Codon optimization of the prM-E coding region generates stable genome-length cDNA clone for a chimeric dengue 2/3 virus that can be propagated in Escherichia coli

Poonsook Keelapang; Sakawdaurn Yasom; Chunya Puttikhunt; Chutithorn Ketloy; Nopporn Sittisombut

Authors

Poonsook Keelapang Department of Microbiology, Faculty of Medicine, Chiang Mai University, Thailand
Sakawdaurn Yasom Department of Microbiology, Faculty of Medicine, Chiang Mai University, Thailand
Chunya Puttikhunt Medical Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Thailand
Chutithorn Ketloy Faculty of Medicine, Chulalongkorn University, Thailand
Nopporn Sittisombut Faculty of Medicine, Chulalongkorn University, Thailand

Keywords:

dengue, serotype 3, cDNA, chimeric virus

Abstract

Abstract In an effort to generate a chimeric dengue virus containing the prM-E sequence from DENV-3 and with the rest of the viral genome derived from a DENV-2 strain 16681-3pm by in vitro ligation of subgenomic fragments, it was found that the ligated chimeric genome could not be stably propagated as genome-length cDNA clone in Escherichia coli. Native DENV-3 prM-E coding region may contain certain nucleotide sequence that results in plasmid instability when ampli fi ed in E. coli. In this study, we modi fi ed codons in the DENV-3 viral sequence into those that are most frequently used in human genome. Employing the Gibson assembly method, the modi fi ed prM-E fragment was introduced into the full-length cDNA clone of strain 16681-3pm to replace the corresponding sequence in a DENV-2 genome. The full-length plasmid cDNA clone intended for the generation of a chimeric virus, D3 prMEopt/16661-3pm, was successfully propagated in E. coli. In vitro transcription of this clone produced full-length RNA transcripts that generated infectious viruses when introduced into cultured mammalian cells. Based on kinetics experiments, the replication of D3 prMEopt/16661-3pm with modi fi ed codons in the prM-E gene was comparable to the one generated by the in vitro ligation approach. By combining the use of modi fi ed codons and the Gibson assembly method, a full-length cDNA clone of a chimeric virus containing the DENV-3 prM-E sequence can be stably propagated in E. coli, facilitating further manipulation of the viral sequences. Availability of
such an infectious cDNA clone would provide a valuable tool for investigating the role of nucleotide/amino acid variations in the DENV-3 prM-E gene in viral replication, immunogenicity, and pathogenicity as well as accelerating dengue vaccine development.

References

Bhatt S, Gething PW, Brady OJ, Messina JP, Far-low AW, Moyes CL, et al. The global distribution and burden of dengue. Nature. 2013;496(7446):504-7.

Calisher CH, Karabatsos N, Dalrymple JM, Shope RE, Porterfi eld JS, Westaway EG, et al. Antigenic relationships between fl aviviruses as determined by cross-neutralization tests with polyclonal anti-sera. J. Gen. Virol. 1989;70(Pt1):37-43.

Nimmannitya S. Clinical spectrum and manage-ment of dengue haemorrhagic fever. Southeast Asian J Trop Med Public Health. 1987;18:392-7.

Torresi J, Ebert G, Pellegrini M. Vaccines licensed and in clinical trials for the prevention of dengue. Hum Vaccin Immunother. 2017;14:1-14.

Hadinegoro SR, Arredondo-García JL, Capeding MR, Deseda C, Chotpitayasunondh T, Dietze R, et al. Effi cacy and long-term safety of a dengue vaccine in regions of endemic disease. N Engl J Med. 2015;373:1195-206.

Pierson TC, Diamond MS. Flaviviruses. In: Knipe DM, Howley DE, editors. Fields Virology. 6th ed. Philadelphia: Lippincott Williams & Wilkins; 2013. p. 747-94.

Lai CJ, Zhao BT, Hori H, Bray M. Infectious RNA transcribed from stably cloned full-length cDNA of dengue type 4 virus. Proc Natl Acad Sci USA. 1991;88:5139-43.

Rice CM, Grakoui A, Galler R, Chambers TJ. Transcription of infectious yellow fever RNA from full-length cDNA templates produced by in vitro li-gation. New Biol.1989;1:285-96.

Sumiyoshi H, Hoke CH, Trent DW. Infectious Japa-nese encephalitis virus RNA can be synthesized from in vitro-ligated cDNA templates. J Virol. 1992; 66:5425-31.

Chen W, Kawano H, Men R, Clark D, Lai CJ. Con-struction of intertypic chimeric dengue viruses ex-hibiting type 3 antigenicity and neurovirulence for mice. J Virol. 1995;69:5186-90.

Kapoor M, Zhang L, Mohan PM, Padmanabhan R. Synthesis and characterization of an infectious dengue virus type-2 RNA genome (New Guinea C strain). Gene. 1995;162:175-80.

Polo S, Ketner G, Levis R, Falgout B. Infectious RNA transcripts from full-length dengue virus type 2 cDNA clones made in yeast. J Virol. 1997;71: 5366-74.

Khromykh AA,Westaway EG. Completion of Kun-jin virus RNA sequence and recovery of an in-fectious RNA transcribed from stably cloned full-length cDNA. J Virol. 1994;68:4580-8.

Gualano RC, Pryor MJ, Cauchi MR, Wright PJ, Davidson AD. Identifi cation of a major determi-nant of mouse neurovirulence of dengue virus type 2 using stably cloned genomic-length cDNA. J Gen Virol. 1998;79(Pt 3):437-46.

Puri B, Polo S, Hayes CG, Falgout B. Construction of a full length infectious clone for dengue-1 virus Western Pacifi c,74 strain. Virus Genes. 2000;20:57-63

Usme-Ciro JA, Lopera JA, Enjuanes L, Almazán F, Gallego-Gomez JC. Development of a novel DNA-launched dengue virus type 2 infectious clone assembled in a bacterial artifi cial chromo-some. Virus Res. 2014;180:12-22.

Yamshchikov V, Mishin V, Cominelli F. A new strat-egy in design of +RNA virus infectious clones ena-bling their stable propagation in E. coli. Virology. 2001;281:272-80.

Blaney JE Jr, Hanson CT, Firestone CY, Hanley KA, Murphy BR, Whitehead SS. Genetically modi-fi ed, live attenuated dengue virus type 3 vaccine candidates. Am J Trop Med Hyg. 2004;71:811-2.

Pu SY, Wu RH, Yang CC, Jao TM, Tsai MH, Wang JC, Lin HM, Chao YS, Yueh A, et al. Successful propagation of fl avivirus infectious cDNAs by a novel method to reduce the cryptic bacterial pro-moter activity of virus genomes. J Virol. 2011;85: 2927-41.

Li D, Aaskov J, Lott WB. Identifi cation of a cryptic prokaryotic promoter within the cDNA encoding the 5′ end of dengue virus RNA genome. PLoS One. 2011;6:e18197.

Sriburi R, Keelapang P, Duangchinda T, Pruksa-korn S, Maneekarn N, Malasit P, et al. Construc-tion of infectious dengue 2 cDNA clones using high copy number plasmid. J Virol Methods. 2001; 92:71-82.

Keelapang P, Nitatpattana N, Suphatrakul A, Pu-nyahathaikul S, Sriburi R, Pulmanausahakul R, et al. Generation and preclinical evaluation of a DENV-1/2 prM+E chimeric live attenuated vaccine candidate with enhanced prM cleavage. Vaccine. 2013;31:5134-40.

Chaiwino K, Keelapang P, Sittisombut N. Infl u-ence of codon usage optimization of dengue virus envelope genes on protein expression and virus replication. Journal of the Medical Technologist Association of Thailand 2013;41(Suppl):s76-88.

Guirakhoo F, Arroyo J, Pugachev KV, Miller C, Zhang ZX, Weltzin R, et al. Construction, safety, and immunogenicity in nonhuman primates of a chimeric yellow fever-dengue virus tetravalent vaccine. J Virol. 2001;75:7290-304.

Santos JJ, Cordeiro MT, Bertani GR, Marques ET, Gil LH. Construction and characterisation of a complete reverse genetics system of dengue virus type 3. Mem Inst Oswaldo Cruz. 2013;108:983-91.

Santos JJ, Cordeiro MT, Bertani GR, Marques ET, Gil LH. A two-plasmid strategy for engineering a dengue virus type 3 infectious clone from primary Brazilian isolate. An Acad Bras Cienc. 2014;86: 1749-59.

Keelapang P, Sriburi R, Supasa S, Panyadee N, Songjaeng A, Jairungsri A, et al. Alterations of pr-M cleavage and virus export in pr-M junction chimeric dengue viruses. J Virol. 2004;78:2367-81.

Prompetchara E, Ketloy C, Keelapang P, Sittisom-but N, Ruxrungtham K. Induction of neutralizing antibody response against four dengue viruses in mice by intramuscular electroporation of tetrava-lent DNA vaccines. PLoS ONE 2014;9: e92643.

Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO. Enzymatic assem-bly of DNA molecules up to several hundred kilo-bases. Nat Methods. 2009;6:343-5.

Wahala WMPB, Kraus AA, Haymore LB, Accav-itti-Loper MA, Aravinda M. de Silva AM. Dengue virus neutralization by human immune sera: role of envelope protein domain III - reactive antibody. Virology. 2009;392:103–13.

Edmonds J, van Grinsven E, Prow N, Bosco-Lauth A, Brault AC, Bowen RA, et al. A novel bacterium-free method for generation of fl avivirus infectious DNA by circular polymerase extension reaction al-lows accurate recapitulation of viral heterogeneity. J Virol. 2013;87:2367–72.

Siridechadilok B, Gomutsukhavadee M, Sawaeng-pol T, Sangiambut S, Puttikhunt C, Chin-inmanu K, et al. A simplifi ed positive-sense-RNA virus con-struction approach that enhances analysis throughput. J Virol. 2013;87:12667-74.

Quax TE, Claassens NJ, Söll D, van der Oost J. Codon Bias as a means to fi ne-tune gene expres-sion. Mol Cell. 2015;59:149-61