Negative-Strand RNA Virus L Proteins: One Machine, Many Activities
2015; Cell Press; Volume: 162; Issue: 2 Linguagem: Inglês
10.1016/j.cell.2015.06.063
ISSN1097-4172
Autores Tópico(s)Viral Infections and Outbreaks Research
ResumoStructures of L proteins from La Crosse orthobunyavirus and vesicular stomatitis virus reveal insights into RNA synthesis and distinctive mRNA capping mechanisms of segmented and non-segmented negative-sense single-strand RNA viruses. Structures of L proteins from La Crosse orthobunyavirus and vesicular stomatitis virus reveal insights into RNA synthesis and distinctive mRNA capping mechanisms of segmented and non-segmented negative-sense single-strand RNA viruses. Negative-strand RNA viruses (NSVs) are responsible for a wide range of diseases in plants, animals, and humans, and can be broadly categorized as segmented and non-segmented viruses. Orthomyxoviruses such as influenza A contain six to eight RNA genomic segments, Bunyaviruses such as hantavirus contain three, and Arenaviruses such as Lassa virus contain two segmented RNAs. Non-segmented NSVs (Mononegavirales) include many deadly sporadic human pathogens such as Ebola, measles, and rabies viruses; vesicular stomatitis virus (VSV) is a widely studied prototype. All NSVs carry L proteins, the viral RNA-dependent RNA polymerases (RdRps), which are responsible for replication and transcription of viral genes. The structure of VSV L protein reported in this issue of Cell (Liang et al., 2015Liang B. Li Z. Jenni S. Rahmeh A.A. Morin B.M. Grant T. Grigorieff N. Harrison S.C. Whelan S.P.J. Cell. 2015; 162 (this issue): 314-327Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar) by Sean Whelan and colleagues, is the first representative from the non-segmented NSVs. Another recent structural study of L protein from La Crosse orthobunyavirus (LACV), a segmented NSV, by Stephen Cusack and colleagues was also published in Cell (Gerlach et al., 2015Gerlach P. Malet H. Cusack S. Reguera J. Cell. 2015; 161: 1267-1279Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The structures of VSV and LACV L proteins, together with the structures of influenza A and influenza B polymerases (Pflug et al., 2014Pflug A. Guilligay D. Reich S. Cusack S. Nature. 2014; 516: 355-360Crossref PubMed Scopus (329) Google Scholar, Reich et al., 2014Reich S. Guilligay D. Pflug A. Malet H. Berger I. Crépin T. Hart D. Lunardi T. Nanao M. Ruigrok R.W. Cusack S. Nature. 2014; 516: 361-366Crossref PubMed Scopus (309) Google Scholar), reveal that the architecture of the RdRp core remains invariant in NSV L proteins despite their high amino acid sequence divergence (Figures 1A and 1B ). All NSV RdRps share similar structural and functional characteristics required for RNA synthesis, including RNA template entry and exit, product exit, and catalytic addition of ribonucleotides. Both orthobunyavirus and influenza are segmented NSVs but orthobunyavirus has a single chain cytoplasmic L protein, while influenza viruses have a heterotrimeric (PA, PB1, and PB2) polymerase that functions in the host cell nucleus. Despite these notable differences, both polymerases have remarkably similar molecular architectures and domain structures. Segmented RNA genes are packaged as viral ribonucleoproteins (vRNPs) in which both the 3′- and 5′-ends of vRNA interact with the RdRp (Jorba et al., 2009Jorba N. Coloma R. Ortín J. PLoS Pathog. 2009; 5: e1000462Crossref PubMed Scopus (119) Google Scholar). The segmented NSV polymerases form complexes with both 3′- and 5′-vRNA ends, and binding of the 5′ end vRNA induces conformational rearrangements in orthobunyavirus and influenza RdRps. This information together with the distinctly visible channels for template entry/exit, for NTP entry, and product exit in the structures led the authors to propose a model for RNA synthesis by bunyavirus RdRp (Gerlach et al., 2015Gerlach P. Malet H. Cusack S. Reguera J. Cell. 2015; 161: 1267-1279Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) that would likely hold true for influenza (Pflug et al., 2014Pflug A. Guilligay D. Reich S. Cusack S. Nature. 2014; 516: 355-360Crossref PubMed Scopus (329) Google Scholar, Reich et al., 2014Reich S. Guilligay D. Pflug A. Malet H. Berger I. Crépin T. Hart D. Lunardi T. Nanao M. Ruigrok R.W. Cusack S. Nature. 2014; 516: 361-366Crossref PubMed Scopus (309) Google Scholar); the channels are more clearly defined in orthobunyavirus RdRp than those in influenza. Functionally, neighboring RdRps in oligomeric states of influenza polymerase could potentially be associated with RNA replication (Chang et al., 2015Chang S. Sun D. Liang H. Wang J. Li J. Guo L. Wang X. Guan C. Boruah B.M. Yuan L. et al.Mol. Cell. 2015; 57: 925-935Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, Jorba et al., 2009Jorba N. Coloma R. Ortín J. PLoS Pathog. 2009; 5: e1000462Crossref PubMed Scopus (119) Google Scholar). In addition to replicating the viral genome, NSV polymerases also transcribe the positive-sense viral mRNAs using the same vRNA template. To initiate translation from viral mRNAs, addition of an m7Gppp cap at the 5′-end and polyadenylation (polyA) of a 3′-terminal tail are required. Non-segmented and segmented NSVs differ significantly in how this is achieved. All segmented NSVs use a "cap-snatching" mechanism (Plotch et al., 1981Plotch S.J. Bouloy M. Ulmanen I. Krug R.M. Cell. 1981; 23: 847-858Abstract Full Text PDF PubMed Scopus (540) Google Scholar) involving host mRNA binding and cleavage to create a short capped primer (Figure 1C); the influenza polymerase structures help in understanding the structural basis of this unique process in the context of the full polymerase (Reich et al., 2014Reich S. Guilligay D. Pflug A. Malet H. Berger I. Crépin T. Hart D. Lunardi T. Nanao M. Ruigrok R.W. Cusack S. Nature. 2014; 516: 361-366Crossref PubMed Scopus (309) Google Scholar). By contrast, the non-segmented NSV L proteins possess capping enzyme activity, which add an m7Gppp cap to the 5′-end of the nascent mRNA. Eukaryotes, double-strand (ds)RNA viruses such as rotaviruses, and dsDNA viruses such as chlorella virus follow a conventional sequence of enzymatic events of (1) RNA 5′-triphosphatase → (2) guanylyltransferase (GTase) → (3) guanine-N7 methyltransferase (MTase) for adding a m7Gppp cap at the 5′-end of a nascent mRNA. Non-segmented NSVs including VSV, interestingly exhibit an unconventional capping mechanism (Ogino and Banerjee, 2007Ogino T. Banerjee A.K. Mol. Cell. 2007; 25: 85-97Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar) that involves a polyribonucleotidyl transferase (PRNTase) enzyme. In VSV transcription, the capping event outlined in Figure 1D can occur if RdRp is paused after transcription of 31 nucleotides. In the structure of VSV L protein, the PRNTase domain is resting on the polymerase core and positioned away from the MTase domain (Figure 1B). In the PRNTase domain, the conserved GTP-binding site and the GTase active site, which would form a covalent H1277-p-RNA intermediate (Figure 1D), are ∼10 Å apart, appropriate for carrying out the PRNTase reaction. The PRNTase domain, however, must move away from the RdRp core to permit RNA synthesis, probably bringing the PRNTase and MTase into close proximity for 2′-O and N7 methylations of the cap. After the synthesis of an mRNA, the L protein, like influenza polymerase, adds a 3′-polyA tail by iterative transcription of a U7 tract. Both structures of L proteins (Figures 1A and 1B) represent pre-initiation states, and it is expected that the polymerase machineries of all NSVs span multiple conformational states for accomplishing their multiple tasks. The cryo-EM structure determination at 3.8 Å resolution of VSV L protein, an asymmetric protein with molecular weight <250 kDa, represents a significant experimental advance. Dramatic advances in cryo-EM technology, including the deployment of direct electron detectors, have only recently enabled determination of complicated biological structures in atomic detail, which formerly was only possible by X-ray crystallography (Cheng, 2015Cheng Y. Cell. 2015; 161: 450-457Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). Additionally, a pseudo-crystallographic refinement protocol is used for obtaining a reliable model of the VSV L protein. A P1 unit cell is constructed to cover the solvent-corrected EM electron density map, and the structure factors are calculated from the experimental map. The structure factor amplitudes and phases are used to refine the L protein model following a restrained macromolecular crystallographic refinement protocol. Routine use of this procedure in addition to real-space fitting/correlation could enhance the quality of cryo-EM macromolecular structures. An alternative approach that is under development in the Phenix program system is to perform all of these refinement operations in real-space using the cryo-EM-derived experimental electron density map. Viral polymerases are among the leading therapeutic targets for treating infections caused by viruses. The recently approved ribonucleoside analog sofosbuvir is highly successful in treating hepatitis C infection, and HIV-1 reverse transcriptase has been targeted by 13 FDA-approved nucleoside and non-nucleoside drugs (Das and Arnold, 2013Das K. Arnold E. Curr opin virol. 2013; 3: 111-118Crossref PubMed Scopus (108) Google Scholar). Structural insights into L proteins may facilitate targeting these large multifunctional molecular machines with nucleoside analogs, and with specific allosteric inhibitors that would restrict the conformational mobility of an L protein. Capping mechanisms for non-segmented and segmented viral mRNAs are different from capping in eukaryotic including human mRNAs and therefore serve as promising targets for drug discovery. Structural insights into these distinct capping mechanisms by the L proteins of NSVs are particularly useful to develop effective and selective antiviral strategies. Structural Insights into Bunyavirus Replication and Its Regulation by the vRNA PromoterGerlach et al.CellMay 21, 2015In BriefThe structure of the monomeric bunyavirus polymerase reveals that divergent segmented negative-strand RNA virus polymerases have a common overall architecture, explains how viral RNA binding allosterically regulates polymerase activity, and suggests a replication model that could apply to all related RNA viruses. Full-Text PDF Open AccessStructure of the L Protein of Vesicular Stomatitis Virus from Electron CryomicroscopyLiang et al.CellJuly 2, 2015In BriefThe vesicular stomatis virus (VSV) L protein is the prototype of the single-chain RNA-dependent RNA polymerase, 5′ capping enzyme, and methyltransferase in all non-segmented, negative-strand RNA viruses. The structure of VSV-L is now determined by electron cryomicroscopy at 3.8 Å resolution. Full-Text PDF Open Archive
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