Citation: Tao YJ, Ye Q (2010) RNA Virus Replication Complexes. PLoS Pathog 6(7): e1000943. https://doi.org/10.1371/journal.ppat.1000943
Editor: Hiten D. Madhani, University of California San Francisco, United States of America
Published: July 22, 2010
Copyright: © 2010 Tao, Ye. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors received no specific funding for this manuscript.
Competing interests: The authors have declared that no competing interests exist.
The majority of viruses infecting animals and plants today are RNA viruses . There are double-stranded (ds) RNA viruses with dsRNA genomes, as well as (+) and (−)RNA viruses whose genomes are single-stranded (ss) RNA of either positive or negative polarity. RNA viruses have small genomes that rarely exceed 30 kb in size, and a large portion of their genomes is used to encode proteins involved in viral RNA replication. Viral RNA synthesis is catalyzed by the virally encoded RNA-dependent RNA polymerase (RdRp), which lacks any proofreading activity. Although the details of RNA replication greatly vary among viruses, common principles clearly exist in the organization of the replication machinery of different RNA viruses.
(+)RNA Viruses Replicate Their Genomes on the Surface of Host Membranes
For many (+)RNA viruses, RNA replication requires viral enzymes such as RdRp, helicase, capping enzymes, and NTPase, as well as non-enzymatic proteins that participate in the assembly of the viral replication complex. Host cell proteins often play essential roles in (+)RNA virus replication as well. (+)RNA virus replication is asymmetric: the synthesis of (+)RNA, the one that is packaged into the progeny virus, is up to ∼100 times greater than that of (−)RNA . Both the structure of the replicase itself and the cis-acting signals in the viral RNA template have been implicated in regulating the relative levels of genomic and antigenomic RNAs.
Host membranes play an important role in (+)RNA virus replication, as the RNA replication complexes released from membranes during purification generally lose the ability to catalyze true RNA replication, although a limited activity may be retained. In general, the intracellular membranes of cells infected with (+)RNA viruses rearrange to form anchor sites for viral RNA replication complexes . Membrane tethering of the replication complexes is facilitated by non-structural proteins with hydrophobic sequences that enable membrane integration or interaction. In picornavirus-infected cells, viral RNA replication occurs on the cytoplasmic surfaces of double-membrane vesicles derived from the endoplasmic reticulum (ER) . Two-dimensional (2D) arrays of polymerase-containing oligomeric structures have been observed on the surface of these vesicles, and the structural integrity of these 2D arrays correlates with the cooperative RNA binding and RNA elongation activities of the polymerase . In cells infected with the flock house virus, outer mitochondrial membranes invaginate to create open-necked spherules of ∼50 nm in size . On average, each spherule contains ∼100 membrane-spanning, self-interacting protein A molecules (i.e., the multifunctional RNA replication factor) and two to four genomic RNA replication intermediates . Membranes may function to expedite the assembly of replication complexes, to protect/sequester viral RNAs, and also to help segregate the products from templates during replication.
Although little is known about the structure of the membrane-associated RNA replication complexes, (+)RNA virus RdRps have been well characterized to have an overall right-handed shape with three domains, the fingers, palm, and the thumb (reviewed in ) (Figure 1A). The palm domain contains an invariant, central, four-stranded β-sheet, with five highly conserved motifs named A to E. Motifs A and C, which contain the sequence –DxxxxD– and –GDD–, respectively, play important roles in metal ion coordination and nucleotide substrate selection during catalysis. The fingers and thumb domains are located on opposite sides of the active site canyon. Most (+)RNA virus RdRps can initiate RNA synthesis de novo, but a number of them use viral protein primers (VPgs) and produce protein-linked genomes. In the RdRp from the foot-and-mouth disease virus, uridylated VPg occupies a site similar to the typical RNA primer in the active site canyon, with the key residue Tyr3 and its associated uridylate (UMP) projected into the active site .
dsRNA Viruses Replicate and Transcribe Their Genomes in Intact Core Particles
Genome replication by dsRNA viruses occurs in subviral particles. These subviral particles, also called the cores, have an intact viral capsid that encloses the viral genome and RdRp molecules. For complex, multi-layered dsRNA viruses, the core is derived from the virion by removing outer capsid proteins during entry. Simple dsRNA viruses without extracellular life cycles often do not have additional capsid layers beyond the core. Progeny cores are assembled from mRNAs, which are then replicated inside the particle to generate the dsRNA genome. The number of gene segments can vary from one to 12 for different dsRNA viruses.
To date, all seven families of encapsidated dsRNA viruses have been analyzed by cryo-EM or crystallography (Figure 1B) (see – for crystal structures). Except for the birnavirus, dsRNA viruses share structural similarity by having a characteristic, 120-subunit T = 1 capsid surrounding their genome. In viruses with high genomic RNA densities, the genome is packed into liquid crystalline arrays that give rise to concentric shells of density, 25–30 Å apart, in the particle interiors. Due to the lack of icosahedral symmetry, viral polymerase is invisible in the structure of the viruses. Reoviruses have ∼12 copies of the polymerase in each virion, each associated with one of the ten to 12 dsRNA gene segments that make up the viral genome. The polymerases are likely to be tethered to the capsid near 5-fold symmetry axes through noncovalent interactions . In addition to the central polymerase domain, dsRNA virus RdRps often have elaborate N- and C-terminal domains that keep the polymerase in the closed conformation (Figure 1A). In particular, the Reoviridae polymerase forms a rigid, caged structure with four well-defined tunnels that allow the inward and outward trafficking of the template, substrates, and products . Capsid proteins play a key role in regulating polymerase function, as the polymerase alone is either inactive or exhibits only limited activities. This intra-particle mechanism of RNA synthesis coordinates genome packaging with replication during the infectious cycle.
(A) RdRps of hepatitis C virus and reovirus. Hepatitis C virus is a (+)RNA virus from the Flaviviridae family, while reovirus is a dsRNA virus belonging to the Reoviridae family. In both structures, the fingers, palm, and the thumb of the polymerase are colored in blue, red, and green, respectively. Yellow and magenta highlight the N- and C-terminal domains, respectively. The three aspartic acid residues in the polymerase catalytic active site are shown in cyan. Reovirus RdRP has large N- and C-terminal domains with distinct functions: the N-terminal domain maintains the closed conformation of the active site, and the C-terminal domain serves as a processivity factor like a sliding clamp. (B) The yeast L-A virus (Totiviridae family). The two independent sets of capsid protein molecules, 60 copies each, are shown in red and yellow, respectively. The viral RdRP is likely to be tethered to the inner capsid near the 5-fold symmetry axis, and viral transcripts made inside the core are released through channels on or near the 5-fold axes. (C) An influenza A virus (Orthomyxoviridae family) RNP model. Blue spheres represent NP monomers, and the green line shows vRNA. A short duplex formed between the 5′ and the 3′ ends provides the binding site for the heterotrimeric RdRp. Overall, the RNP assumes a rod-shaped, double-helical structure that remains intact even after the vRNA is removed.
A key question about dsRNA virus replication is how the (−)RNA template is selected from the dsRNA genome during transcription. A 5′-cap binding site, identified on the reovirus polymerase, provides a neat solution for some dsRNA viruses of high eukaryotic hosts . Because only the (+)RNA strand is capped, cap binding keeps the polymerase proximal to the 3′-end of the (−)RNA strand, thus allowing efficient initiation of transcription. Negative charges on the inner surface of dsRNA viral capsids may help to facilitate viral genome movement during transcription. Viral transcripts are likely to exit the core through pores on or near the 5-fold symmetry axes.
(−)RNA Viruses Replicate Using Protein-Coated Templates and Generate Protein-Stabilized, Single-Stranded RNAs
(−)RNA viruses have a helical-shaped, protein-coated genome, also known as the ribonucloeprotein complex (RNP), which functions as the sole template for viral RNA replication and transcription (Figure 1C). Newly made viral RNA (vRNA) and the replication intermediate (cRNA) are packaged as RNPs and stabilized in a single-stranded form immediately after their synthesis.
The composition of the RNA replication machinery differs between segmented and non-segmented (−)RNA viruses. The influenza A virus, a segmented (−)RNA virus, contains eight vRNAs, each packaged as a double helical-shaped RNP with multiple nucleoproteins (NPs) and a heterotrimeric polymerase (PA, PB1, and PB2) that is bound to the conserved vRNA termini (Figure 1C). Each NP molecule binds to a 24-nt RNA at the outer periphery of the NP scaffold . The influenza A virus polymerase has a compact structure  with at least four active sites: the polymerase active site and a 5′ vRNA binding site in PB1, an endonuclease active site in PA , , and a cap binding site in PB2 . During transcription, the polymerase uses a short capped RNA fragment snatched from cellular mRNAs to initiate RNA synthesis. To switch from transcription to replication mode, it is necessary to change from capped RNA-primed initiation to unprimed initiation and to prevent pre-mature termination and polyadenylation. Free NP is essential for viral RNA replication, possibly because (1) NP interacts with the polymerase, thus altering its activity , or (2) NP prevents the degradation of the cRNA intermediate by forming RNP structures . It has been proposed that viral RNA transcription is catalyzed by the polymerase associated with the vRNP template, whereas RNA replication is catalyzed by a newly made polymerase molecule . The specific binding of the viral polymerase to the 5′-end of newly synthesized vRNAs presumably helps to recruit additional NPs, thus ensuring specific encapsidation of vRNA.
RNA replication of non-segmented (-)RNA viruses, such as the rabies virus and the vesicular stomatitis virus (VSV), requires an intricate interplay of the nucleoprotein N, the large protein L, a non-enzymatic P, and the N-enwrapped genome, also called the nucleocapsid . In the nucleocapsid, each N protein has two lobes angled together to form a cavity that accommodates seven or nine bases of RNA –. In addition to nucleotide polymerization, L also possesses enzymatic activities for 5′ cap synthesis. During RNA synthesis, P delivers L to the active template through an N-P interaction that involves two adjacent N proteins in the nucleocapsid . L-P binding to the N-RNA template triggers conformational changes in the nucleocapsid and allows the polymerase access to RNA.
Different replication strategies are used by (+), (−), and dsRNA viruses to limit dsRNA exposure, to regulate (+)RNA versus (−)RNA synthesis, and to modulate transcription versus replication throughout the infection cycle. The differences in the functionalities of the replication complexes are reflected in their structural composition, their subcellular localization, and in the interaction of the complex with viral RNA templates. It is expected that studies of RNA virus replication machineries will have a large impact on antiviral drug development due to their specific activities in virus replication.
- 1. Ball LA (2007) Virus Replication Strategies. In: Knipe D, Howley P, editors. Fields Virology. Philadelphia: Lippincott Williams & Wilkins. pp. 120–140.LA Ball2007Virus Replication Strategies.D. KnipeP. HowleyFields VirologyPhiladelphiaLippincott Williams & Wilkins1201405 ed. 5 ed.
- 2. Salonen A, Ahola T, Kaariainen L (2005) Viral RNA replication in association with cellular membranes. Curr Top Microbiol Immunol 285: 139–173.A. SalonenT. AholaL. Kaariainen2005Viral RNA replication in association with cellular membranes.Curr Top Microbiol Immunol285139173
- 3. Egger D, Teterina N, Ehrenfeld E, Bienz K (2000) Formation of the poliovirus replication complex requires coupled viral translation, vesicle production, and viral RNA synthesis. J Virol 74: 6570–6580.D. EggerN. TeterinaE. EhrenfeldK. Bienz2000Formation of the poliovirus replication complex requires coupled viral translation, vesicle production, and viral RNA synthesis.J Virol7465706580
- 4. Lyle JM, Bullitt E, Bienz K, Kirkegaard K (2002) Visualization and functional analysis of RNA-dependent RNA polymerase lattices. Science 296: 2218–2222.JM LyleE. BullittK. BienzK. Kirkegaard2002Visualization and functional analysis of RNA-dependent RNA polymerase lattices.Science29622182222
- 5. Kopek BG, Perkins G, Miller DJ, Ellisman MH, Ahlquist P (2007) Three-dimensional analysis of a viral RNA replication complex reveals a virus-induced mini-organelle. PLoS Biol 5: e220.BG KopekG. PerkinsDJ MillerMH EllismanP. Ahlquist2007Three-dimensional analysis of a viral RNA replication complex reveals a virus-induced mini-organelle.PLoS Biol5e220
- 6. Ng KK, Arnold JJ, Cameron CE (2008) Structure-function relationships among RNA-dependent RNA polymerases. Curr Top Microbiol Immunol 320: 137–156.KK NgJJ ArnoldCE Cameron2008Structure-function relationships among RNA-dependent RNA polymerases.Curr Top Microbiol Immunol320137156
- 7. Ferrer-Orta C, Arias A, Agudo R, Perez-Luque R, Escarmis C, et al. (2006) The structure of a protein primer-polymerase complex in the initiation of genome replication. Embo J 25: 880–888.C. Ferrer-OrtaA. AriasR. AgudoR. Perez-LuqueC. Escarmis2006The structure of a protein primer-polymerase complex in the initiation of genome replication.Embo J25880888
- 8. Reinisch KM, Nibert ML, Harrison SC (2000) Structure of the reovirus core at 3.6 A resolution. Nature 404: 960–967.KM ReinischML NibertSC Harrison2000Structure of the reovirus core at 3.6 A resolution.Nature404960967
- 9. Grimes JM, Burroughs JN, Gouet P, Diprose JM, Malby R, et al. (1998) The atomic structure of the bluetongue virus core. Nature 395: 470–478.JM GrimesJN BurroughsP. GouetJM DiproseR. Malby1998The atomic structure of the bluetongue virus core.Nature395470478
- 10. Nakagawa A, Miyazaki N, Taka J, Naitow H, Ogawa A, et al. (2003) The atomic structure of rice dwarf virus reveals the self-assembly mechanism of component proteins. Structure 11: 1227–1238.A. NakagawaN. MiyazakiJ. TakaH. NaitowA. Ogawa2003The atomic structure of rice dwarf virus reveals the self-assembly mechanism of component proteins.Structure1112271238
- 11. Coulibaly F, Chevalier C, Gutsche I, Pous J, Navaza J, et al. (2005) The birnavirus crystal structure reveals structural relationships among icosahedral viruses. Cell 120: 761–772.F. CoulibalyC. ChevalierI. GutscheJ. PousJ. Navaza2005The birnavirus crystal structure reveals structural relationships among icosahedral viruses.Cell120761772
- 12. Naitow H, Tang J, Canady M, Wickner RB, Johnson JE (2002) L-A virus at 3.4 A resolution reveals particle architecture and mRNA decapping mechanism. Nat Struct Biol 9: 725–728.H. NaitowJ. TangM. CanadyRB WicknerJE Johnson2002L-A virus at 3.4 A resolution reveals particle architecture and mRNA decapping mechanism.Nat Struct Biol9725728
- 13. Pan J, Dong L, Lin L, Ochoa WF, Sinkovits RS, et al. (2009) Atomic structure reveals the unique capsid organization of a dsRNA virus. Proc Natl Acad Sci U S A 106: 4225–4230.J. PanL. DongL. LinWF OchoaRS Sinkovits2009Atomic structure reveals the unique capsid organization of a dsRNA virus.Proc Natl Acad Sci U S A10642254230
- 14. Duquerroy S, Da Costa B, Henry C, Vigouroux A, Libersou S, et al. (2009) The picobirnavirus crystal structure provides functional insights into virion assembly and cell entry. Embo J 28: 1655–1665.S. DuquerroyB. Da CostaC. HenryA. VigourouxS. Libersou2009The picobirnavirus crystal structure provides functional insights into virion assembly and cell entry.Embo J2816551665
- 15. Zhang X, Walker SB, Chipman PR, Nibert ML, Baker TS (2003) Reovirus polymerase lambda 3 localized by cryo-electron microscopy of virions at a resolution of 7.6 A. Nat Struct Biol 10: 1011–1018.X. ZhangSB WalkerPR ChipmanML NibertTS Baker2003Reovirus polymerase lambda 3 localized by cryo-electron microscopy of virions at a resolution of 7.6 A.Nat Struct Biol1010111018
- 16. McDonald SM, Tao YJ, Patton JT (2009) The ins and outs of four-tunneled Reoviridae RNA-dependent RNA polymerases. Curr Opin Struct Biol 19: 775–782.SM McDonaldYJ TaoJT Patton2009The ins and outs of four-tunneled Reoviridae RNA-dependent RNA polymerases.Curr Opin Struct Biol19775782
- 17. Coloma R, Valpuesta JM, Arranz R, Carrascosa JL, Ortin J, et al. (2009) The structure of a biologically active influenza virus ribonucleoprotein complex. PLoS Pathog 5: e1000491.R. ColomaJM ValpuestaR. ArranzJL CarrascosaJ. Ortin2009The structure of a biologically active influenza virus ribonucleoprotein complex.PLoS Pathog5e1000491
- 18. Yuan P, Bartlam M, Lou Z, Chen S, Zhou J, et al. (2009) Crystal structure of an avian influenza polymerase PA(N) reveals an endonuclease active site. Nature 458: 909–913.P. YuanM. BartlamZ. LouS. ChenJ. Zhou2009Crystal structure of an avian influenza polymerase PA(N) reveals an endonuclease active site.Nature458909913
- 19. Dias A, Bouvier D, Crepin T, McCarthy AA, Hart DJ, et al. (2009) The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit. Nature 458: 914–918.A. DiasD. BouvierT. CrepinAA McCarthyDJ Hart2009The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit.Nature458914918
- 20. Guilligay D, Tarendeau F, Resa-Infante P, Coloma R, Crepin T, et al. (2008) The structural basis for cap binding by influenza virus polymerase subunit PB2. Nat Struct Mol Biol 15: 500–506.D. GuilligayF. TarendeauP. Resa-InfanteR. ColomaT. Crepin2008The structural basis for cap binding by influenza virus polymerase subunit PB2.Nat Struct Mol Biol15500506
- 21. Newcomb LL, Kuo RL, Ye Q, Jiang Y, Tao YJ, et al. (2009) Interaction of the influenza a virus nucleocapsid protein with the viral RNA polymerase potentiates unprimed viral RNA replication. J Virol 83: 29–36.LL NewcombRL KuoQ. YeY. JiangYJ Tao2009Interaction of the influenza a virus nucleocapsid protein with the viral RNA polymerase potentiates unprimed viral RNA replication.J Virol832936
- 22. Vreede FT, Jung TE, Brownlee GG (2004) Model suggesting that replication of influenza virus is regulated by stabilization of replicative intermediates. J Virol 78: 9568–9572.FT VreedeTE JungGG Brownlee2004Model suggesting that replication of influenza virus is regulated by stabilization of replicative intermediates.J Virol7895689572
- 23. Jorba N, Coloma R, Ortin J (2009) Genetic trans-complementation establishes a new model for influenza virus RNA transcription and replication. PLoS Pathog 5: e1000462.N. JorbaR. ColomaJ. Ortin2009Genetic trans-complementation establishes a new model for influenza virus RNA transcription and replication.PLoS Pathog5e1000462
- 24. Whelan SP, Barr JN, Wertz GW (2004) Transcription and replication of nonsegmented negative-strand RNA viruses. Curr Top Microbiol Immunol 283: 61–119.SP WhelanJN BarrGW Wertz2004Transcription and replication of nonsegmented negative-strand RNA viruses.Curr Top Microbiol Immunol28361119
- 25. Green TJ, Zhang X, Wertz GW, Luo M (2006) Structure of the vesicular stomatitis virus nucleoprotein-RNA complex. Science 313: 357–360.TJ GreenX. ZhangGW WertzM. Luo2006Structure of the vesicular stomatitis virus nucleoprotein-RNA complex.Science313357360
- 26. Albertini AA, Wernimont AK, Muziol T, Ravelli RB, Clapier CR, et al. (2006) Crystal structure of the rabies virus nucleoprotein-RNA complex. Science 313: 360–363.AA AlbertiniAK WernimontT. MuziolRB RavelliCR Clapier2006Crystal structure of the rabies virus nucleoprotein-RNA complex.Science313360363
- 27. Tawar RG, Duquerroy S, Vonrhein C, Varela PF, Damier-Piolle L, et al. (2009) Crystal structure of a nucleocapsid-like nucleoprotein-RNA complex of respiratory syncytial virus. Science 326: 1279–1283.RG TawarS. DuquerroyC. VonrheinPF VarelaL. Damier-Piolle2009Crystal structure of a nucleocapsid-like nucleoprotein-RNA complex of respiratory syncytial virus.Science32612791283
- 28. Green TJ, Luo M (2009) Structure of the vesicular stomatitis virus nucleocapsid in complex with the nucleocapsid-binding domain of the small polymerase cofactor, P. Proc Natl Acad Sci U S A 106: 11713–11718.TJ GreenM. Luo2009Structure of the vesicular stomatitis virus nucleocapsid in complex with the nucleocapsid-binding domain of the small polymerase cofactor, P.Proc Natl Acad Sci U S A1061171311718