Detection of HIV-1 provirus and RNA by in situ amplification

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.