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. 2010 Feb 24;1(3):284–290. doi: 10.1007/s13238-010-0031-0

A novel non-radioactive assay for HIV-RT (RdDp) based on pyrosequencing for high-throughput drug screening

Chang Zhang 1, Yang Wu 2, Yuna Sun 2, Chuan Hong 1, Kehui Xiang 1, Yu Guo 3, Mark Bartlam 3, Zhiyong Lou 1,✉
PMCID: PMC4875084  PMID: 21203975

Abstract

Current in vitro assays for the activity of HIV-RT (reverse transcriptase) require radio-labeled or chemically modified nucleotides to detect reaction products. However, these assays are inherently end-point measurements and labor intensive. Here we describe a novel non-radioactive assay based on the principle of pyrosequencing coupled-enzyme system to monitor the activity of HIV-RT by indirectly measuring the release of pyrophosphate (PPi), which is generated during nascent strand synthesis. The results show that our assay could monitor HIV-RT activity with high sensitivity and is suitable for rapid high-throughput drug screening targeting anti-HIV therapies due to its high speed and convenience. Moreover, this assay can be used to measure primase activity in an easy and sensitive manner, which suggests that this novel approach could be wildly used to analyze the activity of PPi-generated and ATP-free enzyme reactions.

Keywords: HIV-RT (RdDp), assay, non-radioactive, high throughput drug screening

References

  1. Andre M., Morgeaux S., Fuchs F. Quantitative detection of RTactivity by PERTassay: feasibility and limits to a standardized screening assay for human vaccines. Biologicals. 2000;28:67–80. doi: 10.1006/biol.2000.0243. [DOI] [PubMed] [Google Scholar]
  2. Antoun M.D., Rios Y.R., Mendoza N.T., Proctor G. Reverse transcriptase inhibition as prescreen for potential antiviral bioactives. P R Health Sci J. 1994;13:17–18. [PubMed] [Google Scholar]
  3. Arnold B.A., Hepler R.W., Keller P.M. One-step fluorescent probe product-enhanced reverse transcriptase assay. Biotechniques. 1998;25:98–106. doi: 10.2144/98251st06. [DOI] [PubMed] [Google Scholar]
  4. Chang A., Ostrove J.M., Bird R.E. Development of an improved product enhanced reverse transcriptase assay. J Virol Methods. 1997;65:45–54. doi: 10.1016/S0166-0934(96)02168-4. [DOI] [PubMed] [Google Scholar]
  5. Clark A.D., Jr., Jacobo-Molina A., Clark P., Hughes S.H., Arnold E. Crystallization of human immunodeficiency virus type 1 reverse transcriptase with and without nucleic acid substrates, inhibitors, and an antibody Fab fragment. Methods Enzymol. 1995;262:171–185. doi: 10.1016/0076-6879(95)62017-6. [DOI] [PubMed] [Google Scholar]
  6. Hizi A., Tal R., Shaharabany M., Currens M.J., Boyd M.R., Hughes S.H., McMahon J.B. Specific inhibition of the reverse transcriptase of human immunodeficiency virus type 1 and the chimeric enzymes of human immunodeficiency virus type 1 and type 2 by nonnucleoside inhibitors. Antimicrob Agents Chemother. 1993;37:1037–1042. doi: 10.1128/AAC.37.5.1037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Jacobo-Molina A., Ding J., Nanni R.G., Clark A.D., Jr., Lu X., Tantillo C., Williams R.L., Kamer G., Ferris A.L., Clark P. Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA. Proc Natl Acad Sci U S A. 1993;90:6320–6324. doi: 10.1073/pnas.90.13.6320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kanyara J.N., Njagi E.N. Anti-HIV-1 activities in extracts from some medicinal plants as assessed in an in vitro biochemical HIV-1 reverse transcriptase assay. Phytother Res. 2005;19:287–290. doi: 10.1002/ptr.1536. [DOI] [PubMed] [Google Scholar]
  9. Kohlstaedt L.A., Wang J., Friedman J.M., Rice P.A., Steitz T.A. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 1992;256:1783–1790. doi: 10.1126/science.1377403. [DOI] [PubMed] [Google Scholar]
  10. Lahser F.C., Malcolm B.A. A continuous nonradioactive assay for RNA-dependent RNA polymerase activity. Anal Biochem. 2004;325:247–254. doi: 10.1016/j.ab.2003.10.034. [DOI] [PubMed] [Google Scholar]
  11. Lovatt A., Black J., Galbraith D., Doherty I., Moran M.W., Shepherd A.J., Griffen A., Bailey A., Wilson N., Smith K.T. High throughput detection of retrovirus-associated reverse transcriptase using an improved fluorescent product enhanced reverse transcriptase assay and its comparison to conventional detection methods. J Virol Methods. 1999;82:185–200. doi: 10.1016/S0166-0934(99)00111-1. [DOI] [PubMed] [Google Scholar]
  12. Odawara F., Abe H., Kohno T., Nagai-Fujii Y., Arai K., Imamura S., Misaki H., Azuma H., Ikebuchi K., Ikeda H. A highly sensitive chemiluminescent reverse transcriptase assay for human immunodeficiency virus. J Virol Methods. 2002;106:115–124. doi: 10.1016/S0166-0934(02)00142-8. [DOI] [PubMed] [Google Scholar]
  13. Porstmann T., Meissner K., Glaser R., Dopel S.H., Sydow G. A sensitive non-isotopic assay specific for HIV-1 associated reverse transcriptase. J Virol Methods. 1991;31:181–188. doi: 10.1016/0166-0934(91)90156-T. [DOI] [PubMed] [Google Scholar]
  14. Pyra H., Boni J., Schupbach J. Ultrasensitive retrovirus detection by a reverse transcriptase assay based on product enhancement. Proc Natl Acad Sci U S A. 1994;91:1544–1548. doi: 10.1073/pnas.91.4.1544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ronaghi M., Uhlen M., Nyren P. A sequencing method based on real-time pyrophosphate. Science. 1998;281:363–365. doi: 10.1126/science.281.5375.363. [DOI] [PubMed] [Google Scholar]
  16. Sarafianos S.G., Das K., Hughes S.H., Arnold E. Taking aim at a moving target: designing drugs to inhibit drug-resistant HIV-1 reverse transcriptases. Curr Opin Struct Biol. 2004;14:716–730. doi: 10.1016/j.sbi.2004.10.013. [DOI] [PubMed] [Google Scholar]
  17. Silver J., Maudru T., Fujita K., Repaske R. An RT-PCR assay for the enzyme activity of reverse transcriptase capable of detecting single virions. Nucleic Acids Res. 1993;21:3593–3594. doi: 10.1093/nar/21.15.3593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Suzuki K., Craddock B.P., Kano T., Steigbigel R.T. Colorimetric reverse transcriptase assay for HIV-1. J Virol Methods. 1993;41:21–28. doi: 10.1016/0166-0934(93)90159-O. [DOI] [PubMed] [Google Scholar]
  19. Suzuki K., Craddock B.P., Okamoto N., Kano T., Steigbigel R. T. Detection of human immunodeficiency virus (HIV) by colorimetric assay for reverse transcriptase activity on magnetic beads. Biotechnol Appl Biochem. 1993;18:37–44. [PubMed] [Google Scholar]
  20. Suzuki K., Saito T., Kondo M., Osanai M., Watanabe S., Kano T., Kano K., Imai M. Poly A-linked non-isotopic microtiter plate reverse transcriptase assay for sensitive detection of clinical human immunodeficiency virus isolates. J Virol Methods. 1995;55:347–356. doi: 10.1016/0166-0934(95)00073-5. [DOI] [PubMed] [Google Scholar]
  21. Urabe T., Sano K., Tanno M., Mizoguchi J., Otani M., Lee M.H., Takasaki T., Kusakabe H., Imagawa D.T., Nakai M. A non-radioisotopic reverse transcriptase assay using biotin-11-deoxyuridinetriphosphate on primer-immobilized microtiter plates. J Virol Methods. 1992;40:145–154. doi: 10.1016/0166-0934(92)90063-J. [DOI] [PubMed] [Google Scholar]
  22. Vassiliou W., Epp J.B., Wang B.B., Del Vecchio A.M., Widlanski T., Kao C.C. Exploiting polymerase promiscuity: A simple colorimetric RNA polymerase assay. Virology. 2000;274:429–437. doi: 10.1006/viro.2000.0492. [DOI] [PubMed] [Google Scholar]

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