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.
Abstract
The ongoing Corona Virus Disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has emphasized the urgent need for antiviral therapeutics. ...The viral RNA-dependent-RNA-polymerase (RdRp) is a promising target with polymerase inhibitors successfully used for the treatment of several viral diseases. We demonstrate here that Favipiravir predominantly exerts an antiviral effect through lethal mutagenesis. The SARS-CoV RdRp complex is at least 10-fold more active than any other viral RdRp known. It possesses both unusually high nucleotide incorporation rates and high-error rates allowing facile insertion of Favipiravir into viral RNA, provoking C-to-U and G-to-A transitions in the already low cytosine content SARS-CoV-2 genome. The coronavirus RdRp complex represents an Achilles heel for SARS-CoV, supporting nucleoside analogues as promising candidates for the treatment of COVID-19.
Coronaviruses (CoVs) stand out among RNA viruses because of their unusually large genomes (∼30 kb) associated with low mutation rates. CoVs code for nsp14, a bifunctional enzyme carrying RNA cap ...guanine N7-methyltransferase (MTase) and 3′-5′ exoribonuclease (ExoN) activities. ExoN excises nucleotide mismatches at the RNA 3′-end in vitro, and its inactivation in vivo jeopardizes viral genetic stability. Here, we demonstrate for severe acute respiratory syndrome (SARS)-CoV an RNA synthesis and proofreading pathway through association of nsp14 with the low-fidelity nsp12 viral RNA polymerase. Through this pathway, the antiviral compound ribavirin 5′-monophosphate is significantly incorporated but also readily excised from RNA, which may explain its limited efficacy in vivo. The crystal structure at 3.38 Å resolution of SARS-CoV nsp14 in complex with its cofactor nsp10 adds to the uniqueness of CoVs among RNA viruses: The MTase domain presents a new fold that differs sharply from the canonical Rossmann fold.
With sizes <50 kb, viral RNA genomes are at the crossroads of genetic, biophysical, and biochemical stability in their host cell. Here, we analyze the enzymatic assets accompanying large RNA genome ...viruses, mostly based on recent scientific advances in Coronaviridae. We argue that, in addition to the presence of an RNA exonuclease (ExoN), two markers for the large size of viral RNA genomes are (i) the presence of one or more RNA methyltransferases (MTases) and (ii) a specific architecture of the RNA-dependent RNA polymerase active site. We propose that RNA genome expansion and maintenance are driven by an evolutionary ménage-à-trois made of fast and processive RNA polymerases, RNA repair ExoNs, and RNA MTases that relates to the transition between RNA- to DNA-based life.
Gene expression originated from early protoviruses (‘replicators’) resembling (+)RNA viruses.RNA genome size expansion corelates with specific RNA-dependent RNA polymerase signature sequences.RNA genome size expansion and maintenance requires more than an increasing replication accuracy provided by the acquisition of repair exonucleases (ExoNs).The presence of RNA methyltransferase (MTase) genes corelates with increasing RNA genome size.Large RNA genomes result from the coexistence of at least a processive RNA-dependent RNA polymerase, an RNA repair ExoN, and at least one RNA MTase.
•Bunyavirus and arenavirus are important public health threats.•Bunyavirus and arenavirus molecular biology, common and differential features.•Implications of LACV L protein structure for ...understanding viral RNA synthesis.•Current state and future perspectives on bunya- and arenavirus antivirals.
Bunyaviridae and Arenaviridae virus families include an important number of highly pathogenic viruses for humans. They are enveloped viruses with negative stranded RNA genomes divided into three (bunyaviruses) or two (arenaviruses) segments. Each genome segment is coated by the viral nucleoproteins (NPs) and the polymerase (L protein) to form a functional ribonucleoprotein (RNP) complex. The viral RNP provides the necessary context on which the L protein carries out the biosynthetic processes of RNA replication and gene transcription. Decades of research have provided a good understanding of the molecular processes underlying RNA synthesis, both RNA replication and gene transcription, for these two families of viruses. In this review we will provide a global view of the common features, as well as differences, of the molecular biology of Bunyaviridae and Arenaviridae. We will also describe structures of protein and protein-RNA complexes so far determined for these viral families, mainly focusing on the L protein, and discuss their implications for understanding the mechanisms of viral RNA replication and gene transcription within the architecture of viral RNPs, also taking into account the cellular context in which these processes occur. Finally, we will discuss the implications of these structural findings for the development of antiviral drugs to treat human diseases caused by members of the Bunyaviridae and Arenaviridae families.
•This paper presents an in-depth review of coronavirus Orf1b enzymes.•It is organized in sections on RNA synthesis, RNA degradation, and RNA capping.•It discusses integration of these functions into ...the coronavirus replication/transcription complex.•It reviews published inhibitors of any of these functions.
The SARS (severe acute respiratory syndrome) pandemic caused ten years ago by the SARS-coronavirus (SARS-CoV) has stimulated a number of studies on the molecular biology of coronaviruses. This research has provided significant new insight into many mechanisms used by the coronavirus replication-transcription complex (RTC). The RTC directs and coordinates processes in order to replicate and transcribe the coronavirus genome, a single-stranded, positive-sense RNA of outstanding length (∼27–32kilobases). Here, we review the up-to-date knowledge on SARS-CoV replicative enzymes encoded in the ORF1b, i.e., the main RNA-dependent RNA polymerase (nsp12), the helicase/triphosphatase (nsp13), two unusual ribonucleases (nsp14, nsp15) and RNA-cap methyltransferases (nsp14, nsp16). We also review how these enzymes co-operate with other viral co-factors (nsp7, nsp8, and nsp10) to regulate their activity. These last ten years of research on SARS-CoV have considerably contributed to unravel structural and functional details of one of the most fascinating replication/transcription machineries of the RNA virus world. This paper forms part of a series of invited articles in Antiviral Research on “From SARS to MERS: 10years of research on highly pathogenic human coronaviruses”.
The appearance of genetic variants impacts vaccination efficiency and therapeutic options, generating a need to map and relate mutations observed in the proteome and the genome. We develop an ...user-friendly web service software (Viral Instant Mutation Viewer or VIMVer) which allows a direct identification of mutations in the genome and its counterpart in the viral proteome. Since its emergence in 2019, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), responsible for the COVID-19 pandemic, has generated an overwhelming amount of data while becoming one of the most studied viruses of the Nidovirales order. We originally developed this tool during the COVID pandemic; thus, for any SARS-CoV-2 nucleotide sequence, the web service gives a fast identification, mapping, and display of new mutations simultaneously at the nucleotide and amino acid level in comparison to a reference sequence (Wuhan-1). Furthermore, the lineage or the relative position to the known lineage of the variant of interest is available on the link to Phylogenetic Assignment of Named Global Outbreak LINeages (PANGOLIN COVID-19). The workflow presented here is available online. The source code is released under public license and can be easily adapted for further development to other viruses.
Cells sustain high rates of actin filament elongation by maintaining a large pool of actin monomers above the critical concentration for polymerization. Profilin–actin complexes constitute the ...largest fraction of polymerization‐competent actin monomers. Filament elongation factors such as Ena/VASP and formin catalyze the transition of profilin–actin from the cellular pool onto the barbed end of growing filaments. The molecular bases of this process are poorly understood. Here we present structural and energetic evidence for two consecutive steps of the elongation mechanism: the recruitment of profilin–actin by the last poly‐Pro segment of vasodilator‐stimulated phosphoprotein (VASP) and the binding of profilin–actin simultaneously to this poly‐Pro and to the G‐actin‐binding (GAB) domain of VASP. The actin monomer bound at the GAB domain is proposed to be in position to join the barbed end of the growing filament concurrently with the release of profilin.
Among RNA viruses, the order
stands out for including viruses with the largest RNA genomes currently known. Nidoviruses employ a complex RNA-synthesizing machinery comprising a variety of ...non-structural proteins (nsps). One of the postulated drivers of the expansion of nidovirus genomes is the presence of a proofreading 3'-to-5' exoribonuclease (ExoN) belonging to the DEDDh family. ExoN may enhance the fidelity of RNA synthesis by correcting nucleotide incorporation errors made by the RNA-dependent RNA polymerase. Here, we review our current understanding of ExoN evolution, structure, and function. Most experimental data are derived from studies of the ExoN domain of coronaviruses (CoVs), which were triggered by the bioinformatics-based identification of ExoN in the genome of severe acute respiratory syndrome coronavirus (SARS-CoV) and its relatives in 2003. Although convincing data supporting the proofreading hypothesis have been obtained, from biochemical assays and studies with CoV mutants lacking ExoN functionality, the features of ExoN from most other nidovirus families remain to be characterized. Remarkably, viable ExoN knockout mutants were obtained only for two CoVs, mouse hepatitis virus (MHV) and SARS-CoV, whose RNA synthesis and replication kinetics were mildly affected by the lack of ExoN function. In several other CoV species, ExoN inactivation was not tolerated, and knockout mutants could not be rescued when launched using a reverse genetics system. This suggests that ExoN is also critical for primary viral RNA synthesis, a property that poorly matches the profile of an enzyme that would merely boost long-term replication fidelity. In CoVs, ExoN resides in a bifunctional replicase subunit (nsp14) whose C-terminal part has (N7-guanine)-methyltransferase activity. The crystal structure of SARS-CoV nsp14 has shed light on the interplay between these two domains, and on nsp14's interactions with nsp10, a co-factor that strongly enhances ExoN activity
assays. Further elucidation of the structure-function relationships of ExoN and its interactions with other (viral and/or host) members of the CoV replication machinery will be key to understanding the enzyme's role in viral RNA synthesis and pathogenesis, and may contribute to the design of new approaches to combat emerging nidoviruses.
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