1887

Abstract

Unlike other picornaviruses, hepatitis A virus (HAV) replicates so inefficiently in cell culture that the study of its RNA biosynthesis presents a major experimental challenge. To assess viral RNA replication independent of particle formation, a subgenomic replicon representing a self-replicating RNA was constructed by replacing the P1 domain encoding the capsid proteins with the firefly luciferase sequence. Although translation of the HAV replicon was as efficient as a similar poliovirus replicon, the luciferase activity derived from replication of the HAV construct was more than 100-fold lower than that of poliovirus. The replication capacity of the HAV replicon was clearly demonstrated by its ability to recombine genetically with a non-viable, full-length HAV genome that served as capsid donor and thus to rescue a fully infectious virus. In contrast to a replication-deficient replicon, co-expression of the genetically marked and replication-competent HAV replicon with several lethally mutated HAV genomes resulted in the successful rescue of infectious HAV with a unique genetic marker. Our data suggest: (i) that autonomous HAV RNA replication does not require sequences for the HAV structural proteins; and (ii) that low-level genome replication can unequivocally be demonstrated by the rescue of infectious virus after co-expression with non-viable genomes.

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2002-09-01
2020-09-30
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References

  1. Andino R., Rieckhof G. E., Achacoso P. L., Baltimore D.. 1993; Poliovirus RNA synthesis utilizes an RNP complex formed around the 5′-end of viral RNA. EMBO Journal12:3587–3598
    [Google Scholar]
  2. Barton D. J., Flanegan J. B.. 1993; Coupled translation and replication of poliovirus RNA in vitro: synthesis of functional 3D polymerase and infectious virus. Journal of Virology67:822–831
    [Google Scholar]
  3. Beard M. R., Cohen L., Lemon S. M., Martin A.. 2001; Characterization of recombinant hepatitis A virus genomes containing exogenous sequences at the 2A/2B junction. Journal of Virology75:1414–1426
    [Google Scholar]
  4. Bergamini G., Preiss T., Hentze M. W.. 2000; Picornavirus IRESes and the poly(A) tail jointly promote cap-independent translation in a mammalian cell-free system. RNA6:1781–1790
    [Google Scholar]
  5. Bishop N. E., Anderson D. A.. 2000; Uncoating kinetics of hepatitis A virus virions and provirions. Journal of Virology74:3423–3426
    [Google Scholar]
  6. Duggal R., Cuconati A., Gromeier M., Wimmer E.. 1997; Genetic recombination of poliovirus in a cell-free system. Proceedings of the National Academy of Sciences, USA94:13786–13791
    [Google Scholar]
  7. Elroy-Stein O., Moss B.. 1990; Cytoplasmic expression system based on constitutive synthesis of bacteriophage T7 RNA polymerase in mammalian cells. Proceedings of the National Academy of Sciences, USA87:6743–6747
    [Google Scholar]
  8. Evans D. H., Stuart D., McFadden G.. 1988; High levels of genetic recombination among cotransfected plasmid DNAs in poxvirus-infected mammalian cells. Journal of Virology62:367–375
    [Google Scholar]
  9. Funkhouser A. W., Schultz D. E., Lemon S. M., Purcell R. H., Emerson S. U.. 1999; Hepatitis A virus translation is rate-limiting for virus replication in MRC-5 cells. Virology254:268–278
    [Google Scholar]
  10. Gamarnik A. V., Andino R.. 1998; Switch from translation to RNA replication in a positive-stranded RNA virus. Genes and Development12:2293–2304
    [Google Scholar]
  11. Gmyl A. P., Belousov E. V., Maslova S. V., Khitrina E. V., Chetverin A. B., Agol V. I.. 1999; Nonreplicative RNA recombination in poliovirus. Journal of Virology73:8958–8965
    [Google Scholar]
  12. Graeber I., Tischer J., Heinrich J., Hachula G., Lopez-Pila J. M.. 1998; Persistence of heterologous nucleic acids after uptake by mammalian cells. DNA and Cell Biology17:945–949
    [Google Scholar]
  13. Jarvis T. C., Kirkegaard K.. 1992; Poliovirus RNA recombination: mechanistic studies in the absence of selection. EMBO Journal11:3135–3145
    [Google Scholar]
  14. Jia X. Y., Van Eden M., Busch M. G., Ehrenfeld E., Summers D. F.. 1998; Trans-encapsidation of a poliovirus replicon by different picornavirus capsid proteins. Journal of Virology72:7972–7979
    [Google Scholar]
  15. Kim M. J., Kao C.. 2000; Factors regulating template switch in vitro by viral RNA-dependent RNA polymerases: implications for RNA–RNA recombination. Proceedings of the National Academy of Sciences, USA98:4972–4977
    [Google Scholar]
  16. Kirkegaard K., Baltimore D.. 1986; The mechanism of RNA recombination in poliovirus. Cell47:433–443
    [Google Scholar]
  17. Kusov Y. Y., Gauss-Müller V.. 1999; Improving proteolytic cleavage at the 3A/3B site of the hepatitis A virus polyprotein I impairs processing and particle formation, and the impairment can be complemented in trans by 3AB and 3ABC. Journal of Virology73:9867–9878
    [Google Scholar]
  18. Kusov Y., Shatirishvili G., Klinger M., Gauss-Müller V.. 2002; A vaccinia virus MVA-T7-mediated recovery of infectious hepatitis A virus from full-size cDNA or from two CDNAs, both by themselves unable to complete the virus life cycle. Virus Research 87 (in Press)
    [Google Scholar]
  19. Lai M. M.. 1992; RNA recombination in animal and plant viruses. Microbiological Reviews56:61–79
    [Google Scholar]
  20. Michel Y. M., Borman A. M., Paulous S., Kean K. M.. 2001; Eukaryotic initiation factor 4G–poly(A) binding protein interaction is required for poly(A) tail-mediated stimulation of picornavirus internal ribosome entry segment-driven translation but not for X-mediated stimulation of hepatitis C virus translation. Molecular and Cellular Biology21:4097–4109
    [Google Scholar]
  21. Molla A., Paul A. V., Wimmer E.. 1991; Cell-free, de novo synthesis of poliovirus. Science254:1647–1651
    [Google Scholar]
  22. Nagy P. D., Simon A. E.. 1997; New insights into the mechanisms of RNA recombination. Virology235:1–9
    [Google Scholar]
  23. Nomoto A., Jacobson A., Lee Y. F., Dunn J., Wimmer E.. 1979; Defective interfering particles of poliovirus: mapping of the deletion and evidence that the deletions in the genomes of DI(1), (2) and (3) are located in the same region. Journal of Molecular Biolology128:179–196
    [Google Scholar]
  24. Nuesch J. P., de Chastonay J., Siegl G.. 1989; Detection of defective genomes in hepatitis A virus particles present in clinical specimens. Journal of General Virology70:3475–3480
    [Google Scholar]
  25. Probst C., Jecht M., Gauss-Müller V.. 1998; Processing of proteinase precursors and their effect on hepatitis A virus particle formation. Journal of Virology72:8013–8020
    [Google Scholar]
  26. Probst C., Jecht M., Gauss-Müller V.. 1999; Intrinsic signals for the assembly of hepatitis A virus particles. Role of structural proteins VP4 and 2A. Journal of Biological Chemistry274:4527–4531
    [Google Scholar]
  27. Schultz D. E., Honda M., Whetter L. E., McKnight K. L., Lemon S. M.. 1996; Mutations within the 5′ nontranslated RNA of cell culture-adapted hepatitis A virus which enhance cap-independent translation in cultured African green monkey kidney cells. Journal of Virology70:1041–1049
    [Google Scholar]
  28. Tang R. S., Barton D. J., Flanegan J. B., Kirkegaard K.. 1997; Poliovirus RNA recombination in cell-free extracts. RNA3:624–633
    [Google Scholar]
  29. Whetter L. E., Day S. P., Elroy-Stein O., Brown E. A., Lemon S. M.. 1994; Low efficiency of the 5′ nontranslated region of hepatitis A virus RNA in directing cap-independent translation in permissive monkey kidney cells. Journal of Virology68:5253–5263
    [Google Scholar]
  30. Wimmer E., Hellen C. U., Cao X.. 1993; Genetics of poliovirus. Annual Review of Genetics27:353–436
    [Google Scholar]
  31. Yi M., Lemon S. M.. 2002; Replication of subgenomic hepatitis A virus RNAs expressing firefly luciferase is enhanced by mutations associated with adaptation of virus to growth in cultured cells. Journal of Virology76:1171–1180
    [Google Scholar]
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