Home LiteratureArticle Details
PMID: 10756054 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Stable high-level expression of heterologous genes in vitro and in vivo by noncytopathic DNA-based Kunjin virus replicon vectors.

Journal of virology ·Vol. 74 ·No. 9 ·2000-05-00 ·Pages 4394-403

Varnavski AN, Young PR, Khromykh AA

Abstract

Primary features of the flavivirus Kunjin (KUN) subgenomic replicons include continuous noncytopathic replication in host cell cytoplasm and the ability to be encapsidated into secreted virus-like particles (VLPs). Previously we reported preparation of RNA-based KUN replicon vectors and expression of heterologous genes (HG) in cell culture after RNA transfection or after infection with recombinant KUN VLPs (A. N. Varnavski and A. A. Khromykh, Virology 255:366-375, 1999). In this study we describe the development of the next generation of KUN replicon vectors, which allow synthesis of replicon RNA in vivo from corresponding plasmid DNAs. These DNA-based vectors were able to direct stable expression of beta-galactosidase (beta-Gal) in several mammalian cell lines, and expression remained high ( approximately 150 pg per cell) throughout cell passaging. The applicability of these vectors in vivo was demonstrated by beta-Gal expression in the mouse lung epithelium for at least 8 weeks after intranasal inoculation and induction of anti-beta-Gal antibody response after intramuscular inoculation of the beta-Gal-encoding KUN replicon DNA. The noncytopathic nature of DNA-based KUN replicon vectors combined with high-level and stability of HG expression in a broad range of host cells should prove them to be useful in a variety of applications in vitro and in vivo.

MeSH Terms
Animals Cell Line Cell Line, Transformed Chlorocebus aethiops Cricetinae Cytopathogenic Effect, Viral Encephalitis Viruses, Japanese/genetics,physiology Gene Amplification Gene Expression Genetic Vectors/genetics,physiology Hepatitis Delta Virus/enzymology,genetics Humans Kinetics Mice RNA, Catalytic/genetics Replicon Transcription, Genetic Tumor Cells, Cultured Ubiquitins/genetics Vero Cells
Chemicals
RNA, Catalytic Ubiquitins
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Varnavski A N
Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Herston, Brisbane 4029, Australia.
Young P R
Khromykh A A
References (44)
44 references, click to expand
  1. 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):4580-8 PMID: 8207832
  2. RNA virus vectors: where are we and where do we need to go?
    Proc Natl Acad Sci U S A. 1998 Oct 27;95(22):12750-2 PMID: 9788984
  3. A Sindbis virus mRNA polynucleotide vector achieves prolonged and high level heterologous gene expression in vivo.
    Nucleic Acids Res. 1995 May 11;23(9):1495-501 PMID: 7784202
  4. Up-regulation of MHC class I by flavivirus-induced peptide translocation into the endoplasmic reticulum.
    Immunity. 1995 Aug;3(2):207-14 PMID: 7544229
  5. Sindbis virus DNA-based expression vectors: utility for in vitro and in vivo gene transfer.
    J Virol. 1996 Jan;70(1):508-19 PMID: 8523564
  6. Subgenomic replicons of the flavivirus Kunjin: construction and applications.
    J Virol. 1997 Feb;71(2):1497-505 PMID: 8995675
  7. Humoral, mucosal, and cellular immunity in response to a human immunodeficiency virus type 1 immunogen expressed by a Venezuelan equine encephalitis virus vaccine vector.
    J Virol. 1997 Apr;71(4):3031-8 PMID: 9060663
  8. Replication of flock house virus RNAs from primary transcripts made in cells by RNA polymerase II.
    J Virol. 1997 Apr;71(4):3323-7 PMID: 9060703
  9. A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus.
    Cell. 1985 Jun;41(2):521-30 PMID: 2985280
  10. Expression in mammalian cells of a gene from Streptomyces alboniger conferring puromycin resistance.
    Nucleic Acids Res. 1986 Jun 11;14(11):4617-24 PMID: 3714487
  11. In vivo half-life of a protein is a function of its amino-terminal residue.
    Science. 1986 Oct 10;234(4773):179-86 PMID: 3018930
  12. Interferon-independent increases in class I major histocompatibility complex antigen expression follow flavivirus infection.
    J Gen Virol. 1988 Oct;69 ( Pt 10):2535-43 PMID: 2844965
  13. Murine polyubiquitin mRNA sequence.
    Nucleic Acids Res. 1990 Apr 11;18(7):1907 PMID: 2159627
  14. RNA conformational requirements of self-cleavage of hepatitis delta virus RNA.
    Mol Cell Biol. 1990 Oct;10(10):5575-9 PMID: 2398903
  15. A pseudoknot-like structure required for efficient self-cleavage of hepatitis delta virus RNA.
    Nature. 1991 Apr 4;350(6317):434-6 PMID: 2011192
  16. Cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence.
    J Gen Virol. 1991 Nov;72 ( Pt 11):2727-32 PMID: 1658199
  17. A new generation of animal cell expression vectors based on the Semliki Forest virus replicon.
    Biotechnology (N Y). 1991 Dec;9(12):1356-61 PMID: 1370252
  18. Infectious defective interfering particles of VSV from transcripts of a cDNA clone.
    Cell. 1992 Jun 12;69(6):1011-20 PMID: 1318785
  19. A poliovirus replicon containing the chloramphenicol acetyltransferase gene can be used to study the replication and encapsidation of poliovirus RNA.
    J Virol. 1992 Aug;66(8):5040-6 PMID: 1321286
  20. cis-acting requirements for the replication of flock house virus RNA 2.
    J Virol. 1993 Jun;67(6):3544-51 PMID: 8497063
  21. Encapsidation of genetically engineered poliovirus minireplicons which express human immunodeficiency virus type 1 Gag and Pol proteins upon infection.
    J Virol. 1993 Jul;67(7):3712-9 PMID: 8389902
  22. Sindbis virus expression vectors: packaging of RNA replicons by using defective helper RNAs.
    J Virol. 1993 Nov;67(11):6439-46 PMID: 8411346
  23. Infectious transcripts and cDNA clones of RNA viruses.
    Virology. 1994 Feb;198(2):415-26 PMID: 8291226
  24. Comparison of the effects of Sindbis virus and Sindbis virus replicons on host cell protein synthesis and cytopathogenicity in BHK cells.
    J Virol. 1994 Mar;68(3):1721-7 PMID: 8107233
  25. Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein.
    EMBO J. 1994 Feb 15;13(4):928-33 PMID: 8112307
  26. A double-stranded RNA-activated protein kinase-dependent pathway mediating stress-induced apoptosis.
    Proc Natl Acad Sci U S A. 1997 Apr 1;94(7):3279-83 PMID: 9096384
  27. Ultrastructure of Kunjin virus-infected cells: colocalization of NS1 and NS3 with double-stranded RNA, and of NS2B with NS3, in virus-induced membrane structures.
    J Virol. 1997 Sep;71(9):6650-61 PMID: 9261387
  28. Expression of beta-galactosidase in mouse brain: utilization of a novel nonreplicative Sindbis virus vector as a neuronal gene delivery system.
    Gene Ther. 1997 Aug;4(8):815-22 PMID: 9338010
  29. DNA immunization: ubiquitination of a viral protein enhances cytotoxic T-lymphocyte induction and antiviral protection but abrogates antibody induction.
    J Virol. 1997 Nov;71(11):8497-503 PMID: 9343207
  30. Early E-selectin, VCAM-1, ICAM-1, and late major histocompatibility complex antigen induction on human endothelial cells by flavivirus and comodulation of adhesion molecule expression by immune cytokines.
    J Virol. 1997 Dec;71(12):9323-32 PMID: 9371591
  31. Replicon-helper systems from attenuated Venezuelan equine encephalitis virus: expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo.
    Virology. 1997 Dec 22;239(2):389-401 PMID: 9434729
  32. DNA immunization against herpes simplex virus: enhanced efficacy using a Sindbis virus-based vector.
    J Virol. 1998 Feb;72(2):950-8 PMID: 9444987
  33. Fusion PCR, a one-step variant of the "megaprimer" method of mutagenesis.
    Biotechniques. 1998 May;24(5):736, 740, 742 PMID: 9591118
  34. Encapsidation of the flavivirus kunjin replicon RNA by using a complementation system providing Kunjin virus structural proteins in trans.
    J Virol. 1998 Jul;72(7):5967-77 PMID: 9621059
  35. Enhancing immune responses using suicidal DNA vaccines.
    Nat Biotechnol. 1998 Jun;16(6):562-5 PMID: 9624688
  36. Subcellular localization and some biochemical properties of the flavivirus Kunjin nonstructural proteins NS2A and NS4A.
    Virology. 1998 Jun 5;245(2):203-15 PMID: 9636360
  37. Noncytopathic Sindbis virus RNA vectors for heterologous gene expression.
    Proc Natl Acad Sci U S A. 1998 Oct 27;95(22):12989-94 PMID: 9789028
  38. Noncytopathic flavivirus replicon RNA-based system for expression and delivery of heterologous genes.
    Virology. 1999 Mar 15;255(2):366-75 PMID: 10069962
  39. Selection of RNA replicons capable of persistent noncytopathic replication in mammalian cells.
    J Virol. 1999 May;73(5):3854-65 PMID: 10196280
  40. Cancer therapy using a self-replicating RNA vaccine.
    Nat Med. 1999 Jul;5(7):823-7 PMID: 10395329
  41. Induction and maintenance of autonomous flock house virus RNA1 replication.
    J Virol. 1999 Oct;73(10):7933-42 PMID: 10482540
  42. Rapid, high level protein production using DNA-based Semliki Forest virus vectors.
    J Biol Chem. 1998 Jul 17;273(29):18060-6 PMID: 9660762
  43. trans-Complementation of flavivirus RNA polymerase gene NS5 by using Kunjin virus replicon-expressing BHK cells.
    J Virol. 1998 Sep;72(9):7270-9 PMID: 9696822
  44. Protein expression using cotranslational fusion and cleavage of ubiquitin. Mutagenesis of the glutathione-binding site of human Pi class glutathione S-transferase.
    J Biol Chem. 1994 Oct 14;269(41):25381-6 PMID: 7929235
Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/jvi.74.9.4394-4403.2000
Published
2000-05-00
Pages
4394-403
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC111956
Subset
IM
External Links
PubMed source DOI: 10.1128/jvi.74.9.4394-4403.2000 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2