A^pro cleaves MDA5 and MAVS

The picornavirus 2A^pro modulates the MDA5–MAVS‐mediated antiviral pathway. The Coxsackievirus B3 (CVB3), poliovirus (PV), and EV‐A71 2A^pros were shown to cleave both MDA5 and MAVS, leading to inhibition of IFNβ and type‐III IFN (IFNλ1‐4) production (Fig. 2A) 76, 77, 78. Feng et al. 77 concluded that enteroviruses use a common strategy to antagonize the host IFN response. Furthermore, these authors found that MDA5 and MAVS were degraded by a caspase‐proteasome‐independent pathway in CVB3‐infected cells. In contrast, Barral et al. 80 reported that MDA5 was cleaved via the caspase‐proteasome‐dependent pathway in PV infection.

2A^pro cleaves TRIF

TRIF is an important adaptor in the TLR3 pathway 41. The protein level of TRIF is reduced in CVB3‐infected cells due to the 3C^pro 81 (see below). In 2016, Lind et al. 78 found that CVB3 2A^pro also cleaves TRIF (Fig. 2A), and further antagonizes type‐I and type‐III IFN production.

Does the 2A^pro affect the JAK‐STAT pathway?

EV‐A71 infection leads to increased IFNβ levels but inhibits the transcription of ISGs in vivo 82. Lu et al. 82 found that the 2A^pro reduces IFNAR1 expression levels to impede the JAK‐STAT pathway (Fig. 2B), thereby leading to a decreased production of ISGs. Furthermore, the protease activity of the 2A^pro was essential for downregulating IFNAR1. However, Liu et al. 83 reported that EV‐A71 infection did not alter IFNAR1 but instead JAK1 expression in vivo. Furthermore, overexpressing viral 2A^pro (or 3C^pro) did not affect the JAK1 expression level. Conclusively, these authors demonstrated that the 2A^pro does not act as an antagonist to the JAK‐STAT pathway, although EV‐A71 infection does affect this signaling pathway by inhibiting JAK1 expression 83. Very recently, Wang et al. reported that EV‐A71 infection leads to degradation of karyopherin‐α1 (KPNA1), a nuclear localization signal receptor for phosphorylated STAT1. Thus, STAT1 transport into the nucleus is blocked, thereby shutting off the JAK‐STAT pathway 84. Interestingly, these authors found that neither the 2A^pro nor the 3C^pro is the culprit here; instead, it is caspase‐3 activated by the virus infection that degrades KPNA1 84. However, it is still unclear whether 2A^pro can affect the JAK‐STAT pathway by other mechanisms or does not affect this signaling pathway at all.

Finally, the 2A^pro can degrade PABP (poly(A)‐binding protein) and eIF4G (eukaryotic initiation factor 4G) to shut down the host translation machinery 85, 86, 87, thereby globally inhibiting the production of antiviral host proteins.

3C^pro modulates RIG‐I, MDA5, and MAVS

The picornavirus 3C^pro inhibits the RLR signaling pathway. EV‐A71 3C^pro has been reported to bind the N‐terminal CARDs of RIG‐I without digesting RIG‐I, thereby inhibiting the recruitment of MAVS and disrupting the type‐I IFN response 92. At variance with this report, Feng et al. 77 found that not only the EV‐A71 enzyme but also the CVB3 and PV 3C^pros do cleave RIG‐I in vivo. EMCV 3C^pro can cleave RIG‐I in vitro 93. A caspase‐mediated degradation of RIG‐I was also observed in EMCV‐infected cells 93. The exact mechanism of the RIG‐I regulation by 3C^pro needs to be further investigated; in any case, the partly conflicting observations mentioned above indicate that RIG‐I is an important target antagonized by the picornavirus 3C^pro.

Like RIG‐I, MDA5 binds MAVS to activate downstream cascades of the innate immune system (see above). Lei et al. 92 found by co‐immunoprecipitation (co‐IP) that EV‐A71 3C^pro can bind MDA5 (Fig. 2A). Furthermore, Rui et al. 94 detected by co‐IP that the CV‐A16, CV‐A6, or EV‐D68 3C^pros can also bind MDA5, thus disrupting the MDA5–MAVS interaction. Interestingly, these 3C^pros do not digest MDA5 and a proteolytically inactive, mutated 3C^pro can also prevent MDA5 from activating IFN.

Differently, HAV can cleave MAVS through the 3ABC precursor protein [the 3A (a membrane anchor protein), 3B (VPg) plus protease domain] but not through the mature 3C^pro alone 95. Mukherjee et al. 81 demonstrated that MAVS was cleaved between Gln148 and Ala149 by the 3C^pro in CVB3‐infected cells. Recently, the picornavirus Seneca Valley virus (SVV) 3C^pro was also shown to induce cleavage of MAVS at the same position, Gln148↓Ala149 (↓: cleavage site) 96. Therefore, both viruses suppress the antiviral IFN production through cleavage of MAVS.

3C^pro cleaves TRIF

The picornavirus 3C^pro can also interfere with the TLR3 pathway. As mentioned above, the C‐terminal TIR domains of dimeric TLR3 interact with TRIF to stimulate the downstream IRF3 and NF‐κB activities. CVB3 3C^pro was demonstrated to cleave TRIF (Fig. 2A), thereby blocking the downstream type‐I IFN production in CVB3‐infected cells 81. In total, five cleavage sites in TRIF (Gln190↓Gly191, Gln653↓Ser654, Gln659↓Ser660, Gln672↓Ser673, and Gln702↓Ala703) were found 81. Furthermore, the 3C^pro of EV‐A71 or EV‐D68 can also cleave TRIF, leading to inactivation of the signaling along the IRF3 and NF‐κB pathways in vivo 97, 98. However, the EV‐A71 3C^pro processes only one site between Gln312 and Ser313, while the EV‐D68 protease cleaves two sites (Gln312↓Ser313, Gln653↓Ser654) 97, 98. It seems that the different 3C^pros possess slightly different cleavage patterns on TRIF. Whether these subtleties are linked to any difference in biological response needs to be answered. Similarly to the 3ABC precursor digesting MAVS (see above), the HAV protease can also cleave TRIF in vivo 99, but the degradation appears to be exclusively performed by the 3CD (protease‐polymerase precursor) protein, not by mature 3C protease alone. The two cleavage sites in TRIF are Gln190↓Gly191 and Gln554↓His555 99. The observation that some precursor proteins (such as HAV 3ABC, 3CD) seem to be involved in counteracting the host immune response should motivate further investigations into more protease precursors, instead of looking only into mature proteases.

3C^pro cleaves IKKγ (also named NEMO)

The FMDV 3C^pro can process the porcine IKKγ (also called NEMO) at the unusual cleavage site Gln384↓Arg385, thereby removing the C‐terminal zinc‐finger domain of this protein 100 (these authors erroneously misnumbered Gln384 as Gln383), thus blocking the signaling pathways of NF‐κB and IRF3. From the same group, Wang et al. 101 reported that mature HAV 3C^pro can cleave NEMO at position Gln304↓Ala305, thereby antagonizing type‐I IFN production. These authors also reported that the precursor proteins 3ABC or 3CD of HAV can also cleave NEMO but with less efficiency 101.

3C^pro cleaves TANK

TANK (TRAF family member‐associated NF‐κB activator), as a positive regulator, interacts with the TBK1/IKKε complex to enhance type‐I IFN production 102. However, the role of TANK in regulating the NF‐κB pathway is a matter of debate. Chariot et al. 103 reported that TANK binds IKKγ (NEMO) and upregulates the NF‐κB pathway. Blocking the TANK–IKKγ interaction by deleting the TANK‐binding domain of IKKγ impairs NF‐κB activation 103. However, Lys63‐linked polyubiquitination of TRAF6 is required for NF‐κB activation 104. Wang et al. 105 proposed that TANK acts as a negative regulator of the NF‐κB pathway by inducing TRAF6 deubiquitination.

The EMCV 3C^pro can cleave TANK at two sites, Gln197↓Ala198 and Gln291↓Gly292 106. Huang et al. 106 reported that NF‐κB signaling increased when TANK was processed by EMCV 3C^pro. According to Wang et al.'s proposal 105 (see above), the explanation might be that the degradation of TANK can release TANK–TRAF6‐mediated NF‐κB inhibition. From the same group, Huang et al. 107 reported that the EMCV 3C^pro can disrupt the TANK–TBK1–IKKε–IRF3 complex by cleaving TANK, thus decreasing type‐I IFN production. It is of interest to further investigate the reason why the EMCV 3C^pro processes TANK to stimulate the NF‐κB pathway but downregulate the IRF3 pathway. Very recently, Qian et al. 96 showed that the SVV 3C^pro processes TANK at two cleavage sites, Glu272↓Phe273 and Gln291↓Gly292. Whereas the former site is unusual because of the large P1' residue, the latter is also cleaved by the EMCV 3C^pro, indicating that it may be a conserved target on TANK for different picornavirus 3C^pros.

3C^pro cleaves TAK1/TAB 1/TAB 2/TAB 3

The EV‐A71 3C^pro uses another mechanism to deactivate the NF‐κB pathway. As mentioned above, TAK1 can bind TAB 1 to form a complex 42. This complex recruits TAB 2 and TAB 3, yielding the quaternary TAK1/TAB 1/TAB 2/TAB 3 43, 44. The combination of TAK1 and TABs can activate IKKs (such as IKKα, IKKβ, and IKKγ) 45, thereby upregulating the NF‐κB pathway. The EV‐A71 3C^pro cleaves TAK1 (Gln360↓Ser361), TAB 1 (Gln414↓Gly415 and Gln451↓Ser452), TAB 2 (Gln113↓Ser114), and TAB 3 (Gln173↓Gly174 and Gln343↓Gly344) in vivo (Fig. 2A) 108. Recently, the CV‐A16, CV‐A6, and EV‐D68 3C^pros have also been shown to process TAK1 94. In summary, the enterovirus 3C^pro impairs NF‐κB activation.

3C^pro cleaves IRFs

IRF7 stimulates type‐I IFN production, such as IFNα, thereby activating the JAK‐STAT pathway in adjacent cells 109. The 3C^pro of EV‐A71 can process IRF7 at a cleavage site between Gln189 and Ser190 in vitro and in vivo, while the 3C^pro of EV‐D68 does so at two sites (Gln167↓Ala168 and Gln180↓S190) 110, 111.

Furthermore, IRF9 interacts with STAT1 and STAT2 to form a complex, ISGF3 in the JAK‐STAT pathway, thereby stimulating ISG production (Fig. 2B) 30, 112. The EV‐A71 3C^pro can cleave IRF9 in EV‐A71‐infected cells as well as in an in vitro assay, resulting in reduced IFN signaling 113.

Finally, the 3C^pro can digest PABP to shut off host translation 114, 115, thereby globally inhibiting the production of antiviral proteins.

HCV NS3/NS4A^pro cleaves MAVS

In 2005, RIG‐I was shown to detect the 3′ UTR (untranslated region) of the HCV genome 151. However, later Cao et al. 59 demonstrated that MDA5 plays a major role in recognizing the 3′ UTR of HCV while RIG‐I seems to be less important. Subsequently, MDA5 (or RIG‐I) binds MAVS, thereby activating the downstream IRF3 and NF‐κB pathways. HCV NS3/NS4A^pro cleaves MAVS (also named Cardif, as mentioned above; Fig. 2A) at the cleavage site Cys508↓His509 152, thereby disrupting the host immune response. The NS3/NS4A^pro of GB virus B (GBV‐B), from the same genus as HCV, cleaves MAVS at the same site as the HCV enzyme 153. These authors further found that MAVS was released from the mitochondrial membrane to the cytosol due to this cleavage 153. Because the location of MAVS on the mitochondrial membrane is essential for its functions, this observation could explain how MAVS fails to transmit the signal downstream after being processed by NS3/NS4A^pro 153. Furthermore, NS3/NS4A proteases from Hepaciviruses infecting other animals (such as monkeys, rodents, horses, and cows) can cleave their cognate MAVS proteins 154. The cleavage of MAVS presents a common mechanism used by Hepaciviruses to regulate the host immune response.

NS3/NS4A^pro cleaves Riplet (upstream regulator of RIG‐I)

As mentioned above, RIG‐I is ubiquitinated by TRIM25 for its activation 62. Oshiumi et al. 155 reported that the protein Riplet (an E3 ubiquitin ligase) is a prerequisite for TRIM25 stimulation of RIG‐I signaling. Knocking out Riplet abrogates the expression of type‐I IFN in response to HCV RNA 155. HCV NS3/NS4A^pro can cleave Riplet at position Cys21↓Ile22 in vitro, because the residues 16‐EDDLGC‐21 of Riplet are similar to the consensus cleavage motif D/E‐XXXX‐C/T of HCV NS3/NS4A protease. As a consequence, RIG‐I activation is abolished 155.

NS3/NS4A^pro modulates TRIF of the TLR3 pathway

TLR3 is sensitive to the intermediate dsRNA of HCV replication 156. Upon sensitization, its C‐terminal TIR domain can interact with TRIF to stimulate the downstream cascades. HCV NS3/NS4A^pro cleaves TRIF at the site Cys372↓Ser373 in vitro and in vivo (HEK293 cells) to disrupt the TLR3 pathway 157. In contrast to this observation, Dansako et al. 158 found that HCV NS3/NS4A cannot cleave TRIF in PH5CH8 (immortalized human hepatocytes), HeLa, and Huh‐7‐derived cells. The effects of the NS3/NS4A protease on TRIF need to be further investigated, in order to resolve this ambiguity.

NS3/NS4A^pro modulates importin β1

Very recently, Gagné et al. 159 found that the HCV NS3/NS4A^pro can cleave importin β1 (IMPβ1; Fig. 2A). IMPβ1, a nucleocytoplasmic transport receptor, transports proteins from the cytoplasm to the nucleus. The HCV NS3/NS4A protease triggers the degradation of IMPβ1 and inhibits or disrupts the nucleocytoplasmic trafficking of IRF3 as well as NF‐κB p65, thus preventing the host immune response 159.

The NS2B/NS3^pro cleaves STING

As mentioned above, STING can upregulate IRF3 signaling (Fig. 2A) 130, 131. DENV NS2B/NS3^pro cleaves human STING at the site Arg94‐Arg95↓Gly96 in vivo and thereby inhibits induction of IFNβ 163, 164. However, this protease is unable to cleave RIG‐I, TLR3, TBK1, IKKε, IRF3, and IRF7 163. Recently, Liu et al. 165 reported that the full‐length DENV NS3 (including the C‐terminal helicase), but not the NS2B, can be modified by Lys27‐linked polyubiquitination when co‐transfecting Ub and NS3 in HEK 293T cells. The ubiquitinated NS3 facilitates recruitment of NS2B, that is, the formation of the NS2B/NS3 protease, thereby enhancing the cleavage of STING. Furthermore, these authors found that the ER protein SCAP (sterol regulatory element‐binding protein (SREBP) cleavage‐activating protein) can bind NS2B and inhibit polyubiquitination of NS3, thus disrupting the formation of NS2B/NS3^pro and the cleavage of STING 165.

The NS2B/NS3 protease cleaves two mitofusins (MFN1, MFN2)

MFN1 and MFN2 regulate the mitochondrial fusion; in particular, MFN1 is required for the RLR signaling pathway 166. Yu et al. 167 demonstrated that the DENV NS2B/NS3 protease can cleave MFN1 (Arg539‐Asn540↓Ala541) and MFN2 (Arg563‐Arg564↓Ala565) in cells. Subsequently, the cleaved MFNs suppress mitochondrial fusion and disrupt IFN production. Interestingly, the homologous protease from JEV cannot cleave MFNs for unknown reasons 167. It is worth investigating whether or not other flavivirus proteases modulate MFNs.

N^pro binds IRF3 and IRF7

The N^pros of CSFV and BVDV directly bind IRF3 and induce degradation of the factor by the host proteasome, thus interfering with IFN production 169, 170, 171, 172. According to point mutation experiments, both the protease domain and the zinc‐binding domain are essential for N^pro binding to IRF3 168, 173, 174.

Furthermore, the CSFV N^pro can also bind IRF7 and downregulate the IRF7 protein level in porcine DC cells, thus limiting type‐I IFN production 175. Also, Fiebach et al. 175 found that the zinc‐binding domain but not the protease domain is required to bind IRF7.

N^pro interacts with IκBα (NF‐κB inhibitor)

The CSFV N^pro binds IκBα in vitro and in vivo 176. However, the enzyme does not affect the NF‐κB activity 176. The BVDV N^pro cannot block the NF‐κB pathway either 171. The role of the N^pro‐IκBα interaction, thus, remains unclear for the time being.