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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 .......

Representative 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.......

Acknowledgements

We thank Jonathan Rodenfels, Kazimierz Tycowski, and Johanna B. Withers for advice on data analysis. We also thank G. Hayward for the anti-SSB antibody. This work was supported by grants T32GM007223 and T32AI055403 from the National Institutes of Health (to TKV) and NIH grant (CA16038) (to JAS). JAS is an investigator of the Howard Hughes Medical Institute. Figures 1-3 and Table 1 were reproduced with permission from the American Society for Microbiology under a Creative Commons Attribution license from the following publication: Vallery, T. K., Withers, J. B., Andoh, J. A., Steitz, J. A. Kaposi's Sarcoma-Associated Herpesvirus mRNA Accumulation in Nuclear Fo....

Materials

NameCompanyCatalog NumberComments
AlexaFluor594-5-dUTPLife TechnologiesC1100
anti-DIG FITC Jackson Lab Immunologicals200-092-156
Anti-Rabbit Secondary AlexaFluor594 Monoclonal AntibodyInvitrogenA-11037Goat
Anti-SSB AntibodyN/AN/ARef. Chiou et al. 2002
BLASTnNIH NCBIN/AFree Sequence Alignment Software
Dextran SulfateSigma AldrichD8906Molecular Biology Grade
DIG-Oligonucleotide Tailing KitSigma Roche#033535839102nd Gen
Eight-Chamber SlidesNunc Lab Tek II#154453Blue seal promotes surface tension but separation by clear gel is also available. 
FormamideSigma AldrichF9037Molecular Biology Grade
GAPDH ProbesStellarisSMF-2019-1Compatible with protocol, Quasar 670
ImageJ NIH, Bethesda, MDN/AFree Image Analysis Software, [http:rsb.info.nih.gov/ij/]
OligoAnalyzerIDTN/AFree Oligonucleotide Analyzer 
pcDNA3InvitrogenA-150228
pmaxGFPAmaxaVDF-1012
Poly L-LysineSigma AldrichP8920
Terminal TransferaseSigma Roche#003333574001
Vanadyl Ribonucleoside Complexes NEBS1402S
VectashieldVector Laboratories, Inc. H-1000DAPI within the mounting media scatters the light and reduces contrast. 

References

  1. Amen, M. A., Griffiths, A. Packaging of Non-Coding RNAs into Herpesvirus Virions: Comparisons to Coding RNAs. Frontiers in Genetics. 2, 81 (2011).
  2. Schmid, M., Speiseder, T., Dobner, T., Gonzalez, R. A. DNA virus replication compartments....

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