Detection of HIV-1 Nucleic Acids by Northern Blotting

  • Richard A. McDonald
  • Christopher A. D. Smith
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 17)

Abstract

Northern blotting (1) is one of many tools used in understanding the human immunodeficiency virus type 1 (HIV-1). In the process of Northern blotting, RNA is first separated by size through a denaturing agarose gel and transferred onto a membrane. With this transfer or blotting, and subsequent hybridization with a DNA or RNA probe, quantitation, expression levels of the RNA, size of the RNA, and mapping of the 5′- and 3′-terminal end of the RNA can be determined from primary and cultured cells, blood, and tissue. For HIV research, Northern blotting has been utilized for determining the expression levels and splicing patterns of HIV RNA regulatory genes (2, 3, 4), HIV RNA protease gene (5), cytokine effects on the levels of spliced and genomic HIV RNA (6,7), steady-state transcriptional levels and splicing patterns of HIV RNA as a result of antiviral constructs (8), and receptor studies (9,10). In addition, Northern blotting has be utilized to answer questions on the effect on the HIV RNA level of expression of the envelope protein on calmodulin (11) and carbohydrate binding proteins (12).

References

  1. 1.
    Alwine, J. C., Kemp, D. J., and Stark, G. R. (1977) Method for detection of specific RNAs in agarose gels by transfer to diazobenzylozxymethyl-paper and hybridization with DNA probes. Proc. Natl. Acad. Sci. USA 74, 5350–5354.PubMedCrossRefGoogle Scholar
  2. 2.
    Junker, U., Bevec, D., Barske, C., Kalfoglou, C., Escaich, S., Dobrovnik, M., Hauber, J., and Bohnlein, E. (1996) Intracellular expression of cellular eif-5a mutants inhibits HIV-1 replication in human T cells: a feasibility cells. Hum. Gene Ther. 7(15), 1861–1869.PubMedCrossRefGoogle Scholar
  3. 3.
    Cagnon, L., Cucchiarini, M., Lefebvre, J. C., and Doglio, A. (1995) Protection of a T-cell from human immunodeficiency virus replication by the stable expression of a short antisense RNA sequence carried by a shuttle RNA molecule. J. Acquir. Immune. Defic. Syndr. Hum. Retroviol. 9(4), 349–358.Google Scholar
  4. 4.
    Dyhr-Mikkelsen, H. and Kjems, J. (1995) Inefficient spliceosome assembly and abnormal branch site selection in splicing of an HIV-1 transcript in vitro. J. Biol. Chem. 270(41), 24,060–24,066.PubMedCrossRefGoogle Scholar
  5. 5.
    Zhang, D., Zhang, N., Wick, M. M., and Byrn, R. A. (1995) HTV type 1 protease activation of NF-kappa B within T lymphoid cells. AIDS Res. Hum. Retroviruses. 11(2), 223–230.PubMedCrossRefGoogle Scholar
  6. 6.
    Naif, H., Ho-Shon, M., Chang, J., and Cunningham, A. L. (1994) Molecular mechanisms of IL-4 effect on HIV expression in promonocytic cell lines and primary human monocytes. J. Leukoc. Biol. 56(3), 335–339.PubMedGoogle Scholar
  7. 7.
    Weissman, D., Poli, G., and Fauci, A. S. (1995) IL-10 synergizes with multiple cytokines in enhancing HIV production in cells of monocytic lineage. J. Acquir. Immune. Defic. Syndr. Hum. Retroviol. 9(5), 442–449.Google Scholar
  8. 8.
    Berkhout, B. and van Wamel, J. L. (1995) Inhibition of human immunodeficiency virus expression by sense transcripts encoding the retroviral leader RNA. Antiviral. Res. 26(2), 101–105.PubMedCrossRefGoogle Scholar
  9. 9.
    Biswas, P., Smith, C. A., Goletti, D., Hardy, E. C., Jackson, R. W., and Fauci, A. S. (1995) Cross-linking of CD30 induces HIV expression in chronically infected T cells. Immunity 2(6), 587–596.PubMedCrossRefGoogle Scholar
  10. 10.
    Harrington, R. D. and Geballe, A. P. (1996) Human immunodeficiency virus type-1 susceptible whole cell and microcell hybrids. Ann. Clin. Lab. Sci. 26(6), 522–530.PubMedGoogle Scholar
  11. 11.
    Radding, W., Pan, Z. Q., Hunter, E., Johnston, P., Williams, J. P., and McDonald, J. M. (1996) Expression of HIV-1 envelope glycoprotein alters cellular calmodulin. Biochem. Biophys. Res. Commun. 218(1), 192–197.PubMedCrossRefGoogle Scholar
  12. 12.
    Schroder, H. C., Ushijima, H., Theis, C., Seve, A. P., Hubert, J., and Muller, W. E. (1995) Expression of nuclear lectin carbohydrate-binding protein 35 in human immunodeficiency virus type 1-infected Molt-3 cells. J. Acquir. Immune. Defic. Syndr. Hum. Retroviol. 9(4), 340–348.Google Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1999

Authors and Affiliations

  • Richard A. McDonald
    • 1
  • Christopher A. D. Smith
    • 1
  1. 1.Division of RetrovirologyWalter Reed Army Institute of ResearchRockville

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