Summary
To illustrate the methods employed in gene expression profiling using cDNA microarrays, infection of CD4+ T cell lines with HIV-1LAI is used to identify expression changes relevant to in vitro HIV-1 infection. Cell lines are infected at a high multiplicity of infection to ensure a population of near-synchronously infected cells to be compared to uninfected cells. Infection status is verified using flow cytometry to determine the intracellular expression of the viral gag p24 protein before samples are harvested for total RNA extraction. Total RNA is extracted and amplified using commercially available kits, and RNA quality is verified using Bioanalyzer technology. To obtain fluorescently labeled cDNA probes, the amplified RNA is reverse-transcribed to yield cDNA, using random nonamers in the presence of dye-labeled dCTP. After first-strand cDNA synthesis, RNA is degraded and the probes are purified. For each infection condition (LAI and mock), two slides are hybridized with identical probes generated from the same RNAs, but with fluorescent labels reversed on one of the slides to control for dyespecific effects. Troubleshooting strategies and issues to consider prior to starting the experiment are discussed in detail in the notes section.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Schena, M., Shalon, D., Davis, R. W., and Brown, P. O. (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467–470.
Xiang, C. C. and Chen, Y. (2000) cDNA microarray technology and its applications. Biotechnol. Adv. 18, 35–46.
Duggan, D. J., Bittner, M., Chen, Y., Meltzer, P., and Trent, J. M. (1999). Expression profiling using cDNA microarrays. Nat. Genet. 1, 10–14.
Deyholos, M. K. and Galbraith, D. W. (2001) High-density microarrays for gene expression analysis. Cytometry 43, 229–238.
Quackenbush, J. (2001) Computational analysis of microarray data. Nat. Rev. Genet. 2, 418–427.
Geiss, G. K., Bumgarner, R. E., An, M. C., Agy, M. B., van ’t Wout, A. B., Hammersmark, E., et al. (2000) Large-scale monitoring of host cell gene expression during HIV-1 infection using cDNA microarrays. Virology 266, 8–16.
Corbeil, J., Sheeter, D., Genini, D., Rought, S., Leoni, L., Du, P., et al. (2001) Temporal gene regulation during HIV-1 infection of human CD4+ T cells. Genome Res. 11, 1198–1204.
Vahey, M. T., Nau, M. E., Jagodzinski, L. L., Yalley-Ogunro, J., Taubman, M., Michael, N. L., and Lewis, M. G. (2002) Impact of viral infection on the gene expression profiles of proliferating normal human peripheral blood mononuclear cells infected with HIV type 1 RF. AIDS Res. Hum. Retroviruses 18, 179–192.
van ’t Wout, A. B., Lehrman, G. K., Mikheeva, S. A., O’Keeffe, G. C., Katze, M. G., Bumgarner, R. E., et al. (2003) Cellular gene expression upon human immunodeficiency virus type 1 infection of CD4(+)-T-cell lines. J. Virol. 77, 1392–1402.
Mitchell, R., Chiang, C. Y., Berry, C., and Bushman, F. (2003) Global analysis of cellular transcription following infection with an HIV-based vector. Mol. Ther. 8, 674–687.
Simmons, A., Aluvihare, V., and McMichael, A. (2001) Nef triggers a transcriptional program in T cells imitating single-signal T cell activation and inducing HIV virulence mediators. Immunity 14, 763–777.
Shaheduzzaman, S., Krishnan, V., Petrovic, A., Bittner, M., Meltzer, P., Trent, J., et al. (2002) Effects of HIV-1 Nef on cellular gene expression profiles. J. Biomed. Sci. 9, 82–96.
Galey, D., Becker, K., Haughey, N., Kalehua, A., Taub, D., Woodward, J., et al. (2003) Differential transcriptional regulation by human immunodeficiency virus type 1 and gp120 in human astrocytes. J. Neurovirol. 9, 358–371.
Cicala, C., Arthos, J., Selig, S. M., Dennis, G., Jr., Hosack, D. A., Van Ryk, D., et al. (2002) HIV envelope induces a cascade of cell signals in non-proliferating target cells that favor virus replication. Proc. Natl. Acad. Sci. USA 99, 9380–9385.
de la Fuente, C., Santiago, F., Deng, L., Eadie, C., Zilberman, I., Kehn, K., et al. (2002) Gene expression profile of HIV-1 Tat expressing cells: a close interplay between proliferative and differentiation signals. BMC Biochem. 3, 14.
Gibellini, D., Re, M. C., La Placa, M., and Zauli, G. (2002) Differentially expressed genes in HIV-1 tat-expressing CD4(+) T-cell line. Virus Res. 90, 337–345.
Izmailova, E., Bertley, F. M., Huang, Q., Makori, N., Miller, C. J., Young, R. A., and Aldovini, A. (2003) HIV-1 Tat reprograms immature dendritic cells to express chemoattractants for activated T cells and macrophages. Nat. Med. 9, 191–197.
Patel, C. A., Mukhtar, M., Harley, S., Kulkosky, J., and Pomerantz, R. J. (2002) Lentiviral expression of HIV-1 Vpr induces apoptosis in human neurons. J. Neurovirol. 8, 86–99.
Chun, T. W., Justement, J. S., Lempicki, R. A., Yang, J., Dennis, G., Jr., Hallahan, C. W., et al. (2003) Gene expression and viral prodution in latently infected, resting CD4+ T cells in viremic versus aviremic HIV-infected individuals. Proc. Natl. Acad. Sci. USA 100, 1908–1913.
Guadalupe, M., Reay, E., Sankaran, S., Prindiville, T., Flamm, J., McNeil, A., and Dandekar, S. (2003) Severe CD4+ T-cell depletion in gut lymphoid tissue during primary human immunodeficiency virus type 1 infection and substantial delay in restoration following highly active antiretroviral therapy. J. Virol. 77, 11,708–11,717.
George, M. D., Sankaran, S., Reay, E., Gelli, A. C., and Dandekar, S. (2003) High-throughput gene expression profiling indicates dysregulation of intestinal cell cycle mediators and growth factors during primary simian immunodeficiency virus infection. Virology 312, 84–94.
Vahey, M. T., Nau, M. E., Taubman, M., Yalley-Ogunro, J., Silvera, P., and Lewis, M. G. (2003) Patterns of gene expression in peripheral blood mononuclear cells of rhesus macaques infected with SIVmac251 and exhibiting differential rates of disease progression. AIDS Res. Hum. Retroviruses 19, 369–387.
Roberts, E. S., Zandonatti, M. A., Watry, D. D., Madden, L. J., Henriksen, S. J., Taffe, M. A., and Fox, H. S. (2003) Induction of pathogenic sets of genes in macrophages and neurons in NeuroAIDS. Am. J. Pathol. 162, 2041–2057.
Sui, Y., Potula, R., Pinson, D., Adany, I., Li, Z., Day, J., et al. (2003) Microarray analysis of cytokine and chemokine genes in the brains of macaques with SHIV-encephalitis. J. Med. Primatol. 32, 229–239.
Miller, E. D., Smith, J. A., Lichtinger, M., Wang, L., and Su, L. (2003) Activation of the signal transducer and activator of transcription 1 signaling pathway in thymocytes from HIV-1-infected human thymus. AIDS 17, 1269–1277.
Acknowledgments
These protocols were developed in close collaboration with the members of the Microbiology Array Group at the University of Washington. The author thanks Katie A. Davis for editorial assistance. This work was supported by grants from the National Institutes of Health (AI37984, AI52028, HL072631, and HG002360).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2005 Humana Press Inc.
About this protocol
Cite this protocol
van ’t Wout, A.B. (2005). Gene Expression Profiling of HIV-1 Infection Using cDNA Microarrays. In: Zhu, T. (eds) Human Retrovirus Protocols. Methods in Molecular Biology™, vol 304. Humana Press. https://doi.org/10.1385/1-59259-907-9:455
Download citation
DOI: https://doi.org/10.1385/1-59259-907-9:455
Publisher Name: Humana Press
Print ISBN: 978-1-58829-495-1
Online ISBN: 978-1-59259-907-3
eBook Packages: Springer Protocols