REVIEWS Conventional and unconventional mechanisms for capping viral mRNA Etienne Decroly1, François Ferron1, Julien Lescar1,2 and Bruno Canard1 Abstract | In the eukaryotic cell, capping of mRNA 5′ ends is an essential structural modification that allows efficient mRNA translation, directs pre-mRNA splicing and mRNA export from the nucleus, limits mRNA degradation by cellular 5′–3′ exonucleases and allows recognition of foreign RNAs (including viral transcripts) as ‘non-self’. However, viruses have evolved mechanisms to protect their RNA 5′ ends with either a covalently attached peptide or a cap moiety (7‑methyl-Gppp, in which p is a phosphate group) that is indistinguishable from cellular mRNA cap structures. Viral RNA caps can be stolen from cellular mRNAs or synthesized using either a host- or virus-encoded capping apparatus, and these capping assemblies exhibit a wide diversity in organization, structure and mechanism. Here, we review the strategies used by viruses of eukaryotic cells to produce functional mRNA 5′-caps and escape innate immunity. Pre-mRNA splicing The cap structure found at the 5′ end of eukaryotic reactions involved in the viral RNA-capping process, A post-transcriptional mRNAs consists of a 7‑methylguanosine (m7G) moi‑ and the specific cellular factors that trigger a response modification of pre-mRNA, in ety linked to the first nucleotide of the transcript via a from the innate immune system. which introns are excised and 5′–5′ triphosphate bridge1 (FIG. 1a). The cap has several exons are joined in order to form a translationally important biological roles, such as protecting mRNA Capping, decapping and turnover of host RNA functional, mature mRNA. from degradation by 5′ exoribonucleases and directing ` Nascent cellular mRNAs are generally produced in the pre-mRNA splicing and mRNA export from the nucleus2. nucleus in a 5′-triphosphate form and are modified co- In addition, the cap confers stability to mRNAs and transcriptionally by a set of cap-synthesizing enzymes. ensures their efficient recognition by eukaryotic trans‑ These enzymes are recruited by the DNA-dependent lation initiation factor 4E (eIF4E) for translation3,4. RNA polymerase RNA pol II on pausing, once the Conversely, RNA molecules with unprotected 5′ ends transcript is approximately 20–25 nucleotides long. are degraded in cytoplasmic granular compartments Interestingly, two viruses — vaccinia virus (from the called processing bodies (P-bodies)5. Uncapped RNAs, Poxviridae family of double-stranded DNA (dsDNA) such as nascent viral transcripts, may also be detected viruses) and mammalian orthoreovirus (from the 1Centre National de la as ‘non-self’ by the host cell, triggering (in mammalian dsRNA Reoviridae family) — played a major part in Recherche Scientifique and cells) an antiviral innate immune response through the the discovery of the RNA cap1,8–11, because they produce Aix-Marseille Université, production of interferons6,7. high levels of capped viral mRNAs and encode their UMR 6098, Architecture et During virus–host co-evolution, viral RNA-capping own capping machinery, allowing bona fide RNA cap Fonction des Macromolécules Biologiques, 163 avenue de pathways that lead to the same cap structure as that of synthesis in vitro. Mammalian orthoreovirus mRNAs Luminy, 13288 Marseille host mRNAs have been selected for as efficient mecha‑ and dsRNA genomes were first shown to be blocked cedex 09, France. nisms to ensure both escape from detection by the innate at the 5′ end, preventing phosphorylation by poly­ 2School of Biological Sciences, immune system and efficient production of viral pro‑ nucleotide kinase9,10. Purified mammalian orthoreo­ Nanyang Technological University, 60 Nanyang teins. Compared with canonical eukaryotic mRNA cap‑ virus preparations were then demonstrated to be able Drive, Singapore 637551, ping, viral mRNA capping is highly diverse in terms of to methylate mRNA 5′ ends, and the full mRNA cap Republic of Singapore. its genetic components, protein domain organization, structures were deciphered for both dsRNA ortho­ Correspondence to B.C. enzyme structures and reaction mechanisms. Here, we reoviruses9,10,12 (those targeting humans and insects) e-mail: Bruno.Canard@ review what is known about the various mRNA-capping and the dsDNA species vaccinia virus11, followed by afmb.univ-mrs.fr 1,13,14 doi:10.1038/nrmicro2675 pathways used by viruses that infect eukaryotes, paying many other viral species . Published online particular attention to human pathogens. We also attempt The three canonical capping reactions responsible 5 December 2011 to connect the pathways, machineries, structures and for the synthesis of the cap structure are outlined in NATURE REVIEWS | MICROBIOLOGY VOLUME 10 | JANUARY 2012 | 51 © 2011 Macmillan Publishers Limited. All rights reserved REVIEWS a Chemical structure of the RNA cap Figure 1 | RNA cap structure and canonical capping mechanisms. a | The mRNA cap consists of a O CH3 Triphosphate RNA 5′ end 7‑methylguanosine linked to the 5′ nucleoside of the + HN N mRNA chain through a 5′–5′ triphosphate bridge. The O O O Base 1 methyl group at the N7 position of the guanosine is H2N N N P P P O O O O shaded green, and the 2′-O-methyl groups of the first O– O– O– O O and second nucleotide residues, forming the cap‑1 and O O CH3 the cap-2 structures, respectively, are shaded red. b | The OH OH Base 2 O P O cap‑0 structure is formed on nascent RNA chains by the 7-methylguanosine O– O sequential action of three enzymes. First, the RNA triphosphatase (RTPase) hydrolyses the γ-phosphate of O O CH3 the nascent RNA (pppNp-RNA, in which N denotes the O P O first transcribed nucleotide and p denotes a phosphate – RNA O group) to yield a diphosphate RNA (ppNp-RNA) and inorganic phosphate (P ). Then, guanylyltransferase pppNp-RNA i b Conventional RNA-capping (GTase) reacts with the α-phosphate of GTP (Gppp), pathway releasing pyrophosphate (PPi) and forming a covalent RTPase enzyme–guanylate intermediate (Gp–GTase). The GTase P i then transfers the GMP molecule (Gp) to the 5′-diphosphate ppNp-RNA RNA to create GpppNp-RNA. In the final step, (guanine‑N7)- methyltransferase (N7MTase) transfers the methyl group PP Gp– GTase i from S‑adenosyl‑l-methionine (AdoMet) to the cap guanine to form the cap‑0 structure, 7-methyl-GpppNp (m7GpppNp), and releases S‑adenosyl‑l-homocysteine Gppp GTase (AdoHcy) as a by-product. The capping reaction is completed by methylation of the ribose‑2′-O position GpppNp-RNA of the first nucleotide by the AdoMet-dependent (nucleoside‑2 -O)-methyltransferase (2 OMTase), Significant viruses ′ ′ AdoMet generating the cap‑1 structure (m7GpppNm p). The • HIV (Retroviridae) 2′-O • HBV (Hepadnaviridae) N7MTase box contains examples of viruses that acquire their cap • HSV (Herpesviridae) AdoHcy structures using the cellular capping machinery or encode • Papillomaviruses (Papillomaviridae) their own viral capping machineries that adopt the • Smallpox virus (Poxviridae) m7GpppNp-RNA (cap-0) canonical pathway. Question marks indicate viruses that • Rotaviruses (Reoviridae) are likely to follow this conventional pathway. The RNAs • BTV (Reoviridae) • Yellow fever virus? (Flaviviridae) AdoMet capped by viral enzymes are indistinguishable from • Dengue virus? (Flaviviridae) 2′OMTase cellular mRNA and can thus be translated into proteins • West Nile virus? (Flaviviridae) AdoHcy by the cellular ribosomal machinery. BTV, bluetongue • SARS CoV? (Coronaviridae) virus; HBV, hepatitis B virus; HSV, herpes simplex viruses; m7GpppNmp-RNA (cap-1) SARS CoV, severe acute respiratory syndrome coronavirus. Nature Reviews | Microbiology γ-phosphate FIG. 1b The third phosphate attached . This pathway is found in all eukaryotic species, Following translation, the lifespan of an mRNA at the 5′ end of the ribose as well as in most DNA viruses and members of the molecule is controlled by two main processes in moiety of a nucleotide. family Reoviridae, and consists of the following eukary­otic cells: first, the removal of its poly(A) tail and reactions: hydrolysis of the γ‑phosphate of the primary subsequent 3′-to‑5′ exonucleolytic degradation, ‘Ping-pong’ mechanism ′ A two-step mechanism in transcript by an RNA 5 -triphosphatase (RTPase); and second, an mRNA-decapping step that allows which a substrate molecule transfer of GMP to the 5′-diphosphate RNA 5′-to‑3′ exo­nucleolytic degradation (see REFS 17,18 for first forms a (covalent) link (ppNp-RNA; in which N is the first transcribed a review). Interestingly, decapped RNA may apparently with the enzyme and is then nucleotide and p is a phosphate group) by a guanylyl­ be re-capped by the combined action of an as-yet- transferred to an acceptor transferase (GTase) through a ‘ping-pong’ mechanism, unknown 5′-monophosphate kinase19 interacting with molecule to yield a product. leading to the formation of GpppNp-RNA; and a host cell GTase that is also present in minor amounts REF. 20 Poly(A) tail methyl­ation of the guanosine moiety by a cap- in the cytoplasm (see for a review). Cytoplasmic A string of AMP that is added specific S‑adenosyl‑l-methionine (AdoMet)-dependent decapping is catalysed by DCPS, a member of the to the 3′ end of mRNA. (guanine‑N7)-methyltransferase (N7MTase), pro‑ NUDIX hydrolase superfamily, and stimulated by decap‑ viding the minimal RNA cap chemical structure, ping enhancer proteins. Following RNA decapping in NUDIX hydrolase superfamily named cap‑0 (m7GpppNp), which is recognized by the P‑bodies, transcripts are quickly degraded by 5′-to‑3′ A family of proteins that 15 ′ hydrolyse a wide range of translation factor eIF4E . Further methylation reac‑ exonucleases such as XRN1. Thus, 5 -triphosphate organic pyrophosphates, tions catalysed by (nucleoside‑2′-O)-methyltransferases mRNAs are almost completely absent in the cyto‑ including NDPs, NTPs, (2′OMTases) can occur on the first and second nucleo­ plasm. The eukaryotic cell has consequently evolved dinucleoside and tides 3′ to the triphosphate bridge, yielding cap‑1 mechanisms to sense triphosphate RNA as non-self diphosphoinositol (m7GpppNm ), and cap‑2 (m7GpppNm pNm p) and uses these RNA species to trigger an innate polyphosphates, nucleotide 2′-O 2′-O 2′-O (FIG.