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Volume 85, Issue 2

Introduction of replication-competent hepatitis C virus transcripts using a tetracycline-regulable baculovirus delivery system Free

Abstract

We have developed a baculovirus delivery system that enables tetracycline-regulated expression of polII-derived hepatitis C virus (HCV) transcripts in hepatocyte-derived cell lines ( McCormick , 2002 ). As part of a study to determine whether such transcripts are replication competent, the transcription start site of the tetracycline-regulable promoter was mapped and three baculovirus transfer vectors containing a -expressing culture adapted replicon cDNA were generated. These vectors either had the first nucleotide of the 5′UTR positioned −2 (mkI) and +1 (mkII) with respect to the transcription start site, or included a hammerhead ribozyme at the 5′ end of the transcript (5′HH) that cleaves between the ribozyme–5′UTR boundary. Transfection of all of the culture-adapted replicon constructs into Huh7 cells resulted in the formation of more neomycin-resistant colonies than seen with a polymerase knock-out replicon construct, although this was less pronounced in the mkI group. Furthermore, both the positive- and negative-strands of the replicon could be detected in all neomycin-resistant polyclonal cell lines except for those derived from transfection of the polymerase knock-out construct. Transduction of Huh7 cells with recombinant baculoviruses carrying the same expression cassettes improved replicon delivery, but the relative efficiency of the constructs remained the same. The baculovirus vectors were also used to introduce the replicon transcript into HepG2 cells. Expression of the culture-adapted but not the polymerase knock-out construct induced transcription of the -interferon gene, a response that may contribute to this cell line being unable to maintain the replicon over long-term culture.

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2004-02-01
2024-04-20
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References

  1. Akrigg A., Wilkinson G. W. G., Oram J. D. 1985; The structure of the major immediate early gene of human cytomegalovirus strain AD169. Virus Res 2:107–121
     [Google Scholar]
  2. Almazan F., Gonzalez J. M., Penzes Z., Izeta A., Calvo E., Plana-Duran J., Enjuanes L. 2000; Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Proc Natl Acad Sci U S A 97:5516–5521
     [Google Scholar]
  3. Baigent S. J., Zhang G., Fray M. D., Flick-Smith H., Goodbourn S., McCauley J. W. 2002; Inhibition of beta interferon transcription by noncytopathogenic bovine viral diarrhea virus is through an interferon regulatory factor 3-dependent mechanism. J Virol 76:8979–8988
     [Google Scholar]
  4. Blight K. J., Kolykhalov A. A., Rice C. M. 2000; Efficient initiation of HCV RNA replication in cell culture. Science 290:1972–1974
     [Google Scholar]
  5. Cheney I. W., Lai V. C., Zhong W., Brodhag T., Dempsey S., Lim C., Hong Z., Lau J. Y., Tam R. C. 2002; Comparative analysis of anti-hepatitis C virus activity and gene expression mediated by alpha, beta, and gamma interferons. J Virol 76:11148–11154
     [Google Scholar]
  6. Chung R. T., He W., Saquib A., Contreras A. M., Xavier R. J., Chawla A., Wang T. C., Schmidt E. V. 2001; Hepatitis C virus replication is directly inhibited by IFN-alpha in a full-length binary expression system. Proc Natl Acad Sci U S A 98:9847–9852
     [Google Scholar]
  7. Dubensky T. W., Driver D. A., Polo J. M. 9 other authors 1996; Sindbis virus DNA-based expression vectors: utility for in vitro and in vivo gene transfer. J Virol 70:508–519
     [Google Scholar]
  8. Fredericksen B., Akkaraju G. R., Foy E., Wang C., Pflugheber J., Chen Z. J., Gale M. Jr 2002; Activation of the interferon-beta promoter during hepatitis C virus RNA replication. Viral Immunol 15:29–40
     [Google Scholar]
  9. Gossen M., Bujard H. 1992; Tight control of gene-expression in mammalian-cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A 89:5547–5551
     [Google Scholar]
  10. Gu B., Gates A. T., Isken O., Behrens S. E., Sarisky R. T. 2003; Replication studies using genotype 1a subgenomic hepatitis C virus replicons. J Virol 77:5352–5359
     [Google Scholar]
  11. Herold J., Andino R. 2000; Poliovirus requires a precise 5′ end for efficient positive-strand RNA synthesis. J Virol 74:6394–6400
     [Google Scholar]
  12. Ikeda M., Yi M., Li K., Lemon S. M. 2002; Selectable subgenomic and genome-length dicistronic RNAs derived from an infectious molecular clone of the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells. J Virol 76:2997–3006
     [Google Scholar]
  13. Koob M., Burkiewicz A., Kur J., Szybalski W. 1992; RecA-AC: single-site cleavage of plasmids and chromosomes at any predetermined restriction site. Nucleic Acids Res 20:5831–5836
     [Google Scholar]
  14. Krieger N., Lohmann V., Bartenschlager R. 2001; Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J Virol 75:4614–4624
     [Google Scholar]
  15. Kunkel T. A. 1985; Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A 82:488–492
     [Google Scholar]
  16. Lanford R. E., Sureau C., Jacob J. R., White R., Fuerst T. R. 1994; Demonstration of in vitro infection of chimpanzee hepatocytes with hepatitis C virus using strand-specific RT/PCR. Virology 202:606–614
     [Google Scholar]
  17. Lanford R. E., Guerra B., Lee H., Averett D. R., Pfeiffer B., Chavez D., Notvall L., Bigger C. 2003; Antiviral effect and virus-host interactions in response to alpha interferon, gamma interferon, poly(I)-poly(C), tumor necrosis factor alpha, and ribavirin in hepatitis C virus subgenomic replicons. J Virol 77:1092–1104
     [Google Scholar]
  18. Lim S. P., Soo H. M., Tan Y. H., Brenner S., Horstmann H., MacKenzie J. M., Ng M. L., Lim S. G., Hong W. 2002; Inducible system in human hepatoma cell lines for hepatitis C virus production. Virology 303:79–99
     [Google Scholar]
  19. Lohmann V., Korner F., Koch J. O., Herian U., Theilmann L., Bartenschlager R. 1999; Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285:110–113
     [Google Scholar]
  20. Luckow V. A., Lee S. C., Barry G. F., Olins P. O. 1993; Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli . J Virol 67:4566–4579
     [Google Scholar]
  21. Luo G., Xin S., Cai Z. 2003; Role of the 5′-proximal stem-loop structure of the 5′ untranslated region in replication and translation of hepatitis C virus RNA. J Virol 77:3312–3318
     [Google Scholar]
  22. Macdonald A., Crowder K., Street A., McCormick C., Saksela K., Harris M. 2003; The hepatitis C virus non-structural NS5A protein inhibits activating protein-1 function by perturbing Ras-ERK pathway signaling. J Biol Chem 278:17775–17784
     [Google Scholar]
  23. Malim M. H., Hauber J., Le S. Y., Maizel J. V., Cullen B. R. 1989; The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature 338:254–257
     [Google Scholar]
  24. McCormick C. J., Rowlands D. J., Harris M. 2002; Efficient delivery and regulable expression of hepatitis C virus full-length and minigenome constructs in hepatocyte-derived cell lines using baculovirus vectors. J Gen Virol 83:383–394
     [Google Scholar]
  25. Myung J., Khalap N., Kalkeri G., Garry R., Dash S. 2001; Inducible model to study negative strand RNA synthesis and assembly of hepatitis C virus from a full-length cDNA clone. J Virol Methods 94:55–67
     [Google Scholar]
  26. Pflugheber J., Fredericksen B., Sumpter R. Jr, Wang C., Ware F., Sodora D. L., Gale M. Jr 2002; Regulation of PKR and IRF-1 during hepatitis C virus RNA replication. Proc Natl Acad Sci U S A 99:4650–4655
     [Google Scholar]
  27. Pietschmann T., Lohmann V., Kaul A., Krieger N., Rinck G., Rutter G., Strand D., Bartenschlager R. 2002; Persistent and transient replication of full-length hepatitis C virus genomes in cell culture. J Virol 76:4008–4021
     [Google Scholar]
  28. Tomassini J., Boots E., Gan L., Graham P., Munshi V., Wolanski B., Fay J. F., Getty K., Lafemina R. 2003; An in vitro Flaviviridae replicase system capable of authentic RNA replication. Virology 313:274–285
     [Google Scholar]
  29. Varnavski A. N., Young P. R., Khromykh A. A. 2000; Stable high-level expression of heterologous genes in vitro and in vivo by noncytopathic DNA-based Kunjin virus replicon vectors. J Virol 74:4394–4403
     [Google Scholar]
  30. Yanagi M., Purcell R. H., Emerson S. U., Bukh J. 1997; Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee. Proc Natl Acad Sci U S A 94:8738–8743
     [Google Scholar]
  31. Yanagi M., St Claire M., Shapiro M., Emerson S. U., Purcell R. H., Bukh J. 1998; Transcripts of a chimeric cDNA clone of hepatitis C virus genotype 1b are infectious in vivo. Virology 244:161–172
     [Google Scholar]
  32. Yap C. C., Ishii K., Aoki Y., Aizaki H., Tani H., Shimizu H., Ueno Y., Miyamura T., Matsuura Y. 1997; A hybrid baculovirus-T7 RNA polymerase system for recovery of an infectious virus from cDNA. Virology 231:192–200
     [Google Scholar]
  33. Zhu Q., Guo J. T., Seeger C. 2003; Replication of hepatitis C virus subgenomes in nonhepatic epithelial and mouse hepatoma cells. J Virol 77:9204–9210
     [Google Scholar]
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Introduction of replication-competent hepatitis C virus transcripts using a tetracycline-regulable baculovirus delivery system
J. Gen. Virol. 85, 429 (2004); https://doi.org/10.1099/vir.0.19676-0
/content/journal/jgv/10.1099/vir.0.19676-0
/content/journal/jgv/10.1099/vir.0.19676-0
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