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).