Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1994 Jul;68(7):4580–4588. doi: 10.1128/jvi.68.7.4580-4588.1994

Completion of Kunjin virus RNA sequence and recovery of an infectious RNA transcribed from stably cloned full-length cDNA.

A A Khromykh 1, E G Westaway 1
PMCID: PMC236385  PMID: 8207832

Abstract

Completion of the Kunjin virus (KUN) RNA sequence showed that it is the longest flavivirus sequence reported (11,022 bases), commencing with a 5' noncoding region of 96 bases. The 3' noncoding sequence of 624 nucleotides included a unique insertion sequence of 46 bases adjacent to the stop codon, but otherwise it had properties similar to those of RNAs of closely related flaviviruses. A full-length KUN cDNA clone which could be stably propagated in Escherichia coli DH5 alpha was constructed; SP6 polymerase RNA transcripts from amplified cDNA were infectious when transfected into BHK-21 cells. A mutational change abolishing the BamHI restriction site at position 4049, leading to a conservative amino acid change of Arg-175 to Lys in the NS2A protein, was introduced into the cDNA during construction and was retained in the recovered virus. Extra terminal nucleotides introduced during cloning of the cDNA were shown to be present in the in vitro RNA transcripts but absent in the RNA of recovered virus. Although recovered virus differed from the parental KUN by a smaller plaque phenotype and delayed growth rate in BHK-21 cells and mice, it was very similar as assessed by several other criteria, such as peak titer during growth in cells, infectivity titer in cells and in mice, rate of adsorption and penetration in cells, replication at 39 degrees C, and neurovirulence after intraperitoneal injection in mice. The KUN stably cloned cDNA will provide a useful basis for future studies in defining and characterizing functional roles of all the gene products.

Full text

PDF
4580

Images in this article

4584Image
on p.4584
4585Image
on p.4585
4585Image
on p.4585

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Atkinson T., Barrett A. D., Mackenzie A., Dimmock N. J. Persistence of virulent Semliki Forest virus in mouse brain following co-inoculation with defective interfering particles. J Gen Virol. 1986 Jun;67(Pt 6):1189–1194. doi: 10.1099/0022-1317-67-6-1189. [DOI] [PubMed] [Google Scholar]
  2. Boyer J. C., Haenni A. L. Infectious transcripts and cDNA clones of RNA viruses. Virology. 1994 Feb;198(2):415–426. doi: 10.1006/viro.1994.1053. [DOI] [PubMed] [Google Scholar]
  3. Bray M., Lai C. J. Construction of intertypic chimeric dengue viruses by substitution of structural protein genes. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10342–10346. doi: 10.1073/pnas.88.22.10342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brinton M. A., Dispoto J. H. Sequence and secondary structure analysis of the 5'-terminal region of flavivirus genome RNA. Virology. 1988 Feb;162(2):290–299. doi: 10.1016/0042-6822(88)90468-0. [DOI] [PubMed] [Google Scholar]
  5. Brinton M. A., Fernandez A. V., Dispoto J. H. The 3'-nucleotides of flavivirus genomic RNA form a conserved secondary structure. Virology. 1986 Aug;153(1):113–121. doi: 10.1016/0042-6822(86)90012-7. [DOI] [PubMed] [Google Scholar]
  6. Burns C. C., Richards O. C., Ehrenfeld E. Temperature-sensitive polioviruses containing mutations in RNA polymerase. Virology. 1992 Aug;189(2):568–582. doi: 10.1016/0042-6822(92)90580-i. [DOI] [PubMed] [Google Scholar]
  7. Castle E., Leidner U., Nowak T., Wengler G., Wengler G. Primary structure of the West Nile flavivirus genome region coding for all nonstructural proteins. Virology. 1986 Feb;149(1):10–26. doi: 10.1016/0042-6822(86)90082-6. [DOI] [PubMed] [Google Scholar]
  8. Chambers T. J., Hahn C. S., Galler R., Rice C. M. Flavivirus genome organization, expression, and replication. Annu Rev Microbiol. 1990;44:649–688. doi: 10.1146/annurev.mi.44.100190.003245. [DOI] [PubMed] [Google Scholar]
  9. Chambers T. J., McCourt D. W., Rice C. M. Production of yellow fever virus proteins in infected cells: identification of discrete polyprotein species and analysis of cleavage kinetics using region-specific polyclonal antisera. Virology. 1990 Jul;177(1):159–174. doi: 10.1016/0042-6822(90)90470-c. [DOI] [PubMed] [Google Scholar]
  10. Chambers T. J., Nestorowicz A., Amberg S. M., Rice C. M. Mutagenesis of the yellow fever virus NS2B protein: effects on proteolytic processing, NS2B-NS3 complex formation, and viral replication. J Virol. 1993 Nov;67(11):6797–6807. doi: 10.1128/jvi.67.11.6797-6807.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chambers T. J., Weir R. C., Grakoui A., McCourt D. W., Bazan J. F., Fletterick R. J., Rice C. M. Evidence that the N-terminal domain of nonstructural protein NS3 from yellow fever virus is a serine protease responsible for site-specific cleavages in the viral polyprotein. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8898–8902. doi: 10.1073/pnas.87.22.8898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chu P. W., Westaway E. G. Characterization of Kunjin virus RNA-dependent RNA polymerase: reinitiation of synthesis in vitro. Virology. 1987 Apr;157(2):330–337. doi: 10.1016/0042-6822(87)90275-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chu P. W., Westaway E. G. Molecular and ultrastructural analysis of heavy membrane fractions associated with the replication of Kunjin virus RNA. Arch Virol. 1992;125(1-4):177–191. doi: 10.1007/BF01309636. [DOI] [PubMed] [Google Scholar]
  14. Chu P. W., Westaway E. G. Replication strategy of Kunjin virus: evidence for recycling role of replicative form RNA as template in semiconservative and asymmetric replication. Virology. 1985 Jan 15;140(1):68–79. doi: 10.1016/0042-6822(85)90446-5. [DOI] [PubMed] [Google Scholar]
  15. Coia G., Parker M. D., Speight G., Byrne M. E., Westaway E. G. Nucleotide and complete amino acid sequences of Kunjin virus: definitive gene order and characteristics of the virus-specified proteins. J Gen Virol. 1988 Jan;69(Pt 1):1–21. doi: 10.1099/0022-1317-69-1-1. [DOI] [PubMed] [Google Scholar]
  16. Deubel V., Kinney R. M., Trent D. W. Nucleotide sequence and deduced amino acid sequence of the nonstructural proteins of dengue type 2 virus, Jamaica genotype: comparative analysis of the full-length genome. Virology. 1988 Jul;165(1):234–244. doi: 10.1016/0042-6822(88)90677-0. [DOI] [PubMed] [Google Scholar]
  17. Eager K. B., Sawicki J. A., Ricciardi R. P. Cellular protein differences between nontumorigenic Ad5 and tumorigenic Ad12 transformed mouse cells. Virology. 1986 Jul 30;152(2):487–491. doi: 10.1016/0042-6822(86)90153-4. [DOI] [PubMed] [Google Scholar]
  18. Eastman P. S., Blair C. D. Temperature-sensitive mutants of Japanese encephalitis virus. J Virol. 1985 Sep;55(3):611–616. doi: 10.1128/jvi.55.3.611-616.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Falgout B., Pethel M., Zhang Y. M., Lai C. J. Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins. J Virol. 1991 May;65(5):2467–2475. doi: 10.1128/jvi.65.5.2467-2475.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fu J., Tan B. H., Yap E. H., Chan Y. C., Tan Y. H. Full-length cDNA sequence of dengue type 1 virus (Singapore strain S275/90). Virology. 1992 Jun;188(2):953–958. doi: 10.1016/0042-6822(92)90560-c. [DOI] [PubMed] [Google Scholar]
  21. Gorbalenya A. E., Donchenko A. P., Koonin E. V., Blinov V. M. N-terminal domains of putative helicases of flavi- and pestiviruses may be serine proteases. Nucleic Acids Res. 1989 May 25;17(10):3889–3897. doi: 10.1093/nar/17.10.3889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gorbalenya A. E., Koonin E. V., Donchenko A. P., Blinov V. M. Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Res. 1989 Jun 26;17(12):4713–4730. doi: 10.1093/nar/17.12.4713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hahn C. S., Hahn Y. S., Rice C. M., Lee E., Dalgarno L., Strauss E. G., Strauss J. H. Conserved elements in the 3' untranslated region of flavivirus RNAs and potential cyclization sequences. J Mol Biol. 1987 Nov 5;198(1):33–41. doi: 10.1016/0022-2836(87)90455-4. [DOI] [PubMed] [Google Scholar]
  24. Hahn Y. S., Galler R., Hunkapiller T., Dalrymple J. M., Strauss J. H., Strauss E. G. Nucleotide sequence of dengue 2 RNA and comparison of the encoded proteins with those of other flaviviruses. Virology. 1988 Jan;162(1):167–180. doi: 10.1016/0042-6822(88)90406-0. [DOI] [PubMed] [Google Scholar]
  25. Jones D. H., Howard B. H. A rapid method for recombination and site-specific mutagenesis by placing homologous ends on DNA using polymerase chain reaction. Biotechniques. 1991 Jan;10(1):62–66. [PubMed] [Google Scholar]
  26. Kirkegaard K., Baltimore D. The mechanism of RNA recombination in poliovirus. Cell. 1986 Nov 7;47(3):433–443. doi: 10.1016/0092-8674(86)90600-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Koonin E. V. Computer-assisted identification of a putative methyltransferase domain in NS5 protein of flaviviruses and lambda 2 protein of reovirus. J Gen Virol. 1993 Apr;74(Pt 4):733–740. doi: 10.1099/0022-1317-74-4-733. [DOI] [PubMed] [Google Scholar]
  28. Kulkarni A. B., Müllbacher A., Parrish C. R., Westaway E. G., Coia G., Blanden R. V. Analysis of murine major histocompatibility complex class II-restricted T-cell responses to the flavivirus Kunjin by using vaccinia virus expression. J Virol. 1992 Jun;66(6):3583–3592. doi: 10.1128/jvi.66.6.3583-3592.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lai C. J., Zhao B. T., 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. doi: 10.1073/pnas.88.12.5139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lee E., Fernon C., Simpson R., Weir R. C., Rice C. M., Dalgarno L. Sequence of the 3' half of the Murray Valley encephalitis virus genome and mapping of the nonstructural proteins NS1, NS3, and NS5. Virus Genes. 1990 Sep;4(3):197–213. doi: 10.1007/BF00265630. [DOI] [PubMed] [Google Scholar]
  31. Mandl C. W., Heinz F. X., Puchhammer-Stöckl E., Kunz C. Sequencing the termini of capped viral RNA by 5'-3' ligation and PCR. Biotechniques. 1991 Apr;10(4):484–486. [PubMed] [Google Scholar]
  32. Mandl C. W., Holzmann H., Kunz C., Heinz F. X. Complete genomic sequence of Powassan virus: evaluation of genetic elements in tick-borne versus mosquito-borne flaviviruses. Virology. 1993 May;194(1):173–184. doi: 10.1006/viro.1993.1247. [DOI] [PubMed] [Google Scholar]
  33. Mohan P. M., Padmanabhan R. Detection of stable secondary structure at the 3' terminus of dengue virus type 2 RNA. Gene. 1991 Dec 15;108(2):185–191. doi: 10.1016/0378-1119(91)90433-c. [DOI] [PubMed] [Google Scholar]
  34. Osatomi K., Sumiyoshi H. Complete nucleotide sequence of dengue type 3 virus genome RNA. Virology. 1990 Jun;176(2):643–647. doi: 10.1016/0042-6822(90)90037-r. [DOI] [PubMed] [Google Scholar]
  35. Parrish C. R., Coia G., Hill A., Müllbacher A., Westaway E. G., Blanden R. V. Preliminary analysis of murine cytotoxic T cell responses to the proteins of the flavivirus Kunjin using vaccinia virus expression. J Gen Virol. 1991 Jul;72(Pt 7):1645–1653. doi: 10.1099/0022-1317-72-7-1645. [DOI] [PubMed] [Google Scholar]
  36. Pletnev A. G., Bray M., Huggins J., Lai C. J. Construction and characterization of chimeric tick-borne encephalitis/dengue type 4 viruses. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10532–10536. doi: 10.1073/pnas.89.21.10532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Preugschat F., Yao C. W., Strauss J. H. In vitro processing of dengue virus type 2 nonstructural proteins NS2A, NS2B, and NS3. J Virol. 1990 Sep;64(9):4364–4374. doi: 10.1128/jvi.64.9.4364-4374.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rice C. M., Grakoui A., Galler R., Chambers T. J. 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] [Google Scholar]
  39. Rice C. M., Lenches E. M., Eddy S. R., Shin S. J., Sheets R. L., Strauss J. H. Nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution. Science. 1985 Aug 23;229(4715):726–733. doi: 10.1126/science.4023707. [DOI] [PubMed] [Google Scholar]
  40. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schrader A. P., Westaway E. G. Translation mapping with the flavivirus Kunjin: gene order and anomalies in translation of Ns5. Virus Res. 1988 Mar;9(4):323–333. doi: 10.1016/0168-1702(88)90091-3. [DOI] [PubMed] [Google Scholar]
  42. Speight G., Coia G., Parker M. D., Westaway E. G. Gene mapping and positive identification of the non-structural proteins NS2A, NS2B, NS3, NS4B and NS5 of the flavivirus Kunjin and their cleavage sites. J Gen Virol. 1988 Jan;69(Pt 1):23–34. doi: 10.1099/0022-1317-69-1-23. [DOI] [PubMed] [Google Scholar]
  43. Speight G., Westaway E. G. Carboxy-terminal analysis of nine proteins specified by the flavivirus Kunjin: evidence that only the intracellular core protein is truncated. J Gen Virol. 1989 Aug;70(Pt 8):2209–2214. doi: 10.1099/0022-1317-70-8-2209. [DOI] [PubMed] [Google Scholar]
  44. Speight G., Westaway E. G. Positive identification of NS4A, the last of the hypothetical nonstructural proteins of flaviviruses. Virology. 1989 May;170(1):299–301. doi: 10.1016/0042-6822(89)90383-8. [DOI] [PubMed] [Google Scholar]
  45. Sumiyoshi H., Hoke C. H., Trent D. W. Infectious Japanese encephalitis virus RNA can be synthesized from in vitro-ligated cDNA templates. J Virol. 1992 Sep;66(9):5425–5431. doi: 10.1128/jvi.66.9.5425-5431.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sumiyoshi H., Mori C., Fuke I., Morita K., Kuhara S., Kondou J., Kikuchi Y., Nagamatu H., Igarashi A. Complete nucleotide sequence of the Japanese encephalitis virus genome RNA. Virology. 1987 Dec;161(2):497–510. doi: 10.1016/0042-6822(87)90144-9. [DOI] [PubMed] [Google Scholar]
  47. Suzich J. A., Tamura J. K., Palmer-Hill F., Warrener P., Grakoui A., Rice C. M., Feinstone S. M., Collett M. S. Hepatitis C virus NS3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with the related pestivirus and flavivirus enzymes. J Virol. 1993 Oct;67(10):6152–6158. doi: 10.1128/jvi.67.10.6152-6158.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wang Y. F., Sawicki S. G., Sawicki D. L. Sindbis virus nsP1 functions in negative-strand RNA synthesis. J Virol. 1991 Feb;65(2):985–988. doi: 10.1128/jvi.65.2.985-988.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Warrener P., Tamura J. K., Collett M. S. RNA-stimulated NTPase activity associated with yellow fever virus NS3 protein expressed in bacteria. J Virol. 1993 Feb;67(2):989–996. doi: 10.1128/jvi.67.2.989-996.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wengler G., Czaya G., Färber P. M., Hegemann J. H. In vitro synthesis of West Nile virus proteins indicates that the amino-terminal segment of the NS3 protein contains the active centre of the protease which cleaves the viral polyprotein after multiple basic amino acids. J Gen Virol. 1991 Apr;72(Pt 4):851–858. doi: 10.1099/0022-1317-72-4-851. [DOI] [PubMed] [Google Scholar]
  51. Wengler G., Wengler G. The NS 3 nonstructural protein of flaviviruses contains an RNA triphosphatase activity. Virology. 1993 Nov;197(1):265–273. doi: 10.1006/viro.1993.1587. [DOI] [PubMed] [Google Scholar]
  52. Wengler G., Wengler G. The carboxy-terminal part of the NS 3 protein of the West Nile flavivirus can be isolated as a soluble protein after proteolytic cleavage and represents an RNA-stimulated NTPase. Virology. 1991 Oct;184(2):707–715. doi: 10.1016/0042-6822(91)90440-m. [DOI] [PubMed] [Google Scholar]
  53. Westaway E. G., Brinton M. A., Gaidamovich SYa, Horzinek M. C., Igarashi A., Käriäinen L., Lvov D. K., Porterfield J. S., Russell P. K., Trent D. W. Flaviviridae. Intervirology. 1985;24(4):183–192. doi: 10.1159/000149642. [DOI] [PubMed] [Google Scholar]
  54. Westaway E. G. Flavivirus replication strategy. Adv Virus Res. 1987;33:45–90. doi: 10.1016/s0065-3527(08)60316-4. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES