Summary
The detection of the HIV-1 provirus that can integrate into a host cell nucleus and remain latent for years is problematic. The threshold of in situ hybridization, which is about 10 copies per cell, is too high to detect one integrated copy of the provirus. Although polymerase chain reaction (PCR) can detect 1 provirus per 100,000 cells, it cannot determine the specific cellular localization of the virus. These problems can be resolved with PCR in situ hybridization. Adapting this method to RNA detection (reverse transcriptase [RT] in situ PCR) allows one to determine whether viral infection is latent or productive as well as to detect the host response in the form of cytokine mRNA expression. These methodologies have demonstrated that (1) there is massive infection of CD4 cells by HIV-1 prior to AIDS-defining symptomatology, (2) progression of AIDS is marked by the progressive destruction of CD4 cells, as evidenced by an increased ratio of productively to latently infected cells, (3) the primary target of the virus in the uterine cervix, lung, central nervous system, and skeletal muscle is the macrophage and its derivatives, and (4) AIDS-related diseases such as AIDS dementia are marked by both many viral-infected cells and upregulation of a wide variety of cytokines, primarily in the neighboring noninfected cells. This chapter will describe the methodologies for detecting HIV-1 DNA and RNA in paraffin-embedded tissue sections as well as the colabeling experiments needed to define the host response to the viral invasion.
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References
Nuovo, G. J. (1997) PCR in situ hybridization; 3rd ed. Lippincott-Raven Press, NY.
Nuovo, G. J., Gallery, F., MacConnell, P., Becker, J., and Bloch, W. (1991) An improved technique for the detection of DNA by in situ hybridization after PCR-amplification. Am. J. Pathol. 139, 1239.
Chou, Q., Russell, M., Birch, D. E., Raymond, J., and Bloch, W. (1992) Prevention of pre-PCR mispriming and primer dimerization improves low copy amplification. Nucl. Acid Res. 20, 1717.
Nuovo, G. J., Gallery, F., Hom, R., MacConnell, P., and Bloch, W. (1993) Importance of different variables for optimizing in situ detection of PCR-amplified DNA. PCR Method Applic. 2, 305.
Nuovo, G. J., Becker, J., Margiotta, M., Burke, M., Fuhrer, J., and Steigbigel, R. (1994) In situ detection of PCR-amplified HIV-1 nucleic acids in lymph nodes and peripheral blood in asymptomatic infection and advanced stage AIDS. J. Acquired Immun. Def. 7, 916.
Embretson, J., Zupancic, M., Ribas, J. L., Racz, P., Haase, A. T. (1993) Massive covert infection of helper T lymphocytes and macrophages by HIV during the incubation period of AIDS. Nature 362, 359.
Bagasra, O., Lischer, H. W., Sachs, M., and Pomerantz, R. (1992) Detection of HIV-1 provirus in mononuclear cells by in situ PCR. N. Engl. J. Med. 326, 1385.
Nuovo, G. J., Forde, A., MacConnell, P., and Fahrenwald, R. (1993) In situ detection of PCR-amplified HIV-1 nucleic acids and tumor necrosis factor cDNA in cervical tissues. Am. J. Pathol. 143, 40.
Nuovo, G. J., Becker, J., Simsir, A., Margiotta, M., and Shevchuck, M. (1994) In situ localization of PCR-amplified HIV-1 nucleic acids in the male genital tract. Am. J. Pathol. 144, 1142.
Nuovo, G. J., Gallery, F., MacConnell, P., and Braun, A. (1994) In situ detection of PCR-amplified HIV-1 nucleic acids and tumor necrosis factor RNA in the central nervous system. Am. J. Pathol. 144, 659.
Dubrovsky, L., Ulrich, P., Manogue, K. R., Cerami, A., and Burkinsky, M. (1995) Nuclear localization signal of HIV-1 as a novel target for therapeutic intervention. Mol. Med. 1, 217.
Schmidtmayerova, H., Notte, H. S., Nuovo, G. J., Raabe, T., Flanagan, C. R., Dubrovsky, L., et al. (1996) HIV-1 infection alters chemokine peptide expression in human monocytes: implications for recruitment of leukocytes into brain and lymph nodes. Proc. Natl. Acad. Sci. USA 93, 700.
Euscher, E., Davis, J., Holtzman, I., and Nuovo, G. J. (2001) Coxsackie virus infection of the placenta associated with neurodevelopmental delays in the newborn. Obstet. Gynecol. 98, 1019.
Cioc, A. and Nuovo, G. J. (2002) Correlation of viral detection with histology in cardiac tissue from patients with sudden, unexpected death. Mod. Pathol. 15, 914–922.
Parham, P. (2000) The Immune System. Garland Publishing, NY.
Goldsby, R. A, Kindt, T. J., Kuby, J., and Osborn, B. A. (2000) Immunology, 4th ed. New York: W. H. Freeman and Co.
Manetti, R., Annunziato, F., Giannò, V., Tomasévic, L., Beloni, L., Mavilia, C., and Maggi, E. (1996) Th1 and Th2 cells in HIV infection. In: Th1 and Th2 in Health and Disease (Romagnani, S. ed.) Chem. Immunol. Basel, Karger, 63, 138–157.
Breen, E. C. (2002) Pro-and anti-inflammatory cytokines in human immunodeficiency infection and acquired immunodeficiency syndrome. Pharmacol. Ther. 95, 295–304.
Gage, J. R., Sandhu, A. K., Nihira, M., Bonecini-Almeida, M. G., Cristoforoni, P., Kishimoto, T., et al. (2000) Cervical cancer cell lines and human papillomavirus (HPV)—immortalized keratinocytes induce HIV-1 in the U1 monocyte line. J. Obst. Gynecol. 96, 879–885.
Acknowledgments
The authors greatly appreciate the financial support from the Lewis Foundation; also supported by NIH grant NCI (RO1 HL-00-012) (GJN).
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Nicol, A., Nuovo, G.J. (2005). Detection of HIV-1 Provirus and RNA by In Situ Amplification. In: Zhu, T. (eds) Human Retrovirus Protocols. Methods in Molecular Biology™, vol 304. Humana Press. https://doi.org/10.1385/1-59259-907-9:171
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DOI: https://doi.org/10.1385/1-59259-907-9:171
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