Evading Pattern Recognition Receptors
Following infection viral RNA is sensed by several classes of pattern recognition receptors (PPRs). The retinoic acid-like receptors (RLRs) include retinoid inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5), Toll-like receptors (TLR) – classically 3, 7 and 8 that trigger IFN pathways and cytokines production [figure 2]. Once engaged these PPRs act downstream via the kinases TANK-binding kinase-1 (TBK1) and inhibitor-κB kinases (IKKs). Such triggering leads to activation of the transcription factors interferon-regulatory factor-3 (IRF3) and 7 (IRF7), and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). These subsequently induce expression of type I IFNs (IFNα/β) and interferon stimulated genes (ISGs) [figure 2] many of which have potent antiviral activities, as well as other proinflammatory mediators e.g., cytokines, chemokines and antimicrobial peptides that are essential to initiate the host innate and adaptive immune response. In addition, the absent in melanoma 2 (AIM2)-like receptors and NOD-like receptors (NLRs) trigger the inflammasome and IL-1β and IL-18 production leading to pyroptosis [figure 2]. Other PPRs and downstream factors relevant to SARS-CoV infection subversion of innate immune responses include C-type lectins and the stimulator of interferon genes (STING). While the cGas/STING pathway is commonly associated with sensing cytosolic DNA (22), it is also activated following binding of enveloped viruses to host cells, and cytosolic viral RNA (23,24). Similar to TLRs and RLR, downstream STING engages TBK1 to active IRF3 and/or NFκB inducing type I IFN and/or proinflammatory cytokines [figure 2].
Coronaviruses have evolved several strategies to escape such innate immune recognition allowing widespread replication. Such evasion includes evolution of low genomic CpG, RNA shielding, masking of potential key antigenic epitopes as well as inhibition of steps in the interferon type I/III pathways. Generally, the zinc finger antiviral protein (ZAP) specifically binds to and degrades CpG motifs in genomes of RNA viruses. In comparison with other viruses SARS-CoV-2 has evolved the most extreme CpG deficiency of all betacoronavirus [Table 2, (25)] thereby evading ZAP action. This suggests that SARS-CoV-2 may have evolved under selective pressure in either a new host or tissues expressing high levels of ZAP (25). Another strategy to protect mRNA used by the host and many viruses is the processing of capping the 5′ end. For both host and virus RNA capping limits degradation and importantly blocks recognition by cytosolic PPRs. Like many RNA viruses SARS-CoV-2 has exploited several mechanisms to protect the 5′ ends by a cap structure of RNA generated during replication. While some viruses snatch the caps from host RNA, SARS-CoV-2, like other coronaviruses uses its own capping machinery composed of nsp10, nsp13 and the dedicated enzyme nsp16 to generate 2′-o-methyltransferase caps [suppl figure 1, (26)]. SARS-CoV-2 yields RNA caps indistinguishable from cellular mRNAs caps thereby evading detection by MDA5, and IFIT activity that target RNA for degradation [figure 2]. The importance of such capping and viral replication is supported by studies of SARS-CoV in mice lacking 2′-O-MTase activity underscoring that MDA5 and the IFIT family are critical for IFN signalling (27). While counter-intuitive, SARS-CoV uses its endoribonuclease (nsp15) to cleave its own viral RNA in the cytosol that would otherwise acts as PAMPs thus evading MDA5, protein kinase R (PKR), and OAS/RNAse L (28,29). Yet another strategy used by SARS-CoV-2 to protect the viral RNA and proteins generated during replication [suppl figure 1] is the use of replicase–transcriptase complex (RTC) or replication organelle, formed of double-membrane vesicles (30). The RTCs link with the ER-Golgi intermediate compartment (ERGIC) and Golgi apparatus shielding the virus during maturation [suppl figure 1]. Another immune evasion strategy utilized by coronaviruses is the use glycans, and likely other post translational modifications to mask immunogenic viral protein epitopes [figure 1C and D]. The envelope of SARS-CoV-2 is studded with glycoprotein spikes comprised of homotrimers spike proteins of 8-12 nm length that are heavily decorated with glycans. Each spike protein comprises of two subunits (S1 and S2) that each bear 22 glycan groups (31). Cell entry of the highly glycosylated S protein of SARS-CoV is promoted by DC-SIGN possibly augmenting virus uptake or aiding capture and transmission of SARS-CoV by DCs and macrophages (3,7,8). Similar to the spike protein the other structural, non-structural and accessory proteins are also modified by glycosylation, palmitoylation, phosphorylation, SUMOylation and ADP-ribosylation (32). Conversely, some viral proteins e.g. nsp3, possess deubiquitinating (DUB) and deISGylation activity thereby interfering with host functions targetting those that are critical for signalling transduction of innate immunity (33). Insertion of the spike protein into cell membranes during replication is a key step for virus budding. Whilst this takes place in the RTC [suppl figure 1], receptor-bound spike proteins interact with TMPRSS2 expressed on the uninfected cell surface mediate fusion between infected and uninfected cells promoting the formation of syncytia allowing the virus to spread to adjacent uninfected cells while evading detection by the immune response (32).