Name: functional imaging of viral transcription factories using 3d fluorescence microscopy
Uploaded: 2018-01-18T14:00:00
Description: Watch this Scientific Journal Video about Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy at JoVE.com

This research was supported by National Institutes of Health grant (R01-DE025985) and by an American Cancer Society Research Scholar Award (RSG-13-383-MPC). This work was also supported by grants from the U.S. Department of Agriculture (2015-67015-23268 and 2014-67015-21787) and New Research Initiative Grant from University of California, Davis.

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
<ol>
	<li>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.</li>
	<li>Grow cells at 37 &#176;C with 5% carbon dioxide (CO2), making sure to continuously grow and to split cel...

<ol>
	<li>de Laat, W., Grosveld, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grosveld+Spatial+organization+of+gene+expression:+the+active+chromatin+hub.">Grosveld Spatial organization of gene expression: the active chromatin hub.</a> Chromosome Res. 11 (5), 447-459 (2003).</li><li>Misteli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+dynamics:+implications+for+nuclear+architecture+and+gene+expression.">Protein dynamics: implications for nuclear architecture and gene expression.</a> Science. 291 (5505), 843-847 (2001).</li><li>Bernardi, R., Pandolfi, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure,+dynamics+and+functions+of+promyelocytic+leukaemia+nuclear+bodies.">Structure, dynamics and functions of promyelocytic leukaemia nuclear bodies.</a> Nat Rev Mol Cell Biol. 8 (12), 1006-1016 (2007).</li><li>Cioce, M., Lamond, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cajal+bodies:+a+long+history+of+discovery.">Cajal bodies: a long history of discovery.</a> Annu Rev Cell Dev Biol. 21, 105-131 (2005).</li><li>Boisvert, F. M., van Koningsbruggen, S., Navascues, J., Lamond, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multifunctional+nucleolus.">The multifunctional nucleolus.</a> Nat Rev Mol Cell Biol. 8 (7), 574-585 (2007).</li><li>Sutherland, H., Bickmore, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factories:+gene+expression+in+unions.">Transcription factories: gene expression in unions.</a> Nat Rev Genet. 10 (7), 457-466 (2009).</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> J Virol. 88 (3), 1404-1420 (2014).</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> J Virol. 91 (11), (2017).</li><li>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=KSHV+and+the+pathogenesis+of+Kaposi+sarcoma:+listening+to+human+biology+and+medicine.">KSHV and the pathogenesis of Kaposi sarcoma: listening to human biology and medicine.</a> J Clin Invest. 120 (4), 939-949 (2010).</li><li>Mesri, E. A., Cesarman, E., Boshoff, C. <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+and+its+associated+herpesvirus.">Kaposi's sarcoma and its associated herpesvirus.</a> Nat Rev Cancer. 10 (10), 707-719 (2010).</li><li>Campbell, M., 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=Protein+arginine+methyltransferase+1-directed+methylation+of+Kaposi+sarcoma-associated+herpesvirus+latency-associated+nuclear+antigen.">Protein arginine methyltransferase 1-directed methylation of Kaposi sarcoma-associated herpesvirus latency-associated nuclear antigen.</a> J Biol Chem. 287 (8), 5806-5818 (2012).</li><li>Guito, J., Lukac, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KSHV+Rta+Promoter+Specification+and+Viral+Reactivation.">KSHV Rta Promoter Specification and Viral Reactivation.</a> Front Microbiol. 3, 30 (2012).</li><li>Darst, R. P., Haecker, I., Pardo, C. E., Renne, R., Kladde, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+diversity+of+Kaposi's+sarcoma-associated+herpesvirus.">Epigenetic diversity of Kaposi's sarcoma-associated herpesvirus.</a> Nucleic Acids Res. 41 (5), 2993-3009 (2013).</li><li>Osborne, C. S., 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=Myc+dynamically+and+preferentially+relocates+to+a+transcription+factory+occupied+by+Igh.">Myc dynamically and preferentially relocates to a transcription factory occupied by Igh.</a> PLoS Biol. 5 (8), 192 (2007).</li><li>Fanucchi, S., Shibayama, Y., Burd, S., Weinberg, M. S., Mhlanga, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+contact+permits+transcription+between+coregulated+genes.">Chromosomal contact permits transcription between coregulated genes.</a> Cell. 155 (3), 606-620 (2013).</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> PLoS Pathog. 7 (10), 1002300 (2011).</li><li>Chang, P. C., 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=Histone+demethylase+JMJD2A+regulates+Kaposi's+sarcoma-associated+herpesvirus+replication+and+is+targeted+by+a+viral+transcriptional+factor.">Histone demethylase JMJD2A regulates Kaposi's sarcoma-associated herpesvirus replication and is targeted by a viral transcriptional factor.</a> J Virol. 85 (7), 3283-3293 (2011).</li><li>McAllister, S. C., Hansen, S. G., Messaoudi, I., Nikolich-Zugich, J., Moses, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+efficiency+of+phorbol+ester-induced+lytic+reactivation+of+Kaposi's+sarcoma-associated+herpesvirus+during+S+phase.">Increased efficiency of phorbol ester-induced lytic reactivation of Kaposi's sarcoma-associated herpesvirus during S phase.</a> J Virol. 79 (4), 2626-2630 (2005).</li><li>Miller, G., El-Guindy, A., Countryman, J., Ye, J., Gradoville, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lytic+cycle+switches+of+oncogenic+human+gammaherpesviruses.">Lytic cycle switches of oncogenic human gammaherpesviruses.</a> Adv Cancer Res. 97, 81-109 (2007).</li><li>Guito, J., Lukac, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KSHV+Rta+Promoter+Specification+and+Viral+Reactivation.">KSHV Rta Promoter Specification and Viral Reactivation.</a> Front Microbiol. 3, 30 (2012).</li></ol>

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...

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 active molecules can traverse the nucleus rather quickly via diffusion2. As a consequence of this ephemeral property, most DNA binding proteins &#39;jump&#39; from binding site to binding site, feeling their way around the nuclear space, which allows for a highly adaptive and versatile nucleus2.
Despite this dynamic behavior of biomolecules within the nucleus, nuclear bodies without membranes such as (but not limited to) the nucleolus, Cajal bodies, and promyelocytic leukemia nuclear bodies (PML-NB) still exist. It is through a variety of mechanisms such as tandem DNA repeats (nucleolus), rRNA (nucleolus) and structural proteins such as coilin (Cajal bodies) or PML proteins (PML-NB) that hold these constructs together3,4,5. These structures and other congregations such as transcription factories serve as scaffolds that not only increase the local concentration of required components, but also regulate the composition of proteins and nucleic acids within them to ultimately create a central site for efficient cellular functions6.
Visualizing when and where nuclear structures form provides a wealth of information to researchers studying epigenetics. From a virology perspective, the reactivation of latently infected viruses, such as KSHV, significantly alters the landscape of the nucleus and distribution of nuclear enzymes to shift transcription primarily from cellular genes to viral genes, ultimately to produce fully functional viral progeny7,8. How does KSHV manipulate the cellular gene expression machinery to facilitate viral gene expression? Such information could also shed light on temporal cellular gene regulatory mechanisms.
Like all other herpesviruses, KSHV has two life cycles called lytic replication and latency. KSHV primarily resides in the latency stage, in which most of its viral gene expression is silenced, except latency associated genes9,10. During latency KSHV produces latency associated nuclear antigen (LANA), which constitutively binds the viral genomes and tethers viral chromatin to the human chromosome11. Because of LANA&#39;s intimate relationship with the viral genome, it is possible to use IFA and DAPI to stain and locate where the viral episomes were in relation to the host chromatin.
To study KSHV reactivation at the single episome level and the association with other viral episomes in an infected cell, a strategy to locate actively transcribing viral chromatin in situ has been established. Accordingly, LANA and RNAPII IFA with intron RNA-FISH were combined, by generating RNA-FISH probes that bind to the intron region (exact probe sequences can be found in the Izumiya lab&#39;s most recent publication8) of KSHV K-Rta-the key viral protein that is essential and sufficient for KSHV reactivation-it was possible to identify where transcription was actually taking place8,11,12,13,14,15. This intron RNA-FISH technique enables researchers to visualize where mRNA is being transcribed immediately before it is spliced and exported to cytoplasm16,17.
KSHV can be reactivated by various chemical stimuli including phorbol esters such as 12-O-Tetradeconoyl-phorbol-13acetate (TPA) and histone deacetylase inhibitors such as sodium butyrate, additionally KSHV can be induced to reactivate by overexpression of the viral transcription factor, K-Rta19. Researchers have successfully increased the efficiency of KSHV reactivation by synchronizing the cell&#39;s cycles prior to inducing reactivation18. Thus, for these particular studies, cells were synchronized using a double thymidine block (protocol described below) and incubated with TPA and doxycycline (Dox) for a short time. Doxycyline was used because the cell line utilized in these experiments has a doxycycline-inducible K-Rta cassette, which was cloned from cDNA and does not include the intron region of K-Rta. Although it is possible to reactivate KSHV using only induced K-Rta expression, it has been proven by other researchers that due to a variety of different biochemical factors K-Rta expression alone proves to be a feeble reactivation stimuli20. By combining all of these, and by limiting the drug incubations to a short period of time, a robust but not overly artificial KSHV reactivation was achieved for imaging.
After labeling LANA, RNAPII, K-Rta introns, and DNA as described in this paper, 3D fluorescence imaging was performed using a widefield deconvolution microscope. After processing with imaging software, the spatial distribution of active viral episomes can be properly evaluated. Using this technique, the central questions regarding the fundamental nature of the formation of active chromatin hubs and other nuclear structures can be studied. Having identical viral episomes in a single cell that processes the same regulatory elements may represent a unique research tool to deepen the understanding of spatiotemporal gene regulatory mechanisms.
A limitation of imaging multiple cell specimens fixed at different time points to characterize an inherently dynamic molecular process is that subtle or small-scale changes in fluorescence distribution are undetected or deemed insignificant. This is true unless in the rare instance, every cell observed displays the same subtle change. Thus, the full spatiotemporal relationship of active viral transcription and other nuclear structures cannot be critically evaluated using fixed imaging. To address these technical challenges, the best approach is to image live cells that have marked viral episomes and to follow the location of key cellular enzymes over time.

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			<td height="85" style="height:85px;width:216px;">RPMI medium 1650 GlutaMAX</td>
			<td style="width:71px;">Gibco by Life Technologies</td>
			<td style="width:119px;">61870-036</td>
			<td style="width:167px;">Needs to be warmed to 37 &#176;C</td>
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			<td height="64" style="height:64px;width:216px;">Fetal Bovine Serum</td>
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			<td height="43" style="height:43px;width:216px;">Thymidine&#160;</td>
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			<td height="43" style="height:43px;width:216px;">Sodium Butyrate&#160;</td>
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			<td height="64" style="height:64px;width:216px;">Rat monoclonal antibody to KSHV/HHV-8 ORF73</td>
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			<td height="43" style="height:43px;width:216px;">Anti-RNA Polymerase II Antibody clone CTD4H8</td>
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			<td height="43" style="height:43px;width:216px;">Ribonucleic acid, transfer from baker&#8217;s yeast (S. cerevisiae)&#160;</td>
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			<td style="width:119px;">9014-25-9</td>
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			<td height="64" style="height:64px;width:216px;">DAPI (4&#8217;,6-diamindino-2-phenylindole, Dihydrochloride)</td>
			<td style="width:71px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">D1306</td>
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functional imaging of viral transcription factories using 3d fluorescence microscopy

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.

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...

Watch this Scientific Journal Video about Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy at JoVE.com

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

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.

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 ...

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