Name: quantitative fluorescence in situ hybridization (fish) and immunofluorescence (if) of specific gene products in kshv-infected cells
Uploaded: 2019-08-27T14:30:00
Description: Watch this Scientific Journal Video about Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells at JoVE.com

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&#160;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&#39;s Sarcoma-Associated Herpesvirus mRNA Accumulation in Nuclear Foci Is Influenced by Viral DNA Replication and Viral Noncoding Polyadenylated Nuclear RNA. Journal of Virology.&#160;92 (13), doi:10.1128/JVI.00220-18, (2018).

1. Design of fluorescence in situ (FISH) anti-sense oligonucleotides to detect a specific herpesviral transcript
<ol>
	<li>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:
	<ol>
		<li>If the RNA of interest contains a unique repeat region, then capitalize on this feature and design an anti-sense oligonucleotide to target ...

<ol>
	<li>Amen, M. A., Griffiths, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Packaging+of+Non-Coding+RNAs+into+Herpesvirus+Virions:+Comparisons+to+Coding+RNAs.">Packaging of Non-Coding RNAs into Herpesvirus Virions: Comparisons to Coding RNAs.</a> Frontiers in Genetics. 2, 81 (2011).</li><li>Schmid, M., Speiseder, T., Dobner, T., Gonzalez, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+virus+replication+compartments.">DNA virus replication compartments.</a> Journal of Virology. 88 (3), 1404-1420 (2014).</li><li>Pawlicki, J. M., Steitz, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+microRNA+transcript+retention+at+sites+of+transcription+leads+to+enhanced+microRNA+production.">Primary microRNA transcript retention at sites of transcription leads to enhanced microRNA production.</a> Journal of Cell Biology. 182 (1), 61-76 (2008).</li><li>Borah, S., Darricarrere, N., Darnell, A., Myoung, J., Steitz, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+viral+nuclear+noncoding+RNA+binds+re-localized+poly(A)+binding+protein+and+is+required+for+late+KSHV+gene+expression.">A viral nuclear noncoding RNA binds re-localized poly(A) binding protein and is required for late KSHV gene expression.</a> Public Library of Science Pathogens. 7 (10), e1002300 (2011).</li><li>Tycowski, K. T., Shu, M. D., Borah, S., Shi, M., Steitz, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+of+a+triple-helix-forming+RNA+stability+element+in+noncoding+and+genomic+RNAs+of+diverse+viruses.">Conservation of a triple-helix-forming RNA stability element in noncoding and genomic RNAs of diverse viruses.</a> Cell Reports. 2 (1), 26-32 (2012).</li><li>Weinberg, R. A., Penman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecular+weight+monodisperse+nuclear+RNA.">Small molecular weight monodisperse nuclear RNA.</a> Journal of Molecular Biology. 38 (3), 289-304 (1968).</li><li>Myoung, J., Ganem, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+doxycycline-inducible+KSHV+producer+cell+line+of+endothelial+origin:+maintenance+of+tight+latency+with+efficient+reactivation+upon+induction.">Generation of a doxycycline-inducible KSHV producer cell line of endothelial origin: maintenance of tight latency with efficient reactivation upon induction.</a> Journal of Virology Methods. 174 (1-2), 12-21 (2011).</li><li>Brulois, K. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+manipulation+of+a+new+Kaposi's+sarcoma-associated+herpesvirus+bacterial+artificial+chromosome+clone.">Construction and manipulation of a new Kaposi's sarcoma-associated herpesvirus bacterial artificial chromosome clone.</a> Journal of Virology. 86 (18), 9708-9720 (2012).</li><li>Sturzl, M., Gaus, D., Dirks, W. G., Ganem, D., Jochmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kaposi's+sarcoma-derived+cell+line+SLK+is+not+of+endothelial+origin,+but+is+a+contaminant+from+a+known+renal+carcinoma+cell+line.">Kaposi's sarcoma-derived cell line SLK is not of endothelial origin, but is a contaminant from a known renal carcinoma cell line.</a> International Journal of Cancer. 132 (8), 1954-1958 (2013).</li><li>Chiou, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+gene+expression+and+a+transactivation+function+exhibited+by+the+vGCR+(ORF74)+chemokine+receptor+protein+of+Kaposi's+sarcoma-associated+herpesvirus.">Patterns of gene expression and a transactivation function exhibited by the vGCR (ORF74) chemokine receptor protein of Kaposi's sarcoma-associated herpesvirus.</a> Journal of Virology. 76 (7), 3421-3439 (2002).</li><li>Cole, R. W., Jinadasa, T., Brown, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+and+interpreting+point+spread+functions+to+determine+confocal+microscope+resolution+and+ensure+quality+control.">Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control.</a> Nature Protocols. 6 (12), 1929-1941 (2011).</li><li>Nakamura, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+changes+in+Kaposi's+sarcoma-associated+virus+gene+expression+patterns+following+expression+of+a+tetracycline-inducible+Rta+transactivator.">Global changes in Kaposi's sarcoma-associated virus gene expression patterns following expression of a tetracycline-inducible Rta transactivator.</a> Journal of Virology. 77 (7), 4205-4220 (2003).</li><li>Majerciak, V., Yamanegi, K., Zheng, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+structure+and+expression+of+Kaposi's+sarcoma-associated+herpesvirus+ORF56,+ORF57,+ORF58,+and+ORF59.">Gene structure and expression of Kaposi's sarcoma-associated herpesvirus ORF56, ORF57, ORF58, and ORF59.</a> Journal of Virology. 80 (24), 11968-11981 (2006).</li><li>Sun, R., Lin, S. F., Gradoville, L., Miller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyadenylylated+nuclear+RNA+encoded+by+Kaposi+sarcoma-associated+herpesvirus.">Polyadenylylated nuclear RNA encoded by Kaposi sarcoma-associated herpesvirus.</a> Proceedings of the National Academy Sciences U S A. 93 (21), 11883-11888 (1996).</li><li>Vallery, T. K., Withers, J. B., Andoh, J. A., Steitz, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kaposi's+Sarcoma-Associated+Herpesvirus+mRNA+Accumulation+in+Nuclear+Foci+Is+Influenced+by+Viral+DNA+Replication+and+Viral+Noncoding+Polyadenylated+Nuclear+RNA.">Kaposi's Sarcoma-Associated Herpesvirus mRNA Accumulation in Nuclear Foci Is Influenced by Viral DNA Replication and Viral Noncoding Polyadenylated Nuclear RNA.</a> Journal of Virology. 92 (13), (2018).</li><li>Borah, S., Nichols, L. A., Hassman, L. M., Kedes, D. H., Steitz, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+expression+and+subcellular+localization+of+RNA+and+protein+species+using+high-throughput+single+cell+imaging+flow+cytometry.">Tracking expression and subcellular localization of RNA and protein species using high-throughput single cell imaging flow cytometry.</a> RNA. 18 (8), 1573-1579 (2012).</li><li>Bruce, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Analysis+of+the+KSHV+Transcriptome+Following+Primary+Infection+of+Blood+and+Lymphatic+Endothelial+Cells.">Quantitative Analysis of the KSHV Transcriptome Following Primary Infection of Blood and Lymphatic Endothelial Cells.</a> Pathogens. 6 (1), (2017).</li><li>Chen, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kaposi's+Sarcoma-Associated+Herpesvirus+Hijacks+RNA+Polymerase+II+To+Create+a+Viral+Transcriptional+Factory.">Kaposi's Sarcoma-Associated Herpesvirus Hijacks RNA Polymerase II To Create a Viral Transcriptional Factory.</a> Journal of Virology. 91 (11), (2017).</li></ol>

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...

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&#8242;,6-diamidino-2-phenylindole (DAPI)-free nuclear regions called viral replication compartments, comprised of both host and viral proteins, RNAs, and viral DNA2. The cell is overhauled to provide space and resources for the replication compartments and thus assembly of viral capsids. Once the capsid exits the nucleus, how the capsid is enveloped in the cytoplasm to produce a membrane-bound viral particle, also known as a virion, is unclear. Understanding of the localization and spatial shifts of both host and viral biomolecules during the lytic phase provides deeper mechanistic insight into the arrangement of the replication compartment, host shutoff effect, the virion-egress pathway, and other processes related to herpesviral infection and replication.
Currently the best method to detect and study these changes is the visualization of proteins and RNAs in infected cells with immunofluorescence (IF) and fluorescent in situ hybridization (FISH), respectively. Use of a time-course with these techniques reveals the localization of biomolecules at key points of the lytic phase or simply, spatiotemporal data. FISH and IF complement other biochemical techniques, such as inhibition of a cellular process (e.g., inhibition of viral DNA replication), RT-qPCR (real-time polymerase chain reaction), RNA sequencing, Northern blots, mass spectrometry, Western blotting, and analysis of viral DNA production, that may provide a more global picture of cellular activities.
We developed RNA FISH strategies to examine the RNA products from specific genes and a computational analysis that quantitatively calculates the nucleocytoplasmic ratio of a specific gene product. The sample preparation, modified from earlier publications by Steitz and colleagues3,4, is relatively easy and can be used for both adherent and suspended cells. The protocol is also adaptable for simultaneous use of multiple RNA FISH strategies (double RNA FISH) or RNA FISH with IF strategies. Development of a specific FISH strategy is challenging, but suggestions to improve success are outlined. The data analysis described here is quantitative if fluorescent beads and strong markers of compartment boundaries are used and offers additional insight into the micrographs, insight that removes observation bias. The detailed protocol is designed for both latent and lytic cells infected by Kaposi&#39;s sarcoma-associated herpesvirus (KSHV) and can be used with uninfected cells or cells infected by other herpesviruses5. The methods of quantitation are applicable to studies on nucleocytoplasmic shifts or relocalization between subcellular compartments in most cells.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">AlexaFluor594-5-dUTP</td>
			<td style="width:191px;">Life Technologies</td>
			<td style="width:117px;">C1100</td>
			<td style="width:277px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-DIG FITC&#160;</td>
			<td>Jackson Lab Immunologicals</td>
			<td>200-092-156</td>
			<td style="width:277px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Rabbit Secondary AlexaFluor594 Monoclonal Antibody</td>
			<td>Invitrogen</td>
			<td>A-11037</td>
			<td>Goat</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-SSB Antibody</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Ref. Chiou et al. 2002</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BLASTn</td>
			<td>NIH NCBI</td>
			<td>N/A</td>
			<td style="width:277px;">Free Sequence Alignment Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextran Sulfate</td>
			<td>Sigma Aldrich</td>
			<td>D8906</td>
			<td style="width:277px;">Molecular Biology Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DIG-Oligonucleotide Tailing Kit</td>
			<td>Sigma Roche</td>
			<td>#03353583910</td>
			<td style="width:277px;">2nd Gen</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Eight-Chamber Slides</td>
			<td>Nunc Lab Tek II</td>
			<td>#154453</td>
			<td style="width:277px;">Blue seal promotes surface tension but separation by clear gel is also available.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formamide</td>
			<td>Sigma Aldrich</td>
			<td>F9037</td>
			<td style="width:277px;">Molecular Biology Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GAPDH Probes</td>
			<td>Stellaris</td>
			<td>SMF-2019-1</td>
			<td style="width:277px;">Compatible with protocol, Quasar 670</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">ImageJ&#160;</td>
			<td>NIH, Bethesda, MD</td>
			<td>N/A</td>
			<td style="width:277px;">Free Image Analysis Software, [http:rsb.info.nih.gov/ij/]</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OligoAnalyzer</td>
			<td>IDT</td>
			<td>N/A</td>
			<td style="width:277px;">Free Oligonucleotide Analyzer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pcDNA3</td>
			<td>Invitrogen</td>
			<td>A-150228</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pmaxGFP</td>
			<td>Amaxa</td>
			<td>VDF-1012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly L-Lysine</td>
			<td>Sigma Aldrich</td>
			<td>P8920</td>
			<td style="width:277px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Terminal Transferase</td>
			<td>Sigma Roche</td>
			<td>#003333574001</td>
			<td style="width:277px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vanadyl Ribonucleoside Complexes&#160;</td>
			<td>NEB</td>
			<td>S1402S</td>
			<td style="width:277px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Vectashield</td>
			<td>Vector Laboratories, Inc.&#160;</td>
			<td>H-1000</td>
			<td style="width:277px;">DAPI within the mounting media scatters the light and reduces contrast.&#160;</td>
		</tr>
	</tbody>
</table>

quantitative fluorescence in situ hybridization (fish) and immunofluorescence (if) of specific gene products in kshv-infected cells

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.

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...

Watch this Scientific Journal Video about Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells at JoVE.com

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

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).

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

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...

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