Reverse Transcriptase Assay Based on Product Enhancement for Assessing the Drug Susceptibility of Retroviruses

  • Jürg Böni
  • Jörg Schüpbach
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 24)


All replication-competent retroviruses possess a characteristic enzyme, reverse transcriptase (RT), which is present at 20–70 mol/virus particle (1, 2, 3). The enzyme is cleaved, and thereby activated, from an inactive precursor by the action of another retroviral enzyme, the viral protease. All RTs possess three distinct enzymatic activities: (1) an RNA-dependent DNA polymerase, which is the RT in the strict sense of the word, (2) an RNase H, and (3) a DNA-dependent DNA polymerase. After infection of a new host cell, these different activities serve in turn to synthesize a cDNA of the viral RNA, to degrade RNA from the cDNA-RNA heteroduplex, and to duplicate the cDNA strand (reviewed in ref. 4).


Wash Solution Reverse Transcriptase Activity Bovine Serum Albumine Polymerase Chain Reaction Tube Reverse Transcription Primer 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Stromberg, K., Hurley, N. E., Davis, N. L., Rueckert, R. R., and Fleissner, E. (1974) Structural studies of avian myeloblastosis virus: comparison of polypeptides in virion and core component by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Virol. 13, 513–528.PubMedGoogle Scholar
  2. 2.
    Panet, A., Baltimore, D., and Hanafusa, T. (1975) Quantitation of avian RNA tumor virus reverse transcriptase by radioimmunoassay. J. Virol. 16, 146–152.PubMedGoogle Scholar
  3. 3.
    Krakower, J. M., Barbacid, M., and Aaronson, S. A. (1977) Radioimmunoassay for mammalian type C viral reverse transcriptase. J. Virol. 22, 331–339.PubMedGoogle Scholar
  4. 4.
    Coffin, J. M. (1996). Retroviridae: The viruses and their replication, in Fields Virology 3rd ed. (Fields, B. N., Knipe, D. M., Howley, P. M., et al., eds.), Lippincott-Raven, Philadelphia, PA, pp. 1767–1847.Google Scholar
  5. 5.
    Schüpbach, J. and Böni, J. (1993) Verfahren zum Nachweis reverser Transcriptase (Process for detecting reverse transcriptase). 118 pp, 25 figs., PCT International Office Publication Nr. WO 93/23560.Google Scholar
  6. 6.
    Silver, J., Maudru, T., Fujita, K., and Repaske, R. (1993) An RT-PCR assay for the enzyme activity of reverse transcriptase capable of detecting single virions. Nucleic Acids Res. 21, 3593,3594.CrossRefGoogle Scholar
  7. 7.
    Heneine, W., Yamamoto, S., Switzer, W. M., Spira, T. J., and Folks, T. M. (1995) Detection of reverse transcriptase by a highly sensitive assay in sera from persons infected with human immunodeficiency virus type 1. J. Inf. Dis. 171, 1210–1216.Google Scholar
  8. 8.
    Pyra, H., Böni, J., and Schüpbach, J. (1994) Ultrasensitive retrovirus detection by a reverse transcriptase assay based on product enhancement. Proc. Natl. Acad. Sci. USA 91, 1544–1548.CrossRefPubMedGoogle Scholar
  9. 9.
    Böni, J., Pyra, H., and Schüpbach, J. (1996) Sensitive detection and quantification of particle-associated reverse transcriptase in plasma of HIV-1 infected individuals by the product-enhanced reverse transcriptase assay. J. Med. Virol. 49, 23–28.CrossRefPubMedGoogle Scholar
  10. 10.
    Weissmahr, R. N., Schüpbach, J., and Böni, J. (1997) Reverse transcriptase activity in chicken embryo fibroblast culture supernatants is associated with particles containing EAV-0 (endogenous avian retrovirus) RNA. J. Virol. 71, 3005–3012.PubMedGoogle Scholar
  11. 11.
    Conrad, B., Weissmahr, R. N., Böni, J., Arcari, R., Schüpbach, J., and Mach, B. (1997) A human endogenous retroviral superantigen as candidate autoimmunegene in type 1 diabetes. Cell 90, 303–313.CrossRefPubMedGoogle Scholar
  12. 12.
    Schüpbach, J., Pyra, H., Jendis, J., Tomasik, Z., and Böni, J. (1996) Isolation of an in vitro transmissible agent with reverse transcriptase activity from a blood donor with a borderline-positive HIV-1 serology for more than five years. Clin. Diagn. Virol. 5, 197–203.CrossRefPubMedGoogle Scholar
  13. 13.
    Griffiths, D. J., Venables, P. J. W., Weiss, R. A., and Boyd, M. D. (1997) A novel exogenous retrovirus sequence identified in humans. J. Virol. 71, 2866–2872.PubMedGoogle Scholar
  14. 14.
    Böni, J., Opravil, M., Tomasik, Z., Rothen, M., Bisset, L., Grob, P. J., Lüthy, R., and Schüpbach, J. (1997) Simple monitoring of antiretroviral therapy with a signal-amplification-boosted human immunodeficiency virus type 1 p24 antigen assay with heat-denatured plasma. AIDS 11, F47–F52.CrossRefPubMedGoogle Scholar
  15. 15.
    Böni, J., Stalder, J., Reigel, F., and Schüpbach, J. (1996) Detection of reverse transcriptase activity in live attenuated virus vaccines. Clin. Diagn. Virol. 5, 43–53.CrossRefPubMedGoogle Scholar
  16. 16.
    Alaeus, A., Lidman, K., Sönnerborg, A., and Albert, J. (1997) Subtype-specific problems with quantification of plasma HIV-1 RNA. AIDS 11, 859–865.CrossRefPubMedGoogle Scholar
  17. 17.
    Kwok, S. and Higuchi, R. (1989) Avoiding false positives with PCR. Nature 339, 237,238.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2000

Authors and Affiliations

  • Jürg Böni
  • Jörg Schüpbach

There are no affiliations available

Personalised recommendations