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J Virol. Jul 1997; 71(7): 5366–5374.
PMCID: PMC191775

Infectious RNA transcripts from full-length dengue virus type 2 cDNA clones made in yeast.

Abstract

The dengue virus type 2 genomic RNA was amplified by reverse transcription-PCR and cloned as four cDNA fragments. We could not assemble these four fragments into full-length cDNA in Escherichia coli. The full-length dengue virus cDNA was constructed by homologous recombination in yeast, either as part of a yeast artificial chromosome or in a yeast-E. coli shuttle vector. Full-length cDNA clones were propagated once in E. coli to prepare useful quantities of DNA. In vitro transcription of these clones produced full-length RNA transcripts. Introduction of these transcripts into LLC-MK2 cells produced typical dengue infection, as judged by cytopathic effects and indirect immunofluorescence. Infectivity was sensitive to RNase digestion and was dependent on the presence of cap analog in the transcription reaction mixture. Virus in the medium was passaged on C6-36 cells to produce stocks, and these stocks had titers and plaque morphologies similar to those of the parental dengue virus type 2. Intracellular dengue virus RNA from cells infected with transcript-derived virus contained an introduced BstEII site, proving that infectivity was derived from RNA transcripts and not from contamination with parental dengue virus. Transcript-derived virus was comparable to dengue virus type 2 for growth and protein expression in tissue culture cells. Sequence analysis of the dengue virus cDNA in one full-length clone revealed only one unexpected silent mutation. By using yeast technology, it will be easy to introduce specific mutations into the dengue virus cDNA, allowing analysis of the virus phenotype in cells transfected with mutant transcripts.

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Selected References

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  • ADA GL, ANDERSON SG. Yield of infective ribonucleic acid from impure Murray Valley encephalitis virus after different treatments. Nature. 1959 Mar 21;183(4664):799–800. [PubMed]
  • Ahlquist P, French R, Janda M, Loesch-Fries LS. Multicomponent RNA plant virus infection derived from cloned viral cDNA. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7066–7070. [PMC free article] [PubMed]
  • Ball LA. Cellular expression of a functional nodavirus RNA replicon from vaccinia virus vectors. J Virol. 1992 Apr;66(4):2335–2345. [PMC free article] [PubMed]
  • Chambers TJ, Hahn CS, Galler R, Rice CM. Flavivirus genome organization, expression, and replication. Annu Rev Microbiol. 1990;44:649–688. [PubMed]
  • Christianson TW, Sikorski RS, Dante M, Shero JH, Hieter P. Multifunctional yeast high-copy-number shuttle vectors. Gene. 1992 Jan 2;110(1):119–122. [PubMed]
  • Deubel V, Kinney RM, Trent DW. Nucleotide sequence and deduced amino acid sequence of the structural proteins of dengue type 2 virus, Jamaica genotype. Virology. 1986 Dec;155(2):365–377. [PubMed]
  • Falgout B, Chanock R, Lai CJ. Proper processing of dengue virus nonstructural glycoprotein NS1 requires the N-terminal hydrophobic signal sequence and the downstream nonstructural protein NS2a. J Virol. 1989 May;63(5):1852–1860. [PMC free article] [PubMed]
  • Falgout B, Markoff L. Evidence that flavivirus NS1-NS2A cleavage is mediated by a membrane-bound host protease in the endoplasmic reticulum. J Virol. 1995 Nov;69(11):7232–7243. [PMC free article] [PubMed]
  • Gritsun TS, Gould EA. Infectious transcripts of tick-borne encephalitis virus, generated in days by RT-PCR. Virology. 1995 Dec 20;214(2):611–618. [PubMed]
  • Hahn CS, Hahn YS, Rice CM, Lee E, Dalgarno L, Strauss EG, Strauss JH. Conserved elements in the 3' untranslated region of flavivirus RNAs and potential cyclization sequences. J Mol Biol. 1987 Nov 5;198(1):33–41. [PubMed]
  • Huxley C, Green ED, Dunham I. Rapid assessment of S. cerevisiae mating type by PCR. Trends Genet. 1990 Aug;6(8):236–236. [PubMed]
  • Irie K, Mohan PM, Sasaguri Y, Putnak R, Padmanabhan R. Sequence analysis of cloned dengue virus type 2 genome (New Guinea-C strain). Gene. 1989 Feb 20;75(2):197–211. [PubMed]
  • Janda M, Ahlquist P. RNA-dependent replication, transcription, and persistence of brome mosaic virus RNA replicons in S. cerevisiae. Cell. 1993 Mar 26;72(6):961–970. [PubMed]
  • 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 Sep 11;162(2):175–180. [PubMed]
  • Khromykh AA, Westaway EG. Completion of Kunjin virus RNA sequence and recovery of an infectious RNA transcribed from stably cloned full-length cDNA. J Virol. 1994 Jul;68(7):4580–4588. [PMC free article] [PubMed]
  • 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 U S A. 1991 Jun 15;88(12):5139–5143. [PMC free article] [PubMed]
  • Lerner CG, Inouye M. Low copy number plasmids for regulated low-level expression of cloned genes in Escherichia coli with blue/white insert screening capability. Nucleic Acids Res. 1990 Aug 11;18(15):4631–4631. [PMC free article] [PubMed]
  • Mizutani S, Colonno RJ. In vitro synthesis of an infectious RNA from cDNA clones of human rhinovirus type 14. J Virol. 1985 Nov;56(2):628–632. [PMC free article] [PubMed]
  • Monath TP. Dengue: the risk to developed and developing countries. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2395–2400. [PMC free article] [PubMed]
  • Rice CM, Grakoui A, Galler R, Chambers TJ. Transcription of infectious yellow fever RNA from full-length cDNA templates produced by in vitro ligation. New Biol. 1989 Dec;1(3):285–296. [PubMed]
  • Smith DR, Smyth AP, Moir DT. Amplification of large artificial chromosomes. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8242–8246. [PMC free article] [PubMed]
  • Sumiyoshi H, Hoke CH, Trent DW. Infectious Japanese encephalitis virus RNA can be synthesized from in vitro-ligated cDNA templates. J Virol. 1992 Sep;66(9):5425–5431. [PMC free article] [PubMed]
  • Winkler G, Randolph VB, Cleaves GR, Ryan TE, Stollar V. Evidence that the mature form of the flavivirus nonstructural protein NS1 is a dimer. Virology. 1988 Jan;162(1):187–196. [PubMed]

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