Innate immune restriction and antagonism of viral RNA lacking 2׳-O methylation

Abstract

N-7 and 2'-O methylation of host cell mRNA occurs in the nucleus and results in the generation of cap structures (cap 0, m(7)GpppN; cap 1, m(7)GpppNm) that control gene expression by modulating nuclear export, splicing, turnover, and protein synthesis. Remarkably, RNA cap modification also contributes to mammalian cell host defense as viral RNA lacking 2'-O methylation is sensed and inhibited by IFIT1, an interferon (IFN) stimulated gene (ISG). Accordingly, pathogenic viruses that replicate in the cytoplasm have evolved mechanisms to circumvent IFIT1 restriction and facilitate infection of mammalian cells. These include: (a) generating cap 1 structures on their RNA through cap-snatching or virally-encoded 2'-O methyltransferases, (b) using cap-independent means of translation, or (c) using RNA secondary structural motifs to antagonize IFIT1 binding. This review will discuss new insights as to how specific modifications at the 5'-end of viral RNA modulate host pathogen recognition responses to promote infection and disease.

Keywords: Immune evasion; Innate immunity; Interferon; Methylation; RNA structure; Viral pathogenesis.

Fig. 1

Overview of cap 1 structure formation in eukaryotic and viral systems. (A) Chemical structure of the G-cap and methylation sites which form cap 0, cap 1, and cap 2 structures. (B) The eukaryotic canonical capping pathway. Newly transcribed RNA (pppNp-RNA) is cleaved by RNA triphosphatase at the γ-phosphate to yield diphosphate RNA (ppNp-RNA). Guanylyltransferase, which forms part of the bifunctional capping enzyme (hCAP) that also encodes triphosphatase activity, then hydrolyzes the α-phosphate from GTP and transfers the GMP moiety to the ppNp-RNA acceptor to generate GpppNp. GpppNp-RNA is methylated by the N-7 MTase, which transfers a methyl group from an S-adenosyl-l-methionine (AdoMet) donor to the unmethylated G-cap acceptor, producing cap 0 RNA (m⁷GpppNp) and S-adenosyl-l-homocysteine (AdoHcy) as a by-product. The 2′-O-ribose MTase (hMTr1) then transfers a methyl group from the AdoMet donor to the first and second nucleotides of cap 0 RNA to generate cap 1 and cap 2 structures, respectively. (C) Generation of viral cap 1 structures through non-canonical capping and methylation pathways. Analogous to eukaryotic RNA methylation, some viruses (e.g. coronavirus and poxvirus) catalyze cap 0 and cap 1 formation separately via two distinct enzymes or enzymes complexes (left). In contrast, other viruses (e.g. flavivirus and reovirus) catalyze N-7 and 2′-O methylation sequentially via a single enzyme (right). Although the cap 1 end product is identical between host and viral transcripts structural differences in the viral and eukaryotic enzymes that catalyze these reactions and their distinct modes of ligand binding make viral 2′-O MTases potential targets for inhibitor design.

Fig. 2

Ifit1 inhibits diverse aspects of RNA translation. Canonical cap-dependent translation proceeds as follows: (1) the 40S ribosomal subunit complexes with eIF3 and the ternary complex (eIF2-GTP-Met-tRNA) to form the 43S pre-initiation complex. (2) eIF4E associates with the cap and recruits the eIF4F cap-binding complex (eIF4A, eIF4E, eIF4G). (3) eIF4F facilitates unwinding of the RNA and the 43S pre-initiation complex is recruited to form the 48S complex. (4) The 48S complex scans along the mRNA for the AUG initiator codon. (5) The 60S ribosomal subunit is recruited to the 48S complex to form the 80S initiator complex, which translates the mRNA. Ifit1/IFIT1 binds to subunits of the eIF3 complex (a) to prevent ternary complex formation or 43S formation and recruitment (Guo et al., 2000a, Hui et al., 2003, Hui et al., 2005, Kumar et al., 2014, Wang et al., 2003). (b) IFIT1 interaction with the 40S ribosomal subunit abrogates 48S formation (Kumar et al., 2014). (c) Ifit1/IFIT1 directly associates with cap 0 viral RNA and inhibits recruitment of eIF4E and eIF4F to RNA.

Fig. 3

Viral mechanisms of Ifit1 evasion and antagonism. Viruses generate cap 1 structures through (a) cap-snatching (orthomyxoviurses, bunyaviruses, arenaviruses) whereby cap 1 is cleaved from host mRNA transcripts and used as a primer for viral RNA transcription. In contrast to orthomyxoviruses, which cap-snatch from mRNA in the nucleus, bunyaviruses associate with and sequester cap 1 containing nonsense-mRNA transcripts in cellular P bodies where N protein protects cap 1 from degradation. Some viruses mimic cap 1 structures via virally-encoded 2′-O MTases (flaviviruses, coronaviruses, rhabdoviruses, paramyxoviruses, reoviruses, and poxviruses). Other viruses (picornaviruses, caliciviruses, and hepaciviruses) circumvent Ifit1-mediated restriction by using non-canonical cap-like structures (VPg) at their 5′ end or IRES-mediated cap-independent translation (b). Alphaviruses antagonize Ifit1 function directly by inhibiting association with viral RNA through the generation of stable secondary structures in the 5′-UTR (c).