Biology:RNA-dependent RNA polymerase

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Short description: Enzyme that synthesizes RNA from an RNA template
RNA-dependent RNA polymerase
HCV NS5B RdRP stalled 4WTG.png
Stalled HCV RNA replicase (NS5B), in complex with sofosbuvir (PDB 4WTG).
Identifiers
EC number2.7.7.48
CAS number9026-28-2
Databases
IntEnzIntEnz view
BRENDABRENDA entry
ExPASyNiceZyme view
KEGGKEGG entry
MetaCycmetabolic pathway
PRIAMprofile
PDB structuresRCSB PDB PDBe PDBsum
Gene OntologyAmiGO / QuickGO

RNA-dependent RNA polymerase (RdRp) or RNA replicase is an enzyme that catalyzes the replication of RNA from an RNA template. Specifically, it catalyzes synthesis of the RNA strand complementary to a given RNA template. This is in contrast to typical DNA-dependent RNA polymerases, which all organisms use to catalyze the transcription of RNA from a DNA template.

RdRp is an essential protein encoded in the genomes of most RNA-containing viruses with no DNA stage[1][2] including SARS-CoV-2. Some eukaryotes also contain RdRps, which are involved in RNA interference and differ structurally from viral RdRps.

History

Viral RdRps were discovered in the early 1960s from studies on mengovirus and polio virus when it was observed that these viruses were not sensitive to actinomycin D, a drug that inhibits cellular DNA-directed RNA synthesis. This lack of sensitivity suggested that there is a virus-specific enzyme that could copy RNA from an RNA template and not from a DNA template.[citation needed]

Distribution

Structure and replication elongation mechanism of a RdRp

RdRps are highly conserved throughout viruses and are even related to telomerase, though the reason for this is an ongoing question as of 2009.[3] The similarity has led to speculation that viral RdRps are ancestral to human telomerase.[citation needed]

The most famous example of RdRp is that of the polio virus. The viral genome is composed of RNA, which enters the cell through receptor-mediated endocytosis. From there, the RNA is able to act as a template for complementary RNA synthesis, immediately. The complementary strand is then, itself, able to act as a template for the production of new viral genomes that are further packaged and released from the cell ready to infect more host cells. The advantage of this method of replication is that there is no DNA stage; replication is quick and easy. The disadvantage is that there is no 'back-up' DNA copy.[citation needed]

Many RdRps are associated tightly with membranes and are, therefore, difficult to study. The best-known RdRps are polioviral 3Dpol, vesicular stomatitis virus L,[4] and hepatitis C virus NS5B protein.

Many eukaryotes also have RdRps and these are involved in RNA interference: these amplify microRNAs and small temporal RNAs and produce double-stranded RNA using small interfering RNAs as primers.[5] In fact these same RdRps that are used in the defense mechanisms can be usurped by RNA viruses for their benefit.[6] Their evolutionary history has been reviewed.[7]

Replication process

RdRp differs from DNA dependent RNA polymerase as it works to catalyze the synthesis of an RNA strand complementary to a given RNA template, rather than using a DNA template. The RNA replication process is a four-step mechanism, as described.

  1. Nucleoside triphosphate (NTP) binding – initially, the RdRp presents with a vacant active site in which an NTP binds, complementary to the corresponding nucleotide on the template strand. Correct NTP binding causes the RdRp to undergo a conformational change.[8]
  2. Active site closure – the conformational change, initiated by the correct NTP binding, results in the restriction of active site access and produces a catalytically competent state.[8]
  3. Phosphodiester bond formation – two Mg2+ ions are present in the catalytically active state and arrange themselves in such a way around the newly synthesized RNA chain that the substrate NTP is able to undergo a phosphatidyl transfer and form a phosphodiester bond with the newly synthesized chain.[9] Without the use of these Mg2+ ions, the active site is no longer catalytically stable and the RdRp complex changes to an open conformation.[9]
  4. Translocation – once the active site is open, the RNA template strand is able to move by one position through the RdRp protein complex and continue chain elongation by binding a new NTP, unless otherwise specified by the template.[8]

RNA synthesis can be performed by means of a primer-independent (de novo) or a primer-dependent mechanism that utilizes a viral protein genome-linked (VPg) primer.[10] The de novo initiation consists in the addition of a nucleoside triphosphate (NTP) to the 3'-OH of the first initiating NTP.[10] During the following so-called elongation phase, this nucleotidyl transfer reaction is repeated with subsequent NTPs to generate the complementary RNA product. Termination of the nascent RNA chain produced by RdRp is not completely known, however, it has been shown that RdRp termination is sequence-independent.[11]

One major drawback of RNA-dependent RNA polymerase replication is the immense error rate during transcription.[10] RdRps are known to have a lack of fidelity on the order of 104 nucleotides, which is thought to be a direct result of its insufficient proofreading abilities.[10] This high rate of variation is favored in viral genomes as it allows for the pathogen to overcome defenses developed by hosts trying to avoid infection allowing for evolutionary growth.[citation needed]

Structure

Overview of the flavivirus RdRp structure based on West Nile Virus (WNV) NS5Pol

Viral/prokaryotic RNA-directed RNA polymerases, along with many single-subunit DNA-directed polymerases, employ a fold whose organization has been linked to the shape of a right hand with three subdomains termed fingers, palm, and thumb.[12] Only the palm subdomain, composed of a four-stranded antiparallel beta sheet with two alpha helices, is well conserved among all of these enzymes. In RdRp, the palm subdomain comprises three well-conserved motifs (A, B, and C). Motif A (D-x(4,5)-D) and motif C (GDD) are spatially juxtaposed; the aspartic acid residues of these motifs are implied in the binding of Mg2+ and/or Mn2+. The asparagine residue of motif B is involved in selection of ribonucleoside triphosphates over dNTPs and, thus, determines whether RNA rather than DNA is synthesized.[13] The domain organization[14] and the 3D structure of the catalytic centre of a wide range of RdRps, even those with a low overall sequence homology, are conserved. The catalytic center is formed by several motifs containing a number of conserved amino acid residues.[citation needed]

Eukaryotic RNA interference requires a cellular RNA-dependent RNA polymerase (c RdRp). Unlike the "hand" polymerases, they resemble simplified multi-subunit DNA-dependent RNA polymerases (DdRPs), specifically in the catalytic β/β' subunits, in that they use two sets of double-psi β-barrels in the active site. QDE1 (Q9Y7G6) in Neurospora crassa, which has both barrels in the same chain,[15] is an example of such a c RdRp enzyme.[16] Bacteriophage homologs of c RdRp, including the similarly single-chain DdRp yonO (O31945), appear to be closer to c RdRps than DdRPs are.[5][17]

RNA dependent RNA polymerase[lower-alpha 1]
Identifiers
SymbolRdRP_1
PfamPF00680
Pfam clanCL0027
InterProIPR001205
SCOP22jlg / SCOPe / SUPFAM
RNA-dependent RNA polymerase, eukaryotic-type
Identifiers
SymbolRdRP_euk
PfamPF05183
InterProIPR007855
Bunyavirus RNA replicase[lower-alpha 2]
Identifiers
SymbolBunya_RdRp
PfamPF04196
InterProIPR007322

In viruses

Structure and evolution of RdRp in RNA viruses and their superfamilies

There are 4 superfamilies of viruses that cover all RNA-containing viruses with no DNA stage:

Flaviviruses produce a polyprotein from the ssRNA genome. The polyprotein is cleaved to a number of products, one of which is NS5, an RNA-dependent RNA polymerase. This RNA-directed RNA polymerase possesses a number of short regions and motifs homologous to other RNA-directed RNA polymerases.[18]

RNA replicase found in positive-strand ssRNA viruses are related to each other, forming three large superfamilies.[19] Birnaviral RNA replicase is unique in that it lacks motif C (GDD) in the palm.[20] Mononegaviral RdRp (PDB 5A22) has been automatically classified as similar to (+)−ssRNA RdRps, specifically one from Pestivirus and one from Leviviridae.[21] Bunyaviral RdRp monomer (PDB 5AMQ) resembles the heterotrimeric complex of Orthomyxoviral (Influenza; PDB 4WSB) RdRp.[22]

Since it is a protein universal to RNA-containing viruses, RdRp is a useful marker for understanding their evolution.[23] The overall structural evolution of viral RdRps has been reviewed.[24]

Recombination

When replicating its (+)ssRNA genome, the poliovirus RdRp is able to carry out recombination. Recombination appears to occur by a copy choice mechanism in which the RdRp switches (+)ssRNA templates during negative strand synthesis.[25] Recombination frequency is determined in part by the fidelity of RdRp replication.[26] RdRp variants with high replication fidelity show reduced recombination, and low fidelity RdRps exhibit increased recombination.[26] Recombination by RdRp strand switching also occurs frequently during replication in the (+)ssRNA plant carmoviruses and tombusviruses.[27]

Intragenic complementation

Sendai virus (family Paramyxoviridae) has a linear, single stranded, negative-sense, nonsegmented RNA genome. The viral RdRp consists of two virus-encoded subunits, a smaller one P and a larger one L. When different inactive RdRp mutants with defects throughout the length of the L subunit where tested in pairwise combinations, restoration of viral RNA synthesis was observed in some combinations.[28] This positive L–L interaction is referred to as intragenic complementation and indicates that the L protein is an oligomer in the viral RNA polymerase complex.[citation needed]

Drug therapies

  • RdRps can be used as drug targets for viral pathogens as their function is not necessary for eukaryotic survival. By inhibiting RNA-dependent RNA polymerase function, new RNAs cannot be replicated from an RNA template strand, however, DNA-dependent RNA polymerase will remain functional.
  • There are currently antiviral drugs against Hepatitis C and COVID-19 that specifically target RdRp. These include Sofosbuvir and Ribavirin against Hepatitis C[29] and Remdesivir, the only FDA approved drug against COVID-19.
  • GS-441524 triphosphate, is a substrate for RdRp, but not mammalian polymerases. It results in premature chain termination and inhibition of viral replication. GS-441524 triphosphate is the biologically active form of the phosphate pro-drug, Remdesivir. Remdesivir is classified as a nucleotide analog in which it works to inhibit the function of RdRp by covalently binding to and interrupting termination of the nascent RNA through early or delayed termination or preventing further elongation of the RNA polynucleotide.[30][31] This early termination leads to nonfunctional RNA that will be degraded through normal cellular processes.

RNA interference

The use of RNA-dependent RNA polymerase plays a major role in RNA interference in eukaryotes, a process used to silence gene expression via small interfering RNAs (siRNAs) binding to mRNA rendering them inactive.[32] Eukaryotic RdRp becomes active in the presence of dsRNA, and is a less widely distributed compared to other RNAi components as it lost in some animals, though still found in C. elegans and P. tetraurelia[33] and plants.[34] This presence of dsRNA triggers the activation of RdRp and RNAi processes by priming the initiation of RNA transcription through the introduction of siRNAs into the system.[33] In C. elegans, siRNAs are integrated into the RNA-induced silencing complex, RISC, which works alongside mRNAs targeted for interference to recruit more RdRps to synthesize more secondary siRNAs and repress gene expression.[35]

See also

Notes

  1. ↑ See Pfam clan for other (+)ssRNA/dsRNA families.
  2. ↑ A (−)ssRNA polymerase.

References

  1. ↑ "Tentative identification of RNA-dependent RNA polymerases of dsRNA viruses and their relationship to positive strand RNA viral polymerases". FEBS Letters 252 (1–2): 42–6. July 1989. doi:10.1016/0014-5793(89)80886-5. PMID 2759231. 
  2. ↑ "A reevaluation of the higher taxonomy of viruses based on RNA polymerases". Journal of Virology 70 (9): 6083–96. September 1996. doi:10.1128/JVI.70.9.6083-6096.1996. PMID 8709232. 
  3. ↑ "Viruses in the sea". Nature 437 (7057): 356–61. September 2005. doi:10.1038/nature04160. PMID 16163346. Bibcode: 2005Natur.437..356S. 
  4. ↑ "Robust kinetics of an RNA virus: Transcription rates are set by genome levels". Biotechnology and Bioengineering 112 (8): 1655–62. August 2015. doi:10.1002/bit.25578. PMID 25726926. 
  5. ↑ 5.0 5.1 "Evolutionary connection between the catalytic subunits of DNA-dependent RNA polymerases and eukaryotic RNA-dependent RNA polymerases and the origin of RNA polymerases". BMC Structural Biology 3: 1. January 2003. doi:10.1186/1472-6807-3-1. PMID 12553882. 
  6. ↑ "RNAi, a new therapeutic strategy against viral infection". Cell Research 14 (6): 460–6. December 2004. doi:10.1038/sj.cr.7290248. PMID 15625012. 
  7. ↑ "Evolution of the RNA-dependent RNA polymerase (RdRP) genes: duplications and possible losses before and after the divergence of major eukaryotic groups". Gene 447 (1): 29–39. November 2009. doi:10.1016/j.gene.2009.07.004. PMID 19616606. 
  8. ↑ 8.0 8.1 8.2 "Visualizing the Nucleotide Addition Cycle of Viral RNA-Dependent RNA Polymerase". Viruses 10 (1): 24. January 2018. doi:10.3390/v10010024. PMID 29300357. 
  9. ↑ 9.0 9.1 "Structural basis of viral RNA-dependent RNA polymerase catalysis and translocation". Proceedings of the National Academy of Sciences of the United States of America 113 (28): E4005–14. July 2016. doi:10.1073/pnas.1602591113. PMID 27339134. Bibcode: 2016PNAS..113E4005S. 
  10. ↑ 10.0 10.1 10.2 10.3 "RNA Dependent RNA Polymerases: Insights from Structure, Function and Evolution". Viruses 10 (2): 76. February 2018. doi:10.3390/v10020076. PMID 29439438. 
  11. ↑ "Mechanistic analysis of RNA synthesis by RNA-dependent RNA polymerase from two promoters reveals similarities to DNA-dependent RNA polymerase". RNA 4 (4): 455–70. April 1998. PMID 9630251. 
  12. ↑ "Structure of the RNA-dependent RNA polymerase of poliovirus". Structure 5 (8): 1109–22. August 1997. doi:10.1016/S0969-2126(97)00261-X. PMID 9309225. 
  13. ↑ "Poliovirus RNA-dependent RNA polymerase (3Dpol): structural, biochemical, and biological analysis of conserved structural motifs A and B". The Journal of Biological Chemistry 275 (33): 25523–32. August 2000. doi:10.1074/jbc.M002671200. PMID 10827187. 
  14. ↑ "Analysis of RNA-dependent RNA polymerase structure and function as guided by known polymerase structures and computer predictions of secondary structure". Virology 252 (2): 287–303. December 1998. doi:10.1006/viro.1998.9463. PMID 9878607. 
  15. ↑ "The Extended "Two-Barrel" Polymerases Superfamily: Structure, Function and Evolution". Journal of Molecular Biology 431 (20): 4167–4183. September 2019. doi:10.1016/j.jmb.2019.05.017. PMID 31103775. 
  16. ↑ "Evolution of multisubunit RNA polymerases in the three domains of life". Nature Reviews. Microbiology 9 (2): 85–98. February 2011. doi:10.1038/nrmicro2507. PMID 21233849. 
  17. ↑ "Single-peptide DNA-dependent RNA polymerase homologous to multi-subunit RNA polymerase". Nature Communications 8: 15774. June 2017. doi:10.1038/ncomms15774. PMID 28585540. Bibcode: 2017NatCo...815774F. 
  18. ↑ "Recombinant dengue type 1 virus NS5 protein expressed in Escherichia coli exhibits RNA-dependent RNA polymerase activity". Virology 216 (2): 317–25. February 1996. doi:10.1006/viro.1996.0067. PMID 8607261. 
  19. ↑ "The phylogeny of RNA-dependent RNA polymerases of positive-strand RNA viruses". The Journal of General Virology 72 ( Pt 9) (9): 2197–206. September 1991. doi:10.1099/0022-1317-72-9-2197. PMID 1895057. 
  20. ↑ "Birnavirus VP1 proteins form a distinct subgroup of RNA-dependent RNA polymerases lacking a GDD motif". Virology 296 (2): 241–50. May 2002. doi:10.1006/viro.2001.1334. PMID 12069523. 
  21. ↑ Structural Similarities for the Entities in PDB 5A22.
  22. ↑ "Structural Insights into Bunyavirus Replication and Its Regulation by the vRNA Promoter". Cell 161 (6): 1267–79. June 2015. doi:10.1016/j.cell.2015.05.006. PMID 26004069. 
  23. ↑ "Origins and Evolution of the Global RNA Virome". mBio 9 (6). November 2018. doi:10.1128/mBio.02329-18. PMID 30482837. 
  24. ↑ "Evolution of tertiary structure of viral RNA dependent polymerases". PLOS ONE 9 (5): e96070. 2014. doi:10.1371/journal.pone.0096070. PMID 24816789. Bibcode: 2014PLoSO...996070C. 
  25. ↑ "The mechanism of RNA recombination in poliovirus". Cell 47 (3): 433–43. November 1986. doi:10.1016/0092-8674(86)90600-8. PMID 3021340. 
  26. ↑ 26.0 26.1 "Biochemical and genetic analysis of the role of the viral polymerase in enterovirus recombination". Nucleic Acids Research 44 (14): 6883–95. August 2016. doi:10.1093/nar/gkw567. PMID 27317698. 
  27. ↑ "Mechanism of RNA recombination in carmo- and tombusviruses: evidence for template switching by the RNA-dependent RNA polymerase in vitro". Journal of Virology 77 (22): 12033–47. November 2003. doi:10.1128/jvi.77.22.12033-12047.2003. PMID 14581540. 
  28. ↑ "Intragenic complementation and oligomerization of the L subunit of the sendai virus RNA polymerase". Virology 304 (2): 235–45. December 2002. doi:10.1006/viro.2002.1720. PMID 12504565. 
  29. ↑ "RNA dependent RNA polymerase of HCV: a potential target for the development of antiviral drugs". Infection, Genetics and Evolution 14: 247–57. March 2013. doi:10.1016/j.meegid.2012.12.004. PMID 23291407. 
  30. ↑ "Structural basis for inhibition of the RNA-dependent RNA polymerase from SARS-CoV-2 by remdesivir". Science 368 (6498): 1499–1504. June 2020. doi:10.1126/science.abc1560. PMID 32358203. Bibcode: 2020Sci...368.1499Y. 
  31. ↑ "Remdesivir against COVID-19 and Other Viral Diseases". Clinical Microbiology Reviews 34 (1). December 2020. doi:10.1128/CMR.00162-20. PMID 33055231. 
  32. ↑ "Hemodynamic effects of aerosol propellants. II. Pulmonary circulation in the dog". Toxicology 5 (2): 139–46. November 1975. doi:10.1016/0300-483x(75)90110-9. PMID 1873. 
  33. ↑ 33.0 33.1 "Distinct RNA-dependent RNA polymerases are required for RNAi triggered by double-stranded RNA versus truncated transgenes in Paramecium tetraurelia". Nucleic Acids Research 38 (12): 4092–107. July 2010. doi:10.1093/nar/gkq131. PMID 20200046. 
  34. ↑ "The Functions of {RNA-Dependent} {RNA} Polymerases in Arabidopsis". Arabidopsis Book 9: e0146. July 2011. doi:10.1199/tab.0146. PMID 22303271. 
  35. ↑ "New insights into siRNA amplification and RNAi". RNA Biology 9 (8): 1045–9. August 2012. doi:10.4161/rna.21246. PMID 22858672. 

External links

This article incorporates text from the public domain Pfam and InterPro: IPR000208



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