Detection of HIV-1 DNA and Messenger RNA in Individual Cells by PCR-Driven in Situ Hybridization and Flow Cytometry

Abstract

Human immunodeficiency virus type-1 (HIV-1) DNA and messenger RNA sequences in both cell lines and blood obtained directly from HIV-1-infected patients were amplified by polymerase chain reaction and hybridized to fluorescein-labeled probes in situ, and the individually labeled cells were analyzed by flow cytometry. After flow cytometric analysis, heterogeneous cell populations were reproducibly resolved into HIV-1-positive and -negative distributions. Fluorescence microscopy showed that the cellular morphology was preserved and intracellular localization of amplified product DNA was maintained. Retention of nonspecific probe was not observed. Analysis of proviral DNA and viral messenger RNA in cells in the blood of HIV-1-infected patients showed that the HIV-1 genome persists in a large reservoir of latently infected cells. With the use of this technique it is now possible to detect single-copy DNA or low-abundance messenger RNA rapidly and reproducibly in a minor subpopulation of cells in suspension at single-cell resolution and to sort those cells for further characterization.

Stable gene expression occurs from a minority of integrated HIV-1-based vectors: transcriptional silencing is present in the majority

“…8 The virus was replication competent but apparently had not been exposed to the selection pressure exerted by the therapy, implying that the virus was not replicating. Early in situ polymerase chain reaction (PCR) studies on harvested lymphoid tissues [9][10][11] or peripheral blood cells 9 revealed viral RNA in a small percentageoften less than 10% -of cells in which viral DNA was present, indicating that in most cells the provirus is inactive.…”

A novel method for the direct quantification of gene transfer into cells using PCR in situ

“…We did not find it necessary to use other strategies to obviate this problem, such as the use of multiple primer sets, extra-long products, overlapping primers, post-PCR fixation with paraformaldehyde or biotinylated primers. 28,29 A post-hybridization wash of 62°C was used. This is higher than in published protocols but was found to eliminate background without significantly decreasing detectable product.…”

Cell reservoirs in lymph nodes infected with HIV-1 subtype E differ from subtype B: identification by combined in situ polymerase chain reaction and immunohistochemistry

“…Identification of the actual cells that are infected has been carried out using in situ RT-PCR to detect single copies of HIV-1 in infected cells. 18,[21][22][23][24][25][26][27][28] Previous in situ RT-PCR studies (presumably on subtype B) have localized the virus to lymph node germinal centres [29][30][31] either as free virus 30 or in association with CD21 þ follicular dendritic cells or macrophages, 18,25,26,[28][29][30][31][32][33] but not interdigitating dendritic cells. 32 It has been proposed that CD4 þ lymphocytes become infected as they contact follicular dendritic cells in transit through lymph node germinal centers.…”