1887

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Volume 104, Issue 10

A structural and functional analysis of opal stop codon translational readthrough during Chikungunya virus replication Open Access

Abstract

Chikungunya virus (CHIKV) is an alphavirus, transmitted by species mosquitoes. The CHIKV single-stranded positive-sense RNA genome contains two open reading frames, coding for the non-structural (nsP) and structural proteins of the virus. The non-structural polyprotein precursor is proteolytically cleaved to generate nsP1-4. Intriguingly, most isolates of CHIKV (and other alphaviruses) possess an opal stop codon close to the 3′ end of the nsP3 coding sequence and translational readthrough is necessary to produce full-length nsP3 and the nsP4 RNA polymerase. Here we investigate the role of this stop codon by replacing the arginine codon with each of the three stop codons in the context of both a subgenomic replicon and infectious CHIKV. Both opal and amber stop codons were tolerated in mammalian cells, but the ochre was not. In mosquito cells all three stop codons were tolerated. Using SHAPE analysis we interrogated the structure of a putative stem loop 3′ of the stop codon and used mutagenesis to probe the importance of a short base-paired region at the base of this structure. Our data reveal that this stem is not required for stop codon translational readthrough, and we conclude that other factors must facilitate this process to permit productive CHIKV replication.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): Chikungunya virus , genome replication , nsP3 , stop codon and translational readthrough
Funding
This study was supported by the:
  • Medical Research Council (Award MR/N01054X/1)
    • Principle Award Recipient: AndrewTuplin
  • Wellcome Trust (Award 096670)
    • Principle Award Recipient: MarkHarris
© 2023 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2023-10-20
2024-05-10
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References

  1. Schwartz O, Albert ML. Biology and pathogenesis of chikungunya virus. Nat Rev Microbiol 2010; 8:491–500 [View Article] [PubMed]
     [Google Scholar]
  2. Chen R, Mukhopadhyay S, Merits A, Bolling B, Nasar F et al. ICTV Virus Taxonomy Profile: Togaviridae. J Gen Virol 2018; 99:761–762 [View Article] [PubMed]
     [Google Scholar]
  3. Ahola T, Kääriäinen L. Reaction in alphavirus mRNA capping: formation of a covalent complex of nonstructural protein nsP1 with 7-methyl-GMP. Proc Natl Acad Sci U S A 1995; 92:507–511 [View Article] [PubMed]
     [Google Scholar]
  4. Russo AT, White MA, Watowich SJ. The crystal structure of the Venezuelan equine encephalitis alphavirus nsP2 protease. Structure 2006; 14:1449–1458 [View Article] [PubMed]
     [Google Scholar]
  5. Rubach JK, Wasik BR, Rupp JC, Kuhn RJ, Hardy RW et al. Characterization of purified Sindbis virus nsP4 RNA-dependent RNA polymerase activity in vitro. Virology 2009; 384:201–208 [View Article] [PubMed]
     [Google Scholar]
  6. Wang YF, Sawicki SG, Sawicki DL. Alphavirus nsP3 functions to form replication complexes transcribing negative-strand RNA. J Virol 1994; 68:6466–6475 [View Article] [PubMed]
     [Google Scholar]
  7. LaStarza MW, Lemm JA, Rice CM. Genetic analysis of the nsP3 region of Sindbis virus: evidence for roles in minus-strand and subgenomic RNA synthesis. J Virol 1994; 68:5781–5791 [View Article] [PubMed]
     [Google Scholar]
  8. Chen KC, Kam Y-W, Lin RTP, Ng M-L, Ng LF et al. Comparative analysis of the genome sequences and replication profiles of chikungunya virus isolates within the East, Central and South African (ECSA) lineage. Virol J 2013; 10:169 [View Article] [PubMed]
     [Google Scholar]
  9. Kim KH, Rümenapf T, Strauss EG, Strauss JH. Regulation of Semliki Forest virus RNA replication: a model for the control of alphavirus pathogenesis in invertebrate hosts. Virology 2004; 323:153–163 [View Article] [PubMed]
     [Google Scholar]
  10. Varjak M, Zusinaite E, Merits A. Novel functions of the alphavirus nonstructural protein nsP3 C-terminal region. J Virol 2010; 84:2352–2364 [View Article] [PubMed]
     [Google Scholar]
  11. Jones JE, Long KM, Whitmore AC, Sanders W, Thurlow LR et al. Disruption of the opal stop codon attenuates Chikungunya virus-induced arthritis and pathology. mBio 2017; 8:e01456-17 [View Article] [PubMed]
     [Google Scholar]
  12. Firth AE, Wills NM, Gesteland RF, Atkins JF. Stimulation of stop codon readthrough: frequent presence of an extended 3’ RNA structural element. Nucleic Acids Res 2011; 39:6679–6691 [View Article] [PubMed]
     [Google Scholar]
  13. Kendra JA, Advani VM, Chen B, Briggs JW, Zhu J et al. Functional and structural characterization of the Chikungunya virus translational recoding signals. J Biol Chem 2018; 293:17536–17545 [View Article] [PubMed]
     [Google Scholar]
  14. Roberts GC, Zothner C, Remenyi R, Merits A, Stonehouse NJ et al. Evaluation of a range of mammalian and mosquito cell lines for use in Chikungunya virus research. Sci Rep 2017; 7:14641 [View Article] [PubMed]
     [Google Scholar]
  15. Brackney DE, Scott JC, Sagawa F, Woodward JE, Miller NA et al. C6/36 Aedes albopictus cells have a dysfunctional antiviral RNA interference response. PLoS Negl Trop Dis 2010; 4:e856 [View Article] [PubMed]
     [Google Scholar]
  16. Pohjala L, Utt A, Varjak M, Lulla A, Merits A et al. Inhibitors of alphavirus entry and replication identified with a stable Chikungunya replicon cell line and virus-based assays. PLoS One 2011; 6:e28923 [View Article] [PubMed]
     [Google Scholar]
  17. Tsetsarkin K, Higgs S, McGee CE, De Lamballerie X, Charrel RN et al. Infectious clones of Chikungunya virus (La Réunion isolate) for vector competence studies. Vector Borne Zoonotic Dis 2006; 6:325–337 [View Article] [PubMed]
     [Google Scholar]
  18. Gao Y, Goonawardane N, Ward J, Tuplin A, Harris M. Multiple roles of the non-structural protein 3 (nsP3) alphavirus unique domain (AUD) during Chikungunya virus genome replication and transcription. PLoS Pathog 2019; 15:e1007239 [View Article] [PubMed]
     [Google Scholar]
  19. Katoh K, Rozewicki J, Yamada KD. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform 2019; 20:1160–1166 [View Article] [PubMed]
     [Google Scholar]
  20. Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ. Jalview Version 2--a multiple sequence alignment editor and analysis workbench. Bioinformatics 2009; 25:1189–1191 [View Article] [PubMed]
     [Google Scholar]
  21. Kendall C, Khalid H, Müller M, Banda DH, Kohl A et al. Structural and phenotypic analysis of Chikungunya virus RNA replication elements. Nucleic Acids Res 2019; 47:9296–9312 [View Article] [PubMed]
     [Google Scholar]
  22. Karabiber F, McGinnis JL, Favorov OV, Weeks KM. QuShape: rapid, accurate, and best-practices quantification of nucleic acid probing information, resolved by capillary electrophoresis. RNA 2013; 19:63–73 [View Article] [PubMed]
     [Google Scholar]
  23. Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 2003; 31:3406–3415 [View Article] [PubMed]
     [Google Scholar]
  24. Reuter JS, Mathews DH. RNAstructure: software for RNA secondary structure prediction and analysis. BMC Bioinformatics 2010; 11:129 [View Article] [PubMed]
     [Google Scholar]
  25. Deigan KE, Li TW, Mathews DH, Weeks KM. Accurate SHAPE-directed RNA structure determination. Proc Natl Acad Sci U S A 2009; 106:97–102 [View Article] [PubMed]
     [Google Scholar]
  26. Darty K, Denise A, Ponty Y. VARNA: Interactive drawing and editing of the RNA secondary structure. Bioinformatics 2009; 25:1974–1975 [View Article] [PubMed]
     [Google Scholar]
  27. Li G, Rice CM. The signal for translational readthrough of a UGA codon in Sindbis virus RNA involves a single cytidine residue immediately downstream of the termination codon. J Virol 1993; 67:5062–5067 [View Article] [PubMed]
     [Google Scholar]
  28. Singh SK, Unni SK. Chikungunya virus: host pathogen interaction. Rev Med Virol 2011; 21:78–88 [View Article] [PubMed]
     [Google Scholar]
  29. Schlesinger S, Dubensky TW. Alphavirus vectors for gene expression and vaccines. Curr Opin Biotechnol 1999; 10:434–439 [View Article] [PubMed]
     [Google Scholar]
  30. Rodnina MV. Decoding and recoding of mRNA sequences by the ribosome. Annu Rev Biophys 2023; 52:161–182 [View Article] [PubMed]
     [Google Scholar]
  31. Myles KM, Kelly CLH, Ledermann JP, Powers AM. Effects of an opal termination codon preceding the nsP4 gene sequence in the O’Nyong-Nyong virus genome on Anopheles gambiae infectivity. J Virol 2006; 80:4992–4997 [View Article] [PubMed]
     [Google Scholar]
  32. Lanciotti RS, Ludwig ML, Rwaguma EB, Lutwama JJ, Kram TM et al. Emergence of epidemic O’nyong-nyong fever in Uganda after a 35-year absence: genetic characterization of the virus. Virology 1998; 252:258–268 [View Article] [PubMed]
     [Google Scholar]
  33. Mounce BC, Cesaro T, Vlajnić L, Vidiņa A, Vallet T et al. Chikungunya virus overcomes polyamine depletion by mutation of nsP1 and the opal stop codon to confer enhanced replication and fitness. J Virol 2017; 91:e00344-17 [View Article] [PubMed]
     [Google Scholar]
  34. Madden EA, Plante KS, Morrison CR, Kutchko KM, Sanders W et al. Using SHAPE-MaP to model RNA secondary structure and identify 3’UTR variation in Chikungunya virus. J Virol 2020; 94:e00701-20 [View Article] [PubMed]
     [Google Scholar]
  35. Wagner RN, Wiessner M, Friedrich A, Zandanell J, Breitenbach-Koller H et al. Emerging personalized opportunities for enhancing translational readthrough in rare genetic diseases and beyond. Int J Mol Sci 2023; 24:6101 [View Article] [PubMed]
     [Google Scholar]
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A structural and functional analysis of opal stop codon translational readthrough during Chikungunya virus replication
J. Gen. Virol. 104, 001909 (2023); https://doi.org/10.1099/jgv.0.001909
/content/journal/jgv/10.1099/jgv.0.001909
/content/journal/jgv/10.1099/jgv.0.001909
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