Non-gradient and genotype-dependent patterns of RSV gene expression

Autoři: Felipe-Andrés Piedra aff001;  Xueting Qiu aff002;  Michael N. Teng aff003;  Vasanthi Avadhanula aff001;  Annette A. Machado aff001;  Do-Kyun Kim aff004;  James Hixson aff004;  Justin Bahl aff002;  Pedro A. Piedra aff001
Působiště autorů: Department of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, TX, United States of America aff001;  Center for the Ecology of Infectious Diseases, Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA, United States of America aff002;  Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida Morsani College of Medicine, Tampa, FL, United States of America aff003;  Human Genetics Center, School of Public Health, University of Texas Health Science Center, Houston, TX, United States of America aff004;  Program in Emerging Infectious Diseases, Duke-National University of Singapore Graduate Medical School, Singapore aff005;  Department of Pediatrics, Baylor College of Medicine, Houston, TX, United States of America aff006
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article


Respiratory syncytial virus (RSV) is a nonsegmented negative-strand RNA virus (NSV) and a leading cause of severe lower respiratory tract illness in infants and the elderly. Transcription of the ten RSV genes proceeds sequentially from the 3’ promoter and requires conserved gene start (GS) and gene end (GE) signals. Previous studies using the prototypical GA1 genotype Long and A2 strains have indicated a gradient of gene transcription extending across the genome, with the highest level of mRNA coming from the most promoter-proximal gene, the first nonstructural (NS1) gene, and mRNA levels from subsequent genes dropping until reaching a minimum at the most promoter-distal gene, the polymerase (L) gene. However, recent reports show non-gradient levels of mRNA, with higher than expected levels from the attachment (G) gene. It is unknown to what extent different transcript stabilities might shape measured mRNA levels. It is also unclear whether patterns of RSV gene expression vary, or show strain- or genotype-dependence. To address this, mRNA abundances from five RSV genes were measured by quantitative real-time PCR (qPCR) in three cell lines and in cotton rats infected with RSV isolates belonging to four genotypes (GA1, ON, GB1, BA). Relative mRNA levels reached steady-state between four and 24 hours post-infection. Steady-state patterns were non-gradient and genotype-specific, where mRNA levels from the G gene exceeded those from the more promoter-proximal nucleocapsid (N) gene across isolates. Transcript stabilities could not account for the non-gradient patterns observed, indicating that relative mRNA levels more strongly reflect transcription than decay. Our results indicate that gene expression from a small but diverse set of RSV genotypes is non-gradient and genotype-dependent. We propose novel models of RSV transcription that can account for non-gradient transcription.

Klíčová slova:

DNA transcription – Gene expression – Genomic signal processing – Messenger RNA – Oligonucleotides – Polymerases – Transcriptional control – Viral gene expression


1. American Academy of Pediatrics Subcommittee on D, Management of B. Diagnosis and management of bronchiolitis. Pediatrics. 2006;118(4):1774–93. doi: 10.1542/peds.2006-2223 17015575.

2. Hasegawa K, Tsugawa Y, Brown DF, Mansbach JM, Camargo CA Jr. Temporal trends in emergency department visits for bronchiolitis in the United States, 2006 to 2010. Pediatr Infect Dis J. 2014;33(1):11–8. doi: 10.1097/INF.0b013e3182a5f324 23934206; PubMed Central PMCID: PMC3984903.

3. Mansbach JM, Piedra PA, Teach SJ, Sullivan AF, Forgey T, Clark S, et al. Prospective multicenter study of viral etiology and hospital length of stay in children with severe bronchiolitis. Arch Pediatr Adolesc Med. 2012;166(8):700–6. doi: 10.1001/archpediatrics.2011.1669 22473882; PubMed Central PMCID: PMC3394902.

4. Nair H, Nokes DJ, Gessner BD, Dherani M, Madhi SA, Singleton RJ, et al. Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis. Lancet. 2010;375(9725):1545–55. doi: 10.1016/S0140-6736(10)60206-1 20399493; PubMed Central PMCID: PMC2864404.

5. Zorc JJ, Hall CB. Bronchiolitis: recent evidence on diagnosis and management. Pediatrics. 2010;125(2):342–9. doi: 10.1542/peds.2009-2092 20100768.

6. Simoes EA. Environmental and demographic risk factors for respiratory syncytial virus lower respiratory tract disease. J Pediatr. 2003;143(5 Suppl):S118–26. doi: 10.1067/s0022-3476(03)00511-0 14615710.

7. Sommer C, Resch B, Simoes EA. Risk factors for severe respiratory syncytial virus lower respiratory tract infection. Open Microbiol J. 2011;5:144–54. doi: 10.2174/1874285801105010144 22262987; PubMed Central PMCID: PMC3258650.

8. Stein RT, Bont LJ, Zar H, Polack FP, Park C, Claxton A, et al. Respiratory syncytial virus hospitalization and mortality: Systematic review and meta-analysis. Pediatr Pulmonol. 2017;52(4):556–69. doi: 10.1002/ppul.23570 27740723; PubMed Central PMCID: PMC5396299.

9. Collins PL, Melero JA. Progress in understanding and controlling respiratory syncytial virus: still crazy after all these years. Virus Res. 2011;162(1–2):80–99. doi: 10.1016/j.virusres.2011.09.020 21963675; PubMed Central PMCID: PMC3221877.

10. Johnson PR, Spriggs MK, Olmsted RA, Collins PL. The G glycoprotein of human respiratory syncytial viruses of subgroups A and B: extensive sequence divergence between antigenically related proteins. Proc Natl Acad Sci U S A. 1987;84(16):5625–9. doi: 10.1073/pnas.84.16.5625 2441388; PubMed Central PMCID: PMC298915.

11. Zlateva KT, Lemey P, Moes E, Vandamme AM, Van Ranst M. Genetic variability and molecular evolution of the human respiratory syncytial virus subgroup B attachment G protein. J Virol. 2005;79(14):9157–67. doi: 10.1128/JVI.79.14.9157-9167.2005 15994810; PubMed Central PMCID: PMC1168771.

12. Agoti CN, Munywoki PK, Phan MVT, Otieno JR, Kamau E, Bett A, et al. Transmission patterns and evolution of respiratory syncytial virus in a community outbreak identified by genomic analysis. Virus Evol. 2017;3(1):vex006. doi: 10.1093/ve/vex006 28458916; PubMed Central PMCID: PMC5399923.

13. Tapia LI, Shaw CA, Aideyan LO, Jewell AM, Dawson BC, Haq TR, et al. Gene sequence variability of the three surface proteins of human respiratory syncytial virus (HRSV) in Texas. PLoS One. 2014;9(3):e90786. doi: 10.1371/journal.pone.0090786 24625544; PubMed Central PMCID: PMC3953119.

14. Schobel SA, Stucker KM, Moore ML, Anderson LJ, Larkin EK, Shankar J, et al. Respiratory Syncytial Virus whole-genome sequencing identifies convergent evolution of sequence duplication in the C-terminus of the G gene. Sci Rep. 2016;6:26311. doi: 10.1038/srep26311 27212633; PubMed Central PMCID: PMC4876326.

15. Bose ME, He J, Shrivastava S, Nelson MI, Bera J, Halpin RA, et al. Sequencing and analysis of globally obtained human respiratory syncytial virus A and B genomes. PLoS One. 2015;10(3):e0120098. doi: 10.1371/journal.pone.0120098 25793751; PubMed Central PMCID: PMC4368745.

16. Abraham G, Banerjee AK. Sequential transcription of the genes of vesicular stomatitis virus. Proc Natl Acad Sci U S A. 1976;73(5):1504–8. doi: 10.1073/pnas.73.5.1504 179088; PubMed Central PMCID: PMC430325.

17. Ball LA, White CN. Order of transcription of genes of vesicular stomatitis virus. Proc Natl Acad Sci U S A. 1976;73(2):442–6. doi: 10.1073/pnas.73.2.442 174107; PubMed Central PMCID: PMC335925.

18. Collins PL, Hightower LE, Ball LA. Transcriptional map for Newcastle disease virus. J Virol. 1980;35(3):682–93. 7420539; PubMed Central PMCID: PMC288862.

19. Collins PL, Mink MA, Stec DS. Rescue of synthetic analogs of respiratory syncytial virus genomic RNA and effect of truncations and mutations on the expression of a foreign reporter gene. Proc Natl Acad Sci U S A. 1991;88(21):9663–7. doi: 10.1073/pnas.88.21.9663 1946383; PubMed Central PMCID: PMC52778.

20. Dickens LE, Collins PL, Wertz GW. Transcriptional mapping of human respiratory syncytial virus. J Virol. 1984;52(2):364–9. 6492254; PubMed Central PMCID: PMC254535.

21. Noton SL, Fearns R. Initiation and regulation of paramyxovirus transcription and replication. Virology. 2015;479–480:545–54. doi: 10.1016/j.virol.2015.01.014 25683441; PubMed Central PMCID: PMC4424093.

22. Whelan SP, Barr JN, Wertz GW. Transcription and replication of nonsegmented negative-strand RNA viruses. Curr Top Microbiol Immunol. 2004;283:61–119. doi: 10.1007/978-3-662-06099-5_3 15298168.

23. Kuo L, Fearns R, Collins PL. Analysis of the gene start and gene end signals of human respiratory syncytial virus: quasi-templated initiation at position 1 of the encoded mRNA. J Virol. 1997;71(7):4944–53. 9188557; PubMed Central PMCID: PMC191725.

24. Kuo L, Grosfeld H, Cristina J, Hill MG, Collins PL. Effects of mutations in the gene-start and gene-end sequence motifs on transcription of monocistronic and dicistronic minigenomes of respiratory syncytial virus. J Virol. 1996;70(10):6892–901. 8794332; PubMed Central PMCID: PMC190738.

25. Sutherland KA, Collins PL, Peeples ME. Synergistic effects of gene-end signal mutations and the M2-1 protein on transcription termination by respiratory syncytial virus. Virology. 2001;288(2):295–307. doi: 10.1006/viro.2001.1105 11601901.

26. Barik S. The structure of the 5' terminal cap of the respiratory syncytial virus mRNA. J Gen Virol. 1993;74 (Pt 3):485–90. doi: 10.1099/0022-1317-74-3-485 8445369.

27. Liuzzi M, Mason SW, Cartier M, Lawetz C, McCollum RS, Dansereau N, et al. Inhibitors of respiratory syncytial virus replication target cotranscriptional mRNA guanylylation by viral RNA-dependent RNA polymerase. J Virol. 2005;79(20):13105–15. doi: 10.1128/JVI.79.20.13105-13115.2005 16189012; PubMed Central PMCID: PMC1235819.

28. Fearns R, Collins PL. Model for polymerase access to the overlapped L gene of respiratory syncytial virus. J Virol. 1999;73(1):388–97. PubMed Central PMCID: PMC103844. 9847343

29. Barr JN, Tang X, Hinzman E, Shen R, Wertz GW. The VSV polymerase can initiate at mRNA start sites located either up or downstream of a transcription termination signal but size of the intervening intergenic region affects efficiency of initiation. Virology. 2008;374(2):361–70. doi: 10.1016/j.virol.2007.12.023 18241907; PubMed Central PMCID: PMC2593140.

30. Brauburger K, Boehmann Y, Krahling V, Muhlberger E. Transcriptional Regulation in Ebola Virus: Effects of Gene Border Structure and Regulatory Elements on Gene Expression and Polymerase Scanning Behavior. J Virol. 2016;90(4):1898–909. doi: 10.1128/JVI.02341-15 26656691; PubMed Central PMCID: PMC4733972.

31. Kolakofsky D, Le Mercier P, Iseni F, Garcin D. Viral DNA polymerase scanning and the gymnastics of Sendai virus RNA synthesis. Virology. 2004;318(2):463–73. doi: 10.1016/j.virol.2003.10.031 15015496.

32. Boukhvalova MS, Prince GA, Blanco JC. Respiratory syncytial virus infects and abortively replicates in the lungs in spite of preexisting immunity. J Virol. 2007;81(17):9443–50. doi: 10.1128/JVI.00102-07 17596309; PubMed Central PMCID: PMC1951413.

33. Aljabr W, Touzelet O, Pollakis G, Wu W, Munday DC, Hughes M, et al. Investigating the Influence of Ribavirin on Human Respiratory Syncytial Virus RNA Synthesis by Using a High-Resolution Transcriptome Sequencing Approach. J Virol. 2016;90(10):4876–88. doi: 10.1128/JVI.02349-15 26656699; PubMed Central PMCID: PMC4859727.

34. Iverson LE, Rose JK. Localized attenuation and discontinuous synthesis during vesicular stomatitis virus transcription. Cell. 1981;23(2):477–84. doi: 10.1016/0092-8674(81)90143-4 6258804.

35. Barik S. Transcription of human respiratory syncytial virus genome RNA in vitro: requirement of cellular factor(s). J Virol. 1992;66(11):6813–8. 1404620; PubMed Central PMCID: PMC240184.

36. Collins PL, Wertz GW. cDNA cloning and transcriptional mapping of nine polyadenylylated RNAs encoded by the genome of human respiratory syncytial virus. Proc Natl Acad Sci U S A. 1983;80(11):3208–12. doi: 10.1073/pnas.80.11.3208 6190173; PubMed Central PMCID: PMC394009.

37. Levitz R, Gao Y, Dozmorov I, Song R, Wakeland EK, Kahn JS. Distinct patterns of innate immune activation by clinical isolates of respiratory syncytial virus. PLoS One. 2017;12(9):e0184318. doi: 10.1371/journal.pone.0184318 28877226; PubMed Central PMCID: PMC5587315.

38. Piedra FA, Mei M, Avadhanula V, Mehta R, Aideyan L, Garofalo RP, et al. The interdependencies of viral load, the innate immune response, and clinical outcome in children presenting to the emergency department with respiratory syncytial virus-associated bronchiolitis. PLoS One. 2017;12(3):e0172953. doi: 10.1371/journal.pone.0172953 28267794; PubMed Central PMCID: PMC5340370.

39. Collins PL, Huang YT, Wertz GW. Identification of a tenth mRNA of respiratory syncytial virus and assignment of polypeptides to the 10 viral genes. J Virol. 1984;49(2):572–8. 6546401; PubMed Central PMCID: PMC255499.

40. Boyle B, Dallaire N, MacKay J. Evaluation of the impact of single nucleotide polymorphisms and primer mismatches on quantitative PCR. BMC Biotechnol. 2009;9:75. doi: 10.1186/1472-6750-9-75 19715565; PubMed Central PMCID: PMC2741440.

41. Suss B, Flekna G, Wagner M, Hein I. Studying the effect of single mismatches in primer and probe binding regions on amplification curves and quantification in real-time PCR. J Microbiol Methods. 2009;76(3):316–9. doi: 10.1016/j.mimet.2008.12.003 19135484.

42. Bakre AA, Harcourt JL, Haynes LM, Anderson LJ, Tripp RA. The Central Conserved Region (CCR) of Respiratory Syncytial Virus (RSV) G Protein Modulates Host miRNA Expression and Alters the Cellular Response to Infection. Vaccines (Basel). 2017;5(3). doi: 10.3390/vaccines5030016 28671606; PubMed Central PMCID: PMC5620547.

43. Sedeyn K, Schepens B, Saelens X. Respiratory syncytial virus nonstructural proteins 1 and 2: Exceptional disrupters of innate immune responses. PLoS Pathog. 2019;15(10):e1007984. doi: 10.1371/journal.ppat.1007984 31622448; PubMed Central PMCID: PMC6797084.

44. Hardy RW, Harmon SB, Wertz GW. Diverse gene junctions of respiratory syncytial virus modulate the efficiency of transcription termination and respond differently to M2-mediated antitermination. J Virol. 1999;73(1):170–6. 9847319; PubMed Central PMCID: PMC103820.

45. Harmon SB, Wertz GW. Transcriptional termination modulated by nucleotides outside the characterized gene end sequence of respiratory syncytial virus. Virology. 2002;300(2):304–15. doi: 10.1006/viro.2002.1541 12350361.

46. Moudy RM, Sullender WM, Wertz GW. Variations in intergenic region sequences of Human respiratory syncytial virus clinical isolates: analysis of effects on transcriptional regulation. Virology. 2004;327(1):121–33. doi: 10.1016/j.virol.2004.06.013 15327903.

47. Peret TC, Hall CB, Schnabel KC, Golub JA, Anderson LJ. Circulation patterns of genetically distinct group A and B strains of human respiratory syncytial virus in a community. J Gen Virol. 1998;79 (Pt 9):2221–9. doi: 10.1099/0022-1317-79-9-2221 9747732.

48. Avadhanula V, Chemaly RF, Shah DP, Ghantoji SS, Azzi JM, Aideyan LO, et al. Infection with novel respiratory syncytial virus genotype Ontario (ON1) in adult hematopoietic cell transplant recipients, Texas, 2011–2013. J Infect Dis. 2015;211(4):582–9. doi: 10.1093/infdis/jiu473 25156562.

49. Nicholson EG, Schlegel C, Garofalo RP, Mehta R, Scheffler M, Mei M, et al. Robust Cytokine and Chemokine Response in Nasopharyngeal Secretions: Association With Decreased Severity in Children With Physician Diagnosed Bronchiolitis. J Infect Dis. 2016;214(4):649–55. doi: 10.1093/infdis/jiw191 27190183; PubMed Central PMCID: PMC4957440.

50. Stobart CC, Rostad CA, Ke Z, Dillard RS, Hampton CM, Strauss JD, et al. A live RSV vaccine with engineered thermostability is immunogenic in cotton rats despite high attenuation. Nat Commun. 2016;7:13916. doi: 10.1038/ncomms13916 28000669; PubMed Central PMCID: PMC5187593 for the Company. M.L.M., C.C.S., A.L.H., J.M. and C.A.R. are co-inventors of RSV vaccine technology subject to evaluation in this paper. The vaccine technology has been optioned to Meissa by Emory University. The remaining authors declare no competing financial interests.

51. AB StepOne and StepOnePlus Real-Time PCR Systems—Relative Standard Curve and Comparative CT Experiments. 2008.

52. Lo MK, Jordan R, Arvey A, Sudhamsu J, Shrivastava-Ranjan P, Hotard AL, et al. GS-5734 and its parent nucleoside analog inhibit Filo-, Pneumo-, and Paramyxoviruses. Sci Rep. 2017;7:43395. doi: 10.1038/srep43395 28262699; PubMed Central PMCID: PMC5338263.

53. Warren TK, Jordan R, Lo MK, Ray AS, Mackman RL, Soloveva V, et al. Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys. Nature. 2016;531(7594):381–5. doi: 10.1038/nature17180 26934220; PubMed Central PMCID: PMC5551389.

54. Agoti CN, Otieno JR, Munywoki PK, Mwihuri AG, Cane PA, Nokes DJ, et al. Local evolutionary patterns of human respiratory syncytial virus derived from whole-genome sequencing. J Virol. 2015;89(7):3444–54. doi: 10.1128/JVI.03391-14 25609811; PubMed Central PMCID: PMC4403408.

55. Shepard SS, Meno S, Bahl J, Wilson MM, Barnes J, Neuhaus E. Erratum to: Viral deep sequencing needs an adaptive approach: IRMA, the iterative refinement meta-assembler. BMC Genomics. 2016;17(1):801. doi: 10.1186/s12864-016-3138-8 27737640; PubMed Central PMCID: PMC5064889.

56. Shepard SS, Meno S, Bahl J, Wilson MM, Barnes J, Neuhaus E. Viral deep sequencing needs an adaptive approach: IRMA, the iterative refinement meta-assembler. BMC Genomics. 2016;17:708. doi: 10.1186/s12864-016-3030-6 27595578; PubMed Central PMCID: PMC5011931.

Článek vyšel v časopise


2020 Číslo 1
Nejčtenější tento týden