Abstract
The discovery of novel antivirals for HIV and HCV has been a focus of intensive research for many years. Where the inhibition of critical viral enzymes by small molecules has proven effective for many viruses, there is considerable merit in pursuing protein–protein interactions (PPIs) as targets for therapeutic intervention. The mammalian protein–protein interaction trap (MAPPIT) is a two-hybrid system used for the study of PPIs. The bait and prey proteins are linked to deficient cytokine receptor chimeras, where the bait and prey interaction and subsequent ligand stimulation restores JAK-STAT signaling, resulting in reporter gene expression controlled by a STAT3-responsive promoter. We report the use of MAPPIT as a high-throughput screening assay for the discovery of inhibitors or stimulators of the Vif–APOBEC3G interaction and the reverse transcriptase heterodimerization (RTp66-RTp51) for HIV and the NS4A–NS3 interaction for HCV.
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References
Shafer, R. W., and Schapiro, J. M. (2008) HIV-1 drug resistance mutations: an updated framework for the second decade of HAART AIDS Rev 10, 67–84.
Shimakami, T., Lanford, R. E., and Lemon, S. M. (2009) Hepatitis C: recent successes and continuing challenges in the development of improved treatment modalities Curr Opin Pharmacol 9, 537–44.
Eyckerman, S., Verhee, A., Van der Heyden, J., Lemmens, I., Van Ostade, X., Vandekerckhove, J., and Tavernier, J. (2001) Design and application of a cytokine-receptor-based interaction trap Nature Cell Biology 3, 1114–19.
Sheehy, A. M., Gaddis, N. C., Choi, J. D., and Malim, M. H. (2002) Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein Nature 418, 646–50.
Harris, R. S., Bishop, K. N., Sheehy, A. M., Craig, H. M., Petersen-Mahrt, S. K., Watt, I. N., Neuberger, M. S., and Malim, M. H. (2003) DNA deamination mediates innate immunity to retroviral infection Cell 113, 803–9.
Marin, M., Rose, K. M., Kozak, S. L., and Kabat, D. (2003) HIV-1 Vif protein binds the editing enzyme APOBEC3G and induces its degradation Nat Med 9, 1398–403.
Figueiredo, A., Moore, K. L., Mak, J., Sluis-Cremer, N., de Bethune, M. P., and Tachedjian, G. (2006) Potent nonnucleoside reverse transcriptase inhibitors target HIV-1 Gag-Pol PLoS Pathog 2, e119.
Pattyn, E., Lavens, D., Van der Heyden, J., Verhee, A., Lievens, S., Lemmens, I., Hallenberger, S., Jochmans, D., and Tavernier, J. (2008) MAPPIT (MAmmalian Protein-Protein Interaction Trap) as a tool to study HIV reverse transcriptase dimerization in intact human cells Journal of Virological Methods 153, 7–15.
Bartenschlager, R., Ahlborn-Laake, L., Mous, J., and Jacobsen, H. (1993) Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions J Virol 67, 3835–44.
Tomei, L., Failla, C., Vitale, R. L., Bianchi, E., and De Francesco, R. (1996) A central hydrophobic domain of the hepatitis C virus NS4A protein is necessary and sufficient for the activation of the NS3 protease J Gen Virol 77 ( Pt 5), 1065–70.
Lin, K., Kwong, A. D., and Lin, C. (2004) Combination of a hepatitis C virus NS3-NS4A protease inhibitor and alpha interferon synergistically inhibits viral RNA replication and facilitates viral RNA clearance in replicon cells Antimicrob Agents Chemother 48, 4784–92.
Lin, K., Perni, R. B., Kwong, A. D., and Lin, C. (2006) VX-950, a novel hepatitis C virus (HCV) NS3-4A protease inhibitor, exhibits potent antiviral activities in HCv replicon cells Antimicrob Agents Chemother 50, 1813–22.
Craig, J. C., Duncan, I. B., Hockley, D., Grief, C., Roberts, N. A., and Mills, J. S. (1991) Antiviral properties of Ro 31–8959, an inhibitor of human immunodeficiency virus (HIV) proteinase Antiviral Res 16, 295–305.
Young, S. D., Britcher, S. F., Tran, L. O., Payne, L. S., Lumma, W. C., Lyle, T. A., Huff, J. R., Anderson, P. S., Olsen, D. B., Carroll, S. S., and et al. (1995) L-743, 726 (DMP-266): a novel, highly potent nonnucleoside inhibitor of the human immunodeficiency virus type 1 reverse transcriptase Antimicrob Agents Chemother 39, 2602–5.
Qian, K., Morris-Natschke, S. L., and Lee, K. H. (2009) HIV entry inhibitors and their potential in HIV therapy Med Res Rev 29, 369–93.
Chan, L., Pereira, O., Reddy, T. J., Das, S. K., Poisson, C., Courchesne, M., Proulx, M., Siddiqui, A., Yannopoulos, C. G., Nguyen-Ba, N., Roy, C., Nasturica, D., Moinet, C., Bethell, R., Hamel, M., L’Heureux, L., David, M., Nicolas, O., Courtemanche-Asselin, P., Brunette, S., Bilimoria, D., and Bedard, J. (2004) Discovery of thiophene-2-carboxylic acids as potent inhibitors of HCV NS5B polymerase and HCV subgenomic RNA replication. Part 2: tertiary amides Bioorg Med Chem Lett 14, 797–800.
Li, F., Goila-Gaur, R., Salzwedel, K., Kilgore, N. R., Reddick, M., Matallana, C., Castillo, A., Zoumplis, D., Martin, D. E., Orenstein, J. M., Allaway, G. P., Freed, E. O., and Wild, C. T. (2003) PA-457: a potent HIV inhibitor that disrupts core condensation by targeting a late step in Gag processing Proc Natl Acad Sci USA 100, 13555–60.
Zhou, J., Yuan, X., Dismuke, D., Forshey, B. M., Lundquist, C., Lee, K. H., Aiken, C., and Chen, C. H. (2004) Small-molecule inhibition of human immunodeficiency virus type 1 replication by specific targeting of the final step of virion maturation J Virol 78, 922–9.
Yang, W., Zhao, Y., Fabrycki, J., Hou, X., Nie, X., Sanchez, A., Phadke, A., Deshpande, M., Agarwal, A., and Huang, M. (2008) Selection of replicon variants resistant to ACH-806, a novel hepatitis C virus inhibitor with no cross-resistance to NS3 protease and NS5B polymerase inhibitors Antimicrob Agents Chemother 52, 2043–52.
Sandberg, E. M., Ma, X., He, K., Frank, S. J., Ostrov, D. A., and Sayeski, P. P. (2005) Identification of 1,2,3,4,5,6-hexabromocyclohexane as a small molecule inhibitor of jak2 tyrosine kinase autophosphorylation [correction of autophophorylation] J Med Chem 48, 2526–33.
Schust, J., Sperl, B., Hollis, A., Mayer, T. U., and Berg, T. (2006) Stattic: a small-molecule inhibitor of STAT3 activation and dimerization Chem Biol 13, 1235–42.
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Van Schoubroeck, B. et al. (2012). MAPPIT as a High-Throughput Screening Assay for Modulators of Protein–Protein Interactions in HIV and HCV. In: Suter, B., Wanker, E. (eds) Two Hybrid Technologies. Methods in Molecular Biology, vol 812. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-455-1_18
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DOI: https://doi.org/10.1007/978-1-61779-455-1_18
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