Subcellular localization of low-abundance human immunodeficiency virus nucleic acid sequences visualized by fluorescence in situ hybridization.
AUTOR(ES)
Lawrence, J B
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=54336Documentos Relacionados
- Molecular cloning of developmentally regulated, low-abundance mRNA sequences from embryonic muscle.
- Purification of low-abundance messenger RNAs from rat liver by polysome immunoadsorption.
- Additional low-abundance human small nuclear ribonucleoproteins: U11, U12, etc.
- Detection of viral sequences of low reiteration frequency by in situ hybridization.
- Enhanced Function Conferred on Low-Abundance Chemoreceptor Trg by a Methyltransferase-Docking Site