Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

Area of Science:

• Molecular Biology
• Virology
• Cell Biology

Background:

• Gene expression relies on precise spatial and temporal regulation within the nucleus.
• Understanding the location and timing of transcription, especially for viral episomes, is crucial.
• Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic virus with episomal DNA.

Purpose of the Study:

• To develop and apply a method for visualizing actively transcribing KSHV episomes.
• To analyze the spatial regulation of viral gene expression during KSHV reactivation.
• To investigate the relationship between viral episomes and nuclear architecture.

Main Methods:

• Combined immunofluorescence (IFA) and RNA-FISH techniques were employed.
• KSHV latency-associated nuclear antigen (LANA) staining identified episome location.
• RNA-FISH probes targeted viral gene introns to detect nascent RNA transcripts.
• Anti-RNA polymerase II (RNAPII) antibody staining assessed RNAPII association.

Main Results:

• Actively transcribing KSHV episomes were successfully identified and localized within the nucleus.
• The formation of large viral transcription factories during KSHV reactivation was visualized.
• The spatial association of RNA polymerase II aggregation with KSHV transcription was observed.

Conclusions:

• The combined IFA and RNA-FISH method enables visualization of KSHV transcription dynamics.
• Spatial regulation of viral gene expression during KSHV reactivation can be analyzed.
• This approach provides insights into the organization of viral transcription machinery.