Title

Subcellular localization of low-abundance human immunodeficiency virus nucleic acid sequences visualized by fluorescence in situ hybridization

UMMS Affiliation

Department of Cell Biology; Department of Pediatrics

Date

7-1-1990

Document Type

Article

Subjects

Cell Nucleus; Cells, Cultured; Dactinomycin; *Genes, Viral; HIV-1; Humans; Kinetics; Microscopy, Fluorescence; Nucleic Acid Hybridization; RNA, Viral; Ribonuclease, Pancreatic; T-Lymphocytes; Transcription, Genetic

Disciplines

Cell Biology | Life Sciences | Medicine and Health Sciences

Abstract

Detection and subcellular localization of human immunodeficiency virus (HIV) were investigated using sensitive high-resolution in situ hybridization methodology. Lymphocytes infected with HIV in vitro or in vivo were detected by fluorescence after hybridization with either biotin or digoxigenin-labeled probes. At 12 hr after infection in vitro, a single intense signal appeared in the nuclei of individual cells. Later in infection, when cytoplasmic fluorescence became intense, multiple nuclear foci frequently appeared. The nuclear focus consisted of newly synthesized HIV RNA as shown by hybridization in the absence of denaturation and by susceptibility to RNase and actinomycin D. Virus was detected in patient lymphocytes and it was shown that a singular nuclear focus also characterizes cells infected in vivo. The cell line 8E5/LAV containing one defective integrated provirus revealed a similar focus of nuclear RNA, and the single integrated HIV genome was unequivocally visualized on a D-group chromosome. This demonstrates an extremely sensitive single-cell assay for the presence of a single site of HIV transcription in vitro and in vivo and suggests that it derives from one (or very few) viral genomes per cell. In contrast, productive Epstein-Barr virus infection exhibited many foci of nuclear RNA per cell.

Rights and Permissions

Citation: Proc Natl Acad Sci U S A. 1990 Jul;87(14):5420-4. Link to article on publisher's website

Related Resources

Link to Article in PubMed

PubMed ID

2371279