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. 1996 May;70(5):2869–2875. doi: 10.1128/jvi.70.5.2869-2875.1996

Astrovirus ribosomal frameshifting in an infection-transfection transient expression system.

T L Lewis 1, S M Matsui 1
PMCID: PMC190144  PMID: 8627761

Abstract

Different regions of the human astrovirus frameshift signal were cloned into the rhesus rotavirus VP4 gene and evaluated in an infection-transfection transient expression cell culture system. BHK-21 cells, infected with a vaccinia virus that expresses T7 RNA polymerase (vTF7-3), were transfected with the various astrovirus-VP4 constructs. All constructs were driven by a T7 promoter and contained an internal ribosome entry site. Frameshifted and nonframeshifted protein products were immunoprecipitated with VP4 amino- and carboxy-terminal-specific monoclonal antibodies, and their ratios were determined by PhosphorImager analysis. The efficiency of frameshifting was 25 to 28%, significantly greater than the 5 to 7% efficiency reported previously in a cell-free translation system. Coupling of transcription and translation in a cell-free system yielded frameshifting efficiencies threefold greater than that of the uncoupled in vitro system. The presence of the shifty heptamer was an absolute requirement for frameshifting in both cell-free and intact-cell systems, while deletion of the potential downstream pseudoknot region did not affect the efficiency of frameshifting.

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Selected References

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  1. Bredenbeek P. J., Pachuk C. J., Noten A. F., Charité J., Luytjes W., Weiss S. R., Spaan W. J. The primary structure and expression of the second open reading frame of the polymerase gene of the coronavirus MHV-A59; a highly conserved polymerase is expressed by an efficient ribosomal frameshifting mechanism. Nucleic Acids Res. 1990 Apr 11;18(7):1825–1832. doi: 10.1093/nar/18.7.1825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brierley I., Boursnell M. E., Binns M. M., Bilimoria B., Blok V. C., Brown T. D., Inglis S. C. An efficient ribosomal frame-shifting signal in the polymerase-encoding region of the coronavirus IBV. EMBO J. 1987 Dec 1;6(12):3779–3785. doi: 10.1002/j.1460-2075.1987.tb02713.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brierley I., Digard P., Inglis S. C. Characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an RNA pseudoknot. Cell. 1989 May 19;57(4):537–547. doi: 10.1016/0092-8674(89)90124-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brierley I. Ribosomal frameshifting viral RNAs. J Gen Virol. 1995 Aug;76(Pt 8):1885–1892. doi: 10.1099/0022-1317-76-8-1885. [DOI] [PubMed] [Google Scholar]
  5. Brierley I., Rolley N. J., Jenner A. J., Inglis S. C. Mutational analysis of the RNA pseudoknot component of a coronavirus ribosomal frameshifting signal. J Mol Biol. 1991 Aug 20;220(4):889–902. doi: 10.1016/0022-2836(91)90361-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cassan M., Delaunay N., Vaquero C., Rousset J. P. Translational frameshifting at the gag-pol junction of human immunodeficiency virus type 1 is not increased in infected T-lymphoid cells. J Virol. 1994 Mar;68(3):1501–1508. doi: 10.1128/jvi.68.3.1501-1508.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cox G. J., Matsui S. M., Lo R. S., Hinds M., Bowden R. A., Hackman R. C., Meyer W. G., Mori M., Tarr P. I., Oshiro L. S. Etiology and outcome of diarrhea after marrow transplantation: a prospective study. Gastroenterology. 1994 Nov;107(5):1398–1407. doi: 10.1016/0016-5085(94)90542-8. [DOI] [PubMed] [Google Scholar]
  8. Cruz J. R., Bartlett A. V., Herrmann J. E., Cáceres P., Blacklow N. R., Cano F. Astrovirus-associated diarrhea among Guatemalan ambulatory rural children. J Clin Microbiol. 1992 May;30(5):1140–1144. doi: 10.1128/jcm.30.5.1140-1144.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Elroy-Stein O., Fuerst T. R., Moss B. Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5' sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6126–6130. doi: 10.1073/pnas.86.16.6126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Falk H., Mador N., Udi R., Panet A., Honigman A. Two cis-acting signals control ribosomal frameshift between human T-cell leukemia virus type II gag and pro genes. J Virol. 1993 Oct;67(10):6273–6277. doi: 10.1128/jvi.67.10.6273-6277.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fuerst T. R., Niles E. G., Studier F. W., Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122–8126. doi: 10.1073/pnas.83.21.8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Grakoui A., Wychowski C., Lin C., Feinstone S. M., Rice C. M. Expression and identification of hepatitis C virus polyprotein cleavage products. J Virol. 1993 Mar;67(3):1385–1395. doi: 10.1128/jvi.67.3.1385-1395.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grohmann G. S., Glass R. I., Pereira H. G., Monroe S. S., Hightower A. W., Weber R., Bryan R. T. Enteric viruses and diarrhea in HIV-infected patients. Enteric Opportunistic Infections Working Group. N Engl J Med. 1993 Jul 1;329(1):14–20. doi: 10.1056/NEJM199307013290103. [DOI] [PubMed] [Google Scholar]
  14. Herrmann J. E., Taylor D. N., Echeverria P., Blacklow N. R. Astroviruses as a cause of gastroenteritis in children. N Engl J Med. 1991 Jun 20;324(25):1757–1760. doi: 10.1056/NEJM199106203242501. [DOI] [PubMed] [Google Scholar]
  15. Jacks T., Madhani H. D., Masiarz F. R., Varmus H. E. Signals for ribosomal frameshifting in the Rous sarcoma virus gag-pol region. Cell. 1988 Nov 4;55(3):447–458. doi: 10.1016/0092-8674(88)90031-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jacks T., Power M. D., Masiarz F. R., Luciw P. A., Barr P. J., Varmus H. E. Characterization of ribosomal frameshifting in HIV-1 gag-pol expression. Nature. 1988 Jan 21;331(6153):280–283. doi: 10.1038/331280a0. [DOI] [PubMed] [Google Scholar]
  17. Jacks T. Translational suppression in gene expression in retroviruses and retrotransposons. Curr Top Microbiol Immunol. 1990;157:93–124. doi: 10.1007/978-3-642-75218-6_4. [DOI] [PubMed] [Google Scholar]
  18. Jacks T., Varmus H. E. Expression of the Rous sarcoma virus pol gene by ribosomal frameshifting. Science. 1985 Dec 13;230(4731):1237–1242. doi: 10.1126/science.2416054. [DOI] [PubMed] [Google Scholar]
  19. Jiang B., Monroe S. S., Koonin E. V., Stine S. E., Glass R. I. RNA sequence of astrovirus: distinctive genomic organization and a putative retrovirus-like ribosomal frameshifting signal that directs the viral replicase synthesis. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10539–10543. doi: 10.1073/pnas.90.22.10539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148. doi: 10.1093/nar/15.20.8125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kurtz J. B., Lee T. W. Astroviruses: human and animal. Ciba Found Symp. 1987;128:92–107. doi: 10.1002/9780470513460.ch6. [DOI] [PubMed] [Google Scholar]
  22. Lewis T. L., Greenberg H. B., Herrmann J. E., Smith L. S., Matsui S. M. Analysis of astrovirus serotype 1 RNA, identification of the viral RNA-dependent RNA polymerase motif, and expression of a viral structural protein. J Virol. 1994 Jan;68(1):77–83. doi: 10.1128/jvi.68.1.77-83.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lewis T. L., Matsui S. M. An astrovirus frameshift signal induces ribosomal frameshifting in vitro. Arch Virol. 1995;140(6):1127–1135. doi: 10.1007/BF01315421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mackow E. R., Yamanaka M. Y., Dang M. N., Greenberg H. B. DNA amplification-restricted transcription-translation: rapid analysis of rhesus rotavirus neutralization sites. Proc Natl Acad Sci U S A. 1990 Jan;87(2):518–522. doi: 10.1073/pnas.87.2.518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Madeley C. R., Cosgrove B. P. Letter: Viruses in infantile gastroenteritis. Lancet. 1975 Jul 19;2(7925):124–124. doi: 10.1016/s0140-6736(75)90020-3. [DOI] [PubMed] [Google Scholar]
  26. Marczinke B., Bloys A. J., Brown T. D., Willcocks M. M., Carter M. J., Brierley I. The human astrovirus RNA-dependent RNA polymerase coding region is expressed by ribosomal frameshifting. J Virol. 1994 Sep;68(9):5588–5595. doi: 10.1128/jvi.68.9.5588-5595.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Matsui S. M., Kim J. P., Greenberg H. B., Young L. M., Smith L. S., Lewis T. L., Herrmann J. E., Blacklow N. R., Dupuis K., Reyes G. R. Cloning and characterization of human astrovirus immunoreactive epitopes. J Virol. 1993 Mar;67(3):1712–1715. doi: 10.1128/jvi.67.3.1712-1715.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Monroe S. S., Jiang B., Stine S. E., Koopmans M., Glass R. I. Subgenomic RNA sequence of human astrovirus supports classification of Astroviridae as a new family of RNA viruses. J Virol. 1993 Jun;67(6):3611–3614. doi: 10.1128/jvi.67.6.3611-3614.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Monroe S. S., Stine S. E., Gorelkin L., Herrmann J. E., Blacklow N. R., Glass R. I. Temporal synthesis of proteins and RNAs during human astrovirus infection of cultured cells. J Virol. 1991 Feb;65(2):641–648. doi: 10.1128/jvi.65.2.641-648.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Moosmayer D., Reil H., Ausmeier M., Scharf J. G., Hauser H., Jentsch K. D., Hunsmann G. Expression and frameshifting but extremely inefficient proteolytic processing of the HIV-1 gag and pol gene products in stably transfected rodent cell lines. Virology. 1991 Jul;183(1):215–224. doi: 10.1016/0042-6822(91)90134-W. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Moss B., Elroy-Stein O., Mizukami T., Alexander W. A., Fuerst T. R. Product review. New mammalian expression vectors. Nature. 1990 Nov 1;348(6296):91–92. doi: 10.1038/348091a0. [DOI] [PubMed] [Google Scholar]
  32. Oshiro L. S., Haley C. E., Roberto R. R., Riggs J. L., Croughan M., Greenberg H., Kapikian A. A 27-nm virus isolated during an outbreak of acute infectious nonbacterial gastroenteritis in a convalescent hospital: a possible new serotype. J Infect Dis. 1981 Jun;143(6):791–795. doi: 10.1093/infdis/143.6.791. [DOI] [PubMed] [Google Scholar]
  33. Parkin N. T., Chamorro M., Varmus H. E. Human immunodeficiency virus type 1 gag-pol frameshifting is dependent on downstream mRNA secondary structure: demonstration by expression in vivo. J Virol. 1992 Aug;66(8):5147–5151. doi: 10.1128/jvi.66.8.5147-5151.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Reil H., Hauser H. Test system for determination of HIV-1 frameshifting efficiency in animal cells. Biochim Biophys Acta. 1990 Aug 27;1050(1-3):288–292. doi: 10.1016/0167-4781(90)90183-3. [DOI] [PubMed] [Google Scholar]
  35. Reil H., Kollmus H., Weidle U. H., Hauser H. A heptanucleotide sequence mediates ribosomal frameshifting in mammalian cells. J Virol. 1993 Sep;67(9):5579–5584. doi: 10.1128/jvi.67.9.5579-5584.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Shaw R. D., Vo P. T., Offit P. A., Coulson B. S., Greenberg H. B. Antigenic mapping of the surface proteins of rhesus rotavirus. Virology. 1986 Dec;155(2):434–451. doi: 10.1016/0042-6822(86)90205-9. [DOI] [PubMed] [Google Scholar]
  37. Snijder E. J., den Boon J. A., Bredenbeek P. J., Horzinek M. C., Rijnbrand R., Spaan W. J. The carboxyl-terminal part of the putative Berne virus polymerase is expressed by ribosomal frameshifting and contains sequence motifs which indicate that toro- and coronaviruses are evolutionarily related. Nucleic Acids Res. 1990 Aug 11;18(15):4535–4542. doi: 10.1093/nar/18.15.4535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Willcocks M. M., Brown T. D., Madeley C. R., Carter M. J. The complete sequence of a human astrovirus. J Gen Virol. 1994 Jul;75(Pt 7):1785–1788. doi: 10.1099/0022-1317-75-7-1785. [DOI] [PubMed] [Google Scholar]
  39. Willcocks M. M., Carter M. J. Identification and sequence determination of the capsid protein gene of human astrovirus serotype 1. FEMS Microbiol Lett. 1993 Nov 15;114(1):1–7. doi: 10.1111/j.1574-6968.1993.tb06542.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wilson W., Braddock M., Adams S. E., Rathjen P. D., Kingsman S. M., Kingsman A. J. HIV expression strategies: ribosomal frameshifting is directed by a short sequence in both mammalian and yeast systems. Cell. 1988 Dec 23;55(6):1159–1169. doi: 10.1016/0092-8674(88)90260-7. [DOI] [PubMed] [Google Scholar]

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