Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Summary

We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF).

Abstract

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout the cell at specific times can be captured with fluorescence in situ hybridization (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus hijacks the host cell to preferentially express viral genes, causing changes in cell morphology and behavior of biomolecules. Lytic activities are centered in nuclear factories, termed viral replication compartments, which are discernable only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, both adherent and in suspension. The method includes steps for the development of specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and quantitative calculations of fluorescence intensities. This protocol has been successfully applied to multiple cell types, uninfected cells, latent cells, lytic cells, time-courses, and cells treated with inhibitors to analyze the spatiotemporal activities of specific RNAs and proteins from both the human host and KSHV.

Introduction

In their lytic (active) phase, herpesviruses hijack the host cell, causing changes in cell morphology and localization of biological molecules, to produce virions. The base of operations is the nucleus, where the double-stranded DNA viral genome is replicated and packaged into a protein shell, called a capsid¹. To begin, the virus expresses its own proteins, hijacking host machinery and preventing expression of non-essential host genes, a process termed the host shutoff effect. The majority of this activity is localized to specific 4′,6-diamidino-2-phenylindole (DAPI)-free nuclear regions called viral replication compartments, comprised o....

Protocol

1. Design of fluorescence in situ (FISH) anti-sense oligonucleotides to detect a specific herpesviral transcript

1. Select 25 to 40 nt segments from the sequence of RNA of interest and convert to be anti-sense. A successful FISH strategy may contain from one up to ten or more different anti-sense oligonucleotides. When selecting sequences, consider the following:
   1. If the RNA of interest contains a unique repeat region, then capitalize on this feature and design an anti-sense oligonucleotide to target the repeat sequence.
      NOTE: Tycowski and colleagues⁵ provide an example of this strategy with rhesus rhadinovirus (RRV)....

Results

The FISH and IF methods detailed in this manuscript are shown in Figure 1 along with the quantification of results by line traces of fluorescent intensity. The results presented here are semi-quantitative and offer insight into localization, rather than into comparisons between intensities of different fluorescent stains because experiments did not include a fluorescent bead in the slide preparation. Figure 1 also reveals that th.......

Discussion

The protocol described in this report can be adapted to different cell types and includes steps for double RNA FISH and RNA FISH with IF using both monoclonal and polyclonal primary antibodies. Although prepared slides are typically imaged with a confocal microscope, imaging can be performed with a STED (stimulated emission depletion) microscope after modifications of increased antibody concentration and a different mounting medium. For enhanced analysis of individual cells, samples prepared with this protocol may also b.......

Materials

Name	Company	Catalog Number	Comments
AlexaFluor594-5-dUTP	Life Technologies	C1100
anti-DIG FITC	Jackson Lab Immunologicals	200-092-156
Anti-Rabbit Secondary AlexaFluor594 Monoclonal Antibody	Invitrogen	A-11037	Goat
Anti-SSB Antibody	N/A	N/A	Ref. Chiou et al. 2002
BLASTn	NIH NCBI	N/A	Free Sequence Alignment Software
Dextran Sulfate	Sigma Aldrich	D8906	Molecular Biology Grade
DIG-Oligonucleotide Tailing Kit	Sigma Roche	#03353583910	2nd Gen
Eight-Chamber Slides	Nunc Lab Tek II	#154453	Blue seal promotes surface tension but separation by clear gel is also available.
Formamide	Sigma Aldrich	F9037	Molecular Biology Grade
GAPDH Probes	Stellaris	SMF-2019-1	Compatible with protocol, Quasar 670
ImageJ	NIH, Bethesda, MD	N/A	Free Image Analysis Software, [http:rsb.info.nih.gov/ij/]
OligoAnalyzer	IDT	N/A	Free Oligonucleotide Analyzer
pcDNA3	Invitrogen	A-150228
pmaxGFP	Amaxa	VDF-1012
Poly L-Lysine	Sigma Aldrich	P8920
Terminal Transferase	Sigma Roche	#003333574001
Vanadyl Ribonucleoside Complexes	NEB	S1402S
Vectashield	Vector Laboratories, Inc.	H-1000	DAPI within the mounting media scatters the light and reduces contrast.