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Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)
10:16

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Published on: May 5, 2012

Subcellular localization of low-abundance human immunodeficiency virus nucleic acid sequences visualized by

J B Lawrence1, L M Marselle, K S Byron

  • 1Department of Cell Biology, University of Massachusetts Medical Center, Worcester 01655.

Proceedings of the National Academy of Sciences of the United States of America
|July 1, 1990
PubMed

Insights

This study developed a sensitive in situ hybridization method to detect human immunodeficiency virus (HIV) RNA in single cells. The assay revealed a single nuclear focus of HIV transcription in infected cells, indicating minimal viral genome activity.

Area of Science:

  • Virology
  • Molecular Biology
  • Cell Biology

Background:

  • Human immunodeficiency virus (HIV) infection involves complex viral replication cycles within host cells.
  • Understanding the early stages of HIV transcription and localization is crucial for developing effective antiviral strategies.

Purpose of the Study:

  • To develop and validate a sensitive high-resolution in situ hybridization method for detecting and localizing HIV.
  • To investigate the subcellular localization and transcriptional activity of HIV in infected cells, both in vitro and in vivo.

Main Methods:

  • Utilized high-resolution in situ hybridization with biotin or digoxigenin-labeled probes to detect HIV RNA in lymphocytes.
  • Employed techniques including hybridization without denaturation and susceptibility to RNase and actinomycin D to confirm the nature of nuclear RNA foci.
  • Analyzed HIV transcription in infected cell lines (8E5/LAV) and patient-derived lymphocytes.

Main Results:

  • A single, intense nuclear signal of HIV RNA was observed in cells 12 hours post-infection in vitro.
  • Later stages of infection showed intense cytoplasmic fluorescence alongside multiple nuclear foci.
  • Confirmed the presence of a singular nuclear focus of HIV transcription in vivo in patient lymphocytes, originating from one or very few viral genomes.
  • Demonstrated that productive Epstein-Barr virus infection results in numerous nuclear RNA foci per cell, contrasting with HIV.

Conclusions:

  • The developed in situ hybridization method is highly sensitive for detecting single-cell HIV transcription.
  • HIV transcription in infected cells, both in vitro and in vivo, is characterized by a singular nuclear focus, suggesting transcription from a limited number of viral genomes.
  • This finding provides insights into the transcriptional regulation and early events of HIV infection.

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