Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

Summary

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

Abstract

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.

Introduction

It has become increasingly clear that the spatiotemporal organization of the nucleus plays an important role in modulating finely-tuned gene expression in eukaryotes. Most tissue specific genes are distributed over many chromosomes and need to be regulated synchronously in order to respond to specific stimuli in a coordinated manner¹. Cells construct active chromatin hubs (ACH) as a way of bringing together genes and their cis-regulatory components to a specific nuclear space¹.

It has also been demonstrated by various studies that although the nucleus seems very dense and viscous, biologically....

Protocol

CAUTION: Cell lines used in this procedure contain infectious viruses, be careful and only proceed in level 2 BSL facility or higher.

1. Cell Preparation and Maintenance

1. Culture TREx-K-RTA BCBL-1 cells (or another KSHV infected PEL cell lines) in RPMI1640 medium supplemented with 15% fetal bovine serum (FBS) and 1% penicillin streptomycin glutamine solution.
2. Grow cells at 37 °C with 5% carbon dioxide (CO₂), making sure to continuously grow and to split cells by a ratio of 1:4 every 2–4 days.
3. Monitor cell cultures with a light microscope every day to confirm good cell growth and morpholog....

Results

A slightly abridged protocol was performed where BCBL-1 cells were used and only LANA and K-Rta RNA were stained (Figure 1). This experiment allows us to examine where actively transcribing viral episomes are and the heterogeneity of response to KSHV reactivation stimuli in a population of cells. In the cell indicated by the arrow in Figure 1, the distinct K-Rta fluorescence in regions that closely match the distribution of viral.......

Discussion

There are some aspects of the protocol that can be altered to accommodate unusual circumstances. The choice of fixation and permeabilization buffers can also be altered. For fixation buffers, paraformaldehyde is also effective and ethanol can be used for permeabilization.

Quenching the formaldehyde with glycine PBS is recommended as it prevents formaldehyde from disabling the antibodies in absence of bovine serum albumin (BSA), but if the coverslips are washed thoroughly enough, the glycine P.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI medium 1650 GlutaMAX	Gibco by Life Technologies	61870-036	Needs to be warmed to 37 °C
Fetal Bovine Serum	Omega Scientific Inc.	FB-11
Pen Strep Glutamine (100x)	Gibco by Life Technologies	10378-016
Thymidine	Sigma Aldrich	T9250
Dulbecco’s Phosphate Buffered Solution	Gibco by Life Technologies	14190-144
TPA	Sigma Aldrich	P1585
Sodium Butyrate	Sigma Aldrich	B5887
Formaldehyde Solution	Sigma Aldrich	252549	Corrosive, toxic, health hazard
Diethyl Pyrocarbonate	Sigma Aldrich	D-5758	Acute toxicity
Glycine	Sigma Aldrich	410225
Acetone	Sigma Aldrich	179124	Flammable, toxic
Methanol	Fisher Chemical	67-56-1	Flammable, toxic, health hazard
Rat monoclonal antibody to KSHV/HHV-8 ORF73	Advanced Biotechnologies	13-210-100
Anti-RNA Polymerase II Antibody clone CTD4H8	EMD Milipore	05-623
Ribonucleic acid, transfer from baker’s yeast (S. cerevisiae)	Sigma Aldrich	9014-25-9
Saline sodium citrate buffer 20x (SSC)	Thermo Fisher Scientific	15557044
Formamide	Sigma Aldrich	295876
Omnipur Dextran Sulfate, 50% solution	EMD Milipore	1916C093
DAPI (4’,6-diamindino-2-phenylindole, Dihydrochloride)	Thermo Fisher Scientific	D1306
slowFade Gold antifade reagent	Life Technologies	S36936
Micro cover glass 22x22	Matsunami	C022221
VoloCITY		3D imaging software
DeltaVision microscope
Superfrost/Plus microscope slides	Fisher Scientific	12-550-15