1887

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Volume 91, Issue 4

Self-priming of reverse transcriptase impairs strand-specific detection of dengue virus RNA Free

Abstract

Dengue virus infection is the most frequent arthropod-borne infection affecting humans in the world. Our understanding of the pathophysiological events leading to mild or severe outcomes of the disease remains limited by the fact that viral target cells in the human body are poorly characterized. One of the most sensitive strategies for detecting cells supporting active replication of this positive-strand RNA virus is the search for the replicative intermediate, an antigenome of negative polarity, by RT-PCR. However, a phenomenon described as ‘false priming' of the reverse transcriptase (RT) prevents strand-specific detection. The results of the current study showed that this event corresponds to cDNA synthesis that is independent of any primer addition. This property was general to all RNAs tested and was not associated with small free nucleic acids, such as tRNAs and microRNAs. Rather, it corresponded to initiation of cDNA synthesis from the 3′ end of the RNA template, and a model is proposed in which the template RNA snaps back upon itself and creates a transient RNA primer suitable for the RT. Such a property would explain why many assays proposed for detection of a replicative intermediate are not specific, and may help in the development of a molecular biology protocol that could allow replication studies of RNA viruses of human interest, such as dengue virus, hepatitis C virus and enteroviruses.

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2010-04-01
2024-04-18
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References

  1. Alvarez, D. E., Lodeiro, M. F., Luduena, S. J., Pietrasanta, L. I. & Gamarnik, A. V.(2005). Long-range RNA–RNA interactions circularize the dengue virus genome. J Virol 79, 6631–6643.[CrossRef] [Google Scholar]
  2. Bessaud, M., Autret, A., Jegouic, S., Balanant, J., Joffret, M. L. & Delpeyroux, F.(2008). Development of a Taqman RT-PCR assay for the detection and quantification of negatively stranded RNA of human enteroviruses: evidence for false-priming and improvement by tagged RT-PCR. J Virol Methods 153, 182–189.[CrossRef] [Google Scholar]
  3. Billam, P., Pierson, F. W., Li, W., LeRoith, T., Duncan, R. B. & Meng, X. J.(2008). Development and validation of a negative-strand-specific reverse transcription-PCR assay for detection of a chicken strain of hepatitis E virus: identification of nonliver replication sites. J Clin Microbiol 46, 2630–2634.[CrossRef] [Google Scholar]
  4. Blackley, S., Kou, Z., Chen, H., Quinn, M., Rose, R. C., Schlesinger, J. J., Coppage, M. & Jin, X.(2007). Primary human splenic macrophages, but not T or B cells, are the principal target cells for dengue virus infection in vitro. J Virol 81, 13325–13334.[CrossRef] [Google Scholar]
  5. Boiziau, C., Moreau, S. & Toulmé, J.(1994). A phosphorothioate oligonucleotide blocks reverse transcription via an antisens mechanism. FEBS Lett 340, 236–240.[CrossRef] [Google Scholar]
  6. Carriere, M., Pene, V., Breiman, A., Conti, F., Chouzenoux, S., Meurs, E., Andrieu, M., Jaffray, P., Grira, L. & other authors(2007). A novel, sensitive, and specific RT-PCR technique for quantitation of hepatitis C virus replication. J Med Virol 79, 155–160.[CrossRef] [Google Scholar]
  7. Cleaves, G. R., Ryan, T. E. & Schlesinger, R. W.(1981). Identification and characterization of type 2 dengue virus replicative intermediate and replicative form RNAs. Virology 111, 73–83.[CrossRef] [Google Scholar]
  8. Craggs, J. K., Ball, J. K., Thomson, B. J., Irving, W. L. & Grabowska, A. M.(2001). Development of a strand-specific RT-PCR based assay to detect the replicative form of hepatitis C virus RNA. J Virol Methods 94, 111–120.[CrossRef] [Google Scholar]
  9. Evander, M., Eriksson, I., Pettersson, L., Juto, P., Ahlm, C., Olsson, G. E., Bucht, G. & Allard, A.(2007). Puumala hantavirus viremia diagnosed by real-time reverse transcriptase PCR using samples from patients with hemorrhagic fever and renal syndrome. J Clin Microbiol 45, 2491–2497.[CrossRef] [Google Scholar]
  10. Falvey, A. K., Weiss, G. B., Krueger, L. J., Kantor, J. A. & Anderson, W. F.(1976). Transcription of single base oligonucleotides by ribonucleic acid-directed deoxyribonucleic acid polymerase. Nucleic Acids Res 3, 79–88.[CrossRef] [Google Scholar]
  11. Harada, F., Sawyer, R. C. & Dahlberg, J. E.(1975). A primer ribonucleic acid for initiation of in vitro Rous sarcarcoma virus deoxyribonucleic acid synthesis. J Biol Chem 250, 3487–3497. [Google Scholar]
  12. Hardestam, J., Petterson, L., Ahlm, C., Evander, M., Lundkvist, A. & Klingström, J.(2008). Antiviral effect of human saliva against hantavirus. J Med Virol 80, 2122–2126.[CrossRef] [Google Scholar]
  13. Hsuih, T. C., Park, Y. N., Zaretsky, C., Wu, F., Tyagi, S., Kramer, F. R., Sperling, R. & Zhang, D. Y.(1996). Novel, ligation-dependent PCR assay for detection of hepatitis C in serum. J Clin Microbiol 34, 501–507. [Google Scholar]
  14. Isel, C., Lanchy, J. M., Le Grice, S. F., Ehresmann, C., Ehresmann, B. & Marquet, R.(1996). Specific initiation and switch to elongation of human immunodeficiency virus type 1 reverse transcription require the post-transcriptional modifications of primer tRNA3Lys. EMBO J 15, 917–924. [Google Scholar]
  15. Jessie, K., Fong, M. Y., Devi, S., Lam, S. K. & Wong, K. T.(2004). Localization of dengue virus in naturally infected human tissues, by immunohistochemistry and in situ hybridization. J Infect Dis 189, 1411–1418.[CrossRef] [Google Scholar]
  16. Koppelhus, U., Zachar, V., Nielsen, P., Liu, X., Eugen-olsen, J. & Ebbesen, P.(1997). Efficient in vitro inhibition of HIV-1 gag reverse transcription by peptide nucleic acid (PNA) at minimal ratios of PNA/RNA. Nucleic Acids Res 25, 2167–2173.[CrossRef] [Google Scholar]
  17. Lanford, R. E., Chavez, D., Chisari, F. V. & Sureau, C.(1995). Lack of detection of negative-strand hepatitis C virus RNA in peripheral blood mononuclear cells and other extrahepatic tissues by the highly strand-specific rTth reverse transcriptase PCR. J Virol 69, 8079–8083. [Google Scholar]
  18. Laskus, T., Radkowski, M., Wang, L. F., Cianciara, J., Vargas, H. & Rakela, J.(1997). Hepatitis C virus negative strand RNA is not detected in peripheral blood mononuclear cells and viral sequences are identical to those in serum: a case against extrahepatic replication. J Gen Virol 78, 2747–2750. [Google Scholar]
  19. Laskus, T., Radkowski, M., Wang, L. F., Vargas, H. & Rakela, J.(1998). Search for hepatitis C virus extrahepatic replication sites in patients with acquired immunodeficiency syndrome: specific detection of negative-strand viral RNA in various tissues. Hepatology 28, 1398–1401.[CrossRef] [Google Scholar]
  20. Laurila, M. R., Salgado, P. S., Stuart, D. I., Grimes, J. M. & Bamford, D. H.(2005). Back-priming mode of φ6 RNA-dependent RNA polymerase. J Gen Virol 86, 521–526.[CrossRef] [Google Scholar]
  21. Leparc-Goffart, I., Baragatti, M., Temmam, S., Tuiskunen, A., Moureau, G., Charrel, R. & de Lamballerie, X.(2009). Development and validation of real-time one-step reverse transcription-PCR for the detection and typing of dengue viruses. J Clin Virol 45, 61–66.[CrossRef] [Google Scholar]
  22. Lerat, H., Berby, F., Trabaud, M. A., Vidalin, O., Major, M., Trepo, C. & Inchauspe, G.(1996). Specific detection of hepatitis C virus minus strand RNA in hematopoietic cells. J Clin Invest 97, 845–851.[CrossRef] [Google Scholar]
  23. Lin, L., Fevery, J. & Hiem Yap, S.(2002). A novel strand-specific RT-PCR for detection of hepatitis C virus negative-strand RNA (replicative intermediate): evidence of absence or very low level of HCV replication in peripheral blood mononuclear cells. J Virol Methods 100, 97–105.[CrossRef] [Google Scholar]
  24. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K. & Green, M. R.(1984). Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res 12, 7035–7056.[CrossRef] [Google Scholar]
  25. Mizutani, T., Ikeda, M., Saito, S., Sugiyama, K., Shimotohno, K. & Kato, N.(1998). Detection of negative-stranded hepatitis C virus RNA using a novel strand-specific reverse transcription-polymerase chain reaction. Virus Res 53, 209–214.[CrossRef] [Google Scholar]
  26. Nouri Aria, K. T., Sallie, R., Sangar, D., Alexander, G. J., Smith, H., Byrne, J., Portmann, B., Eddleston, A. L. & Williams, R.(1993). Detection of genomic and intermediate replicative strands of hepatitis C virus in liver tissue by in situ hybridization. J Clin Invest 91, 2226–2234.[CrossRef] [Google Scholar]
  27. Novak, J. E. & Kirkegaard, K.(1991). Improved method for detecting poliovirus negative strands used to demonstrate specificity of positive-strand encapsidation and the ratio of positive to negative strands in infected cells. J Virol 65, 3384–3387. [Google Scholar]
  28. Peters, G., Harada, F., Dahlberg, J. E., Panet, A., Haseltine, W. A. & Baltimore, D.(1977). Low-molecular-weight RNAs of Moloney murine leukemia virus: identification of the primer for RNA-directed DNA synthesis. J Virol 21, 1031–1041. [Google Scholar]
  29. Peyrefitte, C. N., Pastorino, B., Bessaud, M., Tolou, H. J. & Couissinier-Paris, P.(2003). Evidence for in vitro falsely-primed cDNAs that prevent specific detection of virus negative strand RNAs in dengue-infected cells: improvement by tagged RT-PCR. J Virol Methods 113, 19–28.[CrossRef] [Google Scholar]
  30. Ratner, L., Haseltine, W., Patarca, R., Livak, K. J., Starcich, B., Josephs, S. F., Doran, E. R., Rafalski, J. A., Whitehorn, E. A. & other authors(1985). Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature 313, 277–284.[CrossRef] [Google Scholar]
  31. Romero, T. A., Tumban, E., Jun, J., Lott, W. B. & Hanley, K. A.(2006). Secondary structure of dengue virus type 4 3′ untranslated region: impact of deletion and substitution mutations. J Gen Virol 87, 3291–3296.[CrossRef] [Google Scholar]
  32. Sessions, O. M., Barrows, N. J., Souza-Neto, J. A., Robinson, T. J., Hershey, C. L., Rodgers, M. A., Ramirez, J. L., Dimopoulos, G., Yang, P. L. & other authors(2009). Discovery of insect and human dengue virus host factors. Nature 458, 1047–1050.[CrossRef] [Google Scholar]
  33. Simpson, E. B., Ross, S. L., Marchetti, S. E. & Kennell, J. C.(2004). Relaxed primer specificity associated with reverse transcriptases encoded by the pFOXC retroplasmids of Fusarium oxysporum. Eukaryot Cell 3, 1589–1600.[CrossRef] [Google Scholar]
  34. Wakefield, J. K., Wolf, A. G. & Morrow, C. D.(1995). Human immunodeficiency virus type 1 can use different tRNAs as primers for reverse transcription but selectively maintains a primer binding site complementary to tRNA3Lys. J Virol 69, 6021–6029. [Google Scholar]
  35. Whitcomb, J. M., Ortiz-Conde, B. A. & Hughes, S. H.(1995). Replication of avian leukosis viruses with mutations at the primer binding site: use of alternative tRNAs as primers. J Virol 69, 6228–6238. [Google Scholar]
  36. Williams, J. G., Kubelik, A. R., Livak, K. J., Rafalski, J. A. & Tingey, S. V.(1990). DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res 18, 6531–6535.[CrossRef] [Google Scholar]
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Self-priming of reverse transcriptase impairs strand-specific detection of dengue virus RNA
J. Gen. Virol. 91, 1019 (2010); https://doi.org/10.1099/vir.0.016667-0
/content/journal/jgv/10.1099/vir.0.016667-0
/content/journal/jgv/10.1099/vir.0.016667-0
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vol. , part 4, pp. 1019 –1027

Mfold RNA structure predictions for the DENV2 transcripts used in Fig. 4 [ PDF] (273 KB)

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