We thank financial support from an NIH grant (RO1AI118992) awarded to Haidong Gu. We thank the Microscopy, Imaging &#38; Cytometry Resources (MICR) Core facility at Wayne State University for technical support.

1. Cell Seeding and Virus Infection
<ol>
	<li>At 20&#8211;24 h before the virus infection, seed 5 x 104 of human embryonic lung (HEL) fibroblast cells or other cells to be examined on a 4-well 11 mm staggered slide in growth medium (Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS)). Incubate the cells at 37 &#176;C with 5% carbon dioxide (CO2). 
	NOTE: Each well should have 70-80% cell confluency at the time of infection.</li>
	<li>On the next day, remove the growth medium and infect the cells with viruses in Medium-199 at a range of 4&#8211;10 pfu/cell. Incubate virus-infected cells for 1 h at 37 &#176;C. Keep shaking the slide during the incubation period.</li>
	<li>After the 1 h incubation, remove Medium-199 and supplement with growth medium. 
	NOTE: Drugs that interfere with different infection phases can be added at this step or prior to viral absorption.</li>
	<li>Incubate the virus-infected cells at 37 &#176;C with 5% CO2 for various lengths of infection period.</li>
</ol>
2. Fixation and Permeabilization
<ol>
	<li>At proper infection time, quickly wash the infected cells with phosphate-buffered saline (PBS) 3 times and add 200 &#956;L of 4% paraformaldehyde freshly prepared in PBS. Incubate the cells with paraformaldehyde for 8&#8211;10 min at room temperature to fix the cells in each well.</li>
	<li>Aspirate paraformaldehyde and wash the wells with 200 &#956;L of PBS for 3 times. Completely aspirate PBS after the 3rd wash.</li>
	<li>Add 100 &#956;L of 0.2% non-ionic surfactant to each well to permeabilize the cells for 5&#8211;10 min.</li>
	<li>Aspirate the non-ionic surfactant and wash the wells with 200 &#956;L of PBS for 3 times.</li>
</ol>
3. Immunofluorescent Staining
<ol>
	<li>Completely aspirate PBS and add 200 &#956;L of blocking buffer (1% bovine serum albumin (BSA) and 5% horse serum in PBS) in each well and incubate at room temperature for 1 h or at 4 &#176;C overnight.</li>
	<li>Add experimentally determined concentration of primary antibody (rabbit anti-ICP0 polyclonal antibody12) in blocking buffer and incubate primary antibody at room temperature for 2 h or at 4 &#176;C overnight.</li>
	<li>Wash with blocking buffer 3 times with 10 min incubation. Add Alexa 594-conjugated goat anti-rabbit secondary antibody (1:400 diluted in blocking buffer) and incubate the slides at room temperature for 1 h. Then wash the slides 3 times with blocking buffer at 10 min interval.</li>
	<li>Finally wash the slide once with PBS to remove residual BSA and horse serum.</li>
	<li>Add one drop of antifade mounting medium with 4&#39;,6-diamidino-2-phenylindole (DAPI) to mount the slide and seal it with coverslip using transparent nail polish.</li>
</ol>
4. Confocal Imaging
<ol>
	<li>With a confocal microscope, set the wavelength at 590&#8211;650 nm for Alexa 594 and 410&#8211;520 nm for DAPI. Select image format at 1024 x 1024 and line average of 8 to acquire high resolution images.</li>
	<li>Analyze each well on the 4-well slide under confocal microscope. Acquire representative cell images under the 100X objective, as shown in Figure&#160;1 and Figure&#160;2.</li>
	<li>For counting large number of cells, take images of consecutive fields under the 40X objective. 
	NOTE: It requires 5&#8211;10 images to accumulate over 200 infected cells from each time point of each infection.</li>
	<li>In each experiment, take pictures with constant confocal parameters for all samples need to be compared.</li>
</ol>
5. Analyzing Nuclear vs. Cytoplasmic Distribution
<ol>
	<li>Open project with the confocal application software. Select an image from which cells need to be tabulated for nuclear vs. cytoplasmic distribution of ICP0.</li>
	<li>Click the tab &#34;Quantity&#34; from top menu and select &#34;sort ROIs&#34; from tools menu.</li>
	<li>Draw a longitudinal line across the cell to be analyzed by selecting &#34;Draw line&#34; from top menu. 
	NOTE: Histogram will appear showing the fluorescence intensity along the line for both ICP0 and DAPI. In the histogram, blue line represents DAPI pixels and marks the boundary of the nucleus whereas the red line represents ICP0 pixels.</li>
	<li>Based on background staining, set up a constant threshold for ICP0 intensity to analyze ICP0 subcellular distribution in each experiment.
	<ol>
		<li>As exemplified in Figure 2, if the red signal on average is below the threshold in the nuclear region but is above the threshold beyond the blue boundary, categorize the red signal as predominantly located in the cytoplasm.</li>
		<li>If the red signal is above the threshold throughout the nucleus and beyond the boundary of blue signal, group the red signal as nucleus plus cytoplasmic localization.</li>
		<li>If the red signal is above the threshold in the nucleus but on average is below it outside the boundary of blue signal, group the red signal as nuclear localization.</li>
	</ol>
	</li>
	<li>Tabulate more than 200 infected cells from each sample at different infection time and plot in bar graph to illustrate ICP0 movement according to time (Figure 3).</li>
</ol>

<ol>
	<li>Whitley, R., Kimberlin, D. W., Prober, C. G., Arvin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+and+disease.">Pathogenesis and disease.</a> Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis. , (2007).</li><li>Roizman, B., Knipe, D. M., Whitley, R. J., Knipe, D. 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=Herpes+simplex+viruses.">Herpes simplex viruses.</a> Fields' Virology. , 1823-1897 (2013).</li><li>Gu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infected+cell+protein+0+functional+domains+and+their+coordination+in+herpes+simplex+virus+replication.">Infected cell protein 0 functional domains and their coordination in herpes simplex virus replication.</a> World Journal of Virology. 5 (1), 1-13 (2016).</li><li>Lopez, P., Van Sant, C., Roizman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirements+for+the+nuclear-cytoplasmic+translocation+of+infected-cell+protein+0+of+herpes+simplex+virus+1.">Requirements for the nuclear-cytoplasmic translocation of infected-cell protein 0 of herpes simplex virus 1.</a> Journal of Virology. 75 (8), 3832-3840 (2001).</li><li>Kawaguchi, Y., Van Sant, C., Roizman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+1+alpha+regulatory+protein+ICP0+interacts+with+and+stabilizes+the+cell+cycle+regulator+cyclin+D3.">Herpes simplex virus 1 alpha regulatory protein ICP0 interacts with and stabilizes the cell cycle regulator cyclin D3.</a> Journal of Virology. 71 (10), 7328-7336 (1997).</li><li>Mullen, M. A., Ciufo, D. M., Hayward, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+intracellular+localization+domains+and+evidence+for+colocalization+interactions+between+the+IE110+and+IE175+nuclear+transactivator+proteins+of+herpes+simplex+virus.">Mapping of intracellular localization domains and evidence for colocalization interactions between the IE110 and IE175 nuclear transactivator proteins of herpes simplex virus.</a> Journal of Virology. 68 (5), 3250-3266 (1994).</li><li>Maul, G. G., Everett, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nuclear+location+of+PML,+a+cellular+member+of+the+C3HC4+zinc-binding+domain+protein+family,+is+rearranged+during+herpes+simplex+virus+infection+by+the+C3HC4+viral+protein+ICP0.">The nuclear location of PML, a cellular member of the C3HC4 zinc-binding domain protein family, is rearranged during herpes simplex virus infection by the C3HC4 viral protein ICP0.</a> Journal of General Virology. 75 (6), 1223-1233 (1994).</li><li>Chelbi-Alix, M. K., de The, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+virus+induced+proteasome-dependent+degradation+of+the+nuclear+bodies-associated+PML+and+Sp100+proteins.">Herpes virus induced proteasome-dependent degradation of the nuclear bodies-associated PML and Sp100 proteins.</a> Oncogene. 18 (4), 935-941 (1999).</li><li>Zheng, Y., Samrat, S. K., Gu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Tale+of+Two+PMLs:+Elements+Regulating+a+Differential+Substrate+Recognition+by+the+ICP0+E3+Ubiquitin+Ligase+of+Herpes+Simplex+Virus+1.">A Tale of Two PMLs: Elements Regulating a Differential Substrate Recognition by the ICP0 E3 Ubiquitin Ligase of Herpes Simplex Virus 1.</a> Journal of Virology. 90 (23), 10875-10885 (2016).</li><li>Lanfranca, M. P., Mostafa, H. H., Davido, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSV-1+ICP0:+An+E3+Ubiquitin+Ligase+That+Counteracts+Host+Intrinsic+and+Innate+Immunity.">HSV-1 ICP0: An E3 Ubiquitin Ligase That Counteracts Host Intrinsic and Innate Immunity.</a> Cells. 3 (2), 438-454 (2014).</li><li>Zheng, Y., Gu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+three+redundant+segments+responsible+for+herpes+simplex+virus+1+ICP0+to+fuse+with+ND10+nuclear+bodies.">Identification of three redundant segments responsible for herpes simplex virus 1 ICP0 to fuse with ND10 nuclear bodies.</a> Journal of Virology. 89 (8), 4214-4226 (2015).</li><li>Samrat, S. K., Ha, B. L., Zheng, Y., Gu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Elements+Regulating+the+Nuclear-to-Cytoplasmic+Translocation+of+ICP0+in+Late+Herpes+Simplex+Virus+1+Infection.">Characterization of Elements Regulating the Nuclear-to-Cytoplasmic Translocation of ICP0 in Late Herpes Simplex Virus 1 Infection.</a> Journal of Virology. 92 (2), e01673-e01617 (2018).</li><li>Gu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+role+does+cytoplasmic+ICP0+play+in+HSV-1+infection?.">What role does cytoplasmic ICP0 play in HSV-1 infection?.</a> Future Virology. 13 (6), (2018).</li><li>Ettinger, A., Wittmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+live+cell+imaging.">Fluorescence live cell imaging.</a> Methods Cell Biology. 123, 77-94 (2014).</li><li>Frigault, M. M., Lacoste, J., Swift, J. L., 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=Live-cell+microscopy+-+tips+and+tools.">Live-cell microscopy - tips and tools.</a> Journal of Cell Science. 122 (6), 753-767 (2009).</li><li>Chudakov, D. M., Lukyanov, S., Lukyanov, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+proteins+as+a+toolkit+for+in+vivo+imaging.">Fluorescent proteins as a toolkit for in vivo imaging.</a> Trends in Biotechnology. 23 (12), 605-613 (2005).</li><li>Teng, K. W., 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=Labeling+proteins+inside+living+cells+using+external+fluorophores+for+microscopy.">Labeling proteins inside living cells using external fluorophores for microscopy.</a> Elife. 5, e20378 (2016).</li><li>Stephens, D. J., Allan, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+microscopy+techniques+for+live+cell+imaging.">Light microscopy techniques for live cell imaging.</a> Science. 300 (5616), 82-86 (2003).</li></ol>

The authors have nothing to disclose.

This protocol has been used to study the nuclear-to-cytoplasmic translocation of HSV-1 ICP0. ICP0 undergoes subcellular trafficking during HSV-1 infection (Figure 1). Likely, ICP0 interacts with various cell pathways to carry out different functions at different locations. This enables ICP0 to fine tune its multiple functions in the tug-of-war with human host13. However, how ICP0 coordinates the multiple functions in a spatial-temporal manner has not been well studied...

Herpes simplex virus 1 (HSV-1) causes a wide range of mild to severe herpetic diseases including herpes labialis, genital herpes, stromal keratitis, and encephalitis. Once infected, the virus establishes a lifelong latent infection in ganglia neurons. Occasionally, the virus can be reactivated by various reasons such as fever, stress, and immune suppression1, leading to recurrent herpes infection. Infected cell protein 0 (ICP0) is a key viral regulator crucial for both lytic and latent HSV-1 infection. It transactivates downstream virus genes via counteracting the host intrinsic/innate antiviral defenses2,3. ICP0 has an E3 ubiquitin ligase activity, which targets several cell factors for proteasome-dependent degradation3. It also interacts with various cell pathways to regulate their activities and subsequently to offset host antiviral restrictions3. ICP0 is known to locate at different subcellular compartments as the infection proceeds3,4,5. The protein has a lysine/arginine-rich nuclear localization signal (NLS) located at residues 500 to 5066. Upon de novo synthesis at early HSV-1 infection, ICP0 is immediately imported into the nucleus. It is first detected at a dynamic nuclear structure termed nuclear domain 10 (ND10)7. The E3 ubiquitin ligase activity of ICP0 triggers the degradation of ND10 organizer proteins, promyelocytic leukemia (PML) protein, and speckled protein 100 kDa (Sp100)8,9,10. After the loss of organizer proteins, ND10 nuclear bodies are dispersed and ICP0 is diffused to fill the entire nucleus4,11.
Interestingly, after the onset of viral DNA replication, ICP0 disappears from the nucleus. It is solely found in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation late in HSV-1 infection4,12. The requirement of the DNA replication implies the potential involvement of a late viral protein(s) in facilitating the cytoplasmic translocation of HSV-1 ICP04,12. Apparently ICP0 trafficking among different compartments during infection empowers ICP0 to modulate its interactions to various cellular pathways in a spatial-temporal fashion, and therefore coordinate its multiple functions to fine tune the balance between the lytic and latent HSV-1 infection13. To better understand ICP0 multifunctionality and the coordination of ICP0 functional domains throughout the lytic infection, we carefully dissected the molecular basis of the dynamic ICP0 translocation12. To conduct the mechanistic studies previously reported12, we have applied an immunofluorescent staining method to visualize ICP0 subcellular localization at different infection status under confocal microscope. We have also developed a quantitative protocol to analyze the nuclear vs. cytoplasmic distribution of ICP0 using the confocal software. The population of HSV-1 infected cells was tabulated throughout the infection phases and the trends of ICP0 movement were analyzed, under different biochemical treatments12. Here we describe the detailed protocol that documents ICP0 translocation in HSV-1 infection. We propose that this method can be adopted as a general method to study the nuclear vs. cytoplasmic translocation for other viral or cellular proteins, which can serve as an alternative to live imaging when the live imaging technique is inapplicable due to problems such as labeling method, signal intensity, or protein abundance.

<table><tbody><tr><td>&lt;strong&gt;Cells and viruses&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Human Embryonic Lung fibroblasts (HEL Cells)</td><td>Dr. Thomas E. Shenk (Princeton University)</td><td></td><td>HEL cells were grown in DMEM supplemented with 10% FBS</td></tr><tr><td>HSV-1 viral Stock (Strain F)</td><td>Dr. Bernard Roizman Lab</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Medium&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM)</td><td>Invitrogen</td><td>&amp;nbsp;11965-092</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma</td><td>F0926-500ml</td><td></td></tr><tr><td>Medium-199 (10x)</td><td>Gibco</td><td>11825-015</td><td></td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>4- well 11 mm staggered slide</td><td>Cel-Line/Thermofisher Scientific</td><td>&amp;nbsp;30-149H-BLACK</td><td></td></tr><tr><td>16% Paraformaldehyde solution(w/v) Methanol free</td><td>Thermo Scientific</td><td>28908</td><td></td></tr><tr><td>Triton X-100</td><td>Fisher reagents</td><td>BP151-1C0</td><td></td></tr><tr><td>Bovine Serum Abumin (BSA)</td><td>Calbiochem</td><td>CAS 9048-46-8</td><td></td></tr><tr><td>Horse Serum</td><td>Sigma</td><td>H1270</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, pH7.4)</td><td>Dr. Haidong Gu lab</td><td></td><td></td></tr><tr><td>NaCl&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Fisher Bioreagent</td><td>BP358-212</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Fisher Bioreagent</td><td>BP362-500</td><td></td></tr><tr><td>KCl&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>BP366-500</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Fisher Bioreagent</td><td>BP332-500</td><td></td></tr><tr><td>Blocking buffer (PBS with 1% BSA and 5% Horse serum )</td><td>Dr. Haidong Gu lab</td><td></td><td></td></tr><tr><td>Rabiit Anti-ICP0 antibody</td><td>Dr. Haidong Gu lab</td><td></td><td></td></tr><tr><td>PML (PG-M3)-Mouse monoclonal IgG</td><td>santa Cruz Biotechnology</td><td>SC-966</td><td></td></tr><tr><td>Alexa Fluor 594-goat anti-rabbit IgG</td><td>invitrogen</td><td>A11012</td><td></td></tr><tr><td>Alexa Fluor 488-goat anti-mouse IgG</td><td>invitrogen</td><td>A11001</td><td></td></tr><tr><td>Vectashield Mouting medium with DAPI</td><td>Vector laboratories</td><td>H-1200</td><td></td></tr><tr><td>Pasteur pipette</td><td>Fisher Brand</td><td>13-678-20D</td><td></td></tr><tr><td>Nail Polish</td><td>Sally Hansen</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Confocal Microscope</td><td>Leica SP8</td><td></td><td></td></tr><tr><td>Confocal Software</td><td>Leica LAS X Application suite</td><td></td><td></td></tr><tr><td>Excel software</td><td>Microsoft Excel</td><td></td><td></td></tr><tr><td>HERAcell 150i CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Thermo Scientific</td><td>Order code 51026282</td><td></td></tr></tbody></table>

temporal analysis of the nuclear-to-cytoplasmic translocation of a herpes simplex virus 1 protein by immunofluorescent confocal microscopy

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

To understand the molecular basis and biological functions of ICP0 trafficking during HSV-1 infection, we use an immunofluorescent microscopy method to analyze ICP0 subcellular distribution at different infection phases. Figure 1 shows the representative cells with distinctive ICP0 localization as the infection progresses. To quantify the nuclear-to-cytoplasmic translocation of ICP0, we analyze ICP0 distribution relative to the nucleus by categorizing infecte...

Watch this Scientific Journal Video about Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy at JoVE.com

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is ...

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Immunology and Infection

6.2K Views.  Wayne State University. Deze methode kan helpen bij het beantwoorden van belangrijke vragen op het gebied van eiwithandel over nucleaire en cytoplasmische translocatie in het systeem waarbinnen geen lichtbeeldvorming beschikbaar is. Het belangrijkste voordeel van deze techniek is dat het kan worden toegepast als een algemeen protocol voor de studie van temporele eiwitbeweging zonder dat er lichtbeeldvorming is. 20 tot 24 uur voor de virusinfectie zaad 5 keer 10 tot de 4 menselijke embryonale long of HEL vezel blast ...

Video: Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread.

Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.

Advances in live cell fluorescence microscopy  techniques, as well as the construction of recombinant viral strains that  express fluorescent fusion ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Neuroscience

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Biology

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.

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).

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

Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-Dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells

The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical regulators of necroptosis, an inflammatory form of cell death with important antiviral functions. Autophosphorylation of RIPK3 induces phosphorylation and activation of the pore-forming executioner protein of necroptosis MLKL. Trafficking and oligomerization of phosphorylated MLKL at the cell membrane results in cell lysis, characteristic of necroptotic cell death. The nucleic acid sensor ZBP1 is activated by binding to left-handed Z-form double-stranded RNA (Z-RNA) after infection with RNA and DNA viruses. ZBP1 activation restricts virus infection by inducing regulated cell death, including necroptosis, of infected host cells. Immunofluorescence microscopy permits the visualization of different signaling steps downstream of ZBP1-mediated necroptosis on a per-cell basis. However, the sensitivity of standard fluorescence microscopy, using current commercially available phospho-specific antibodies against human RIPK3 and MLKL, precludes reproducible imaging of these markers. Here, we describe an optimized staining procedure for serine (S) phosphorylated RIPK3 (S227) and MLKL (S358) in human HT-29 cells infected with herpes simplex virus 1 (HSV-1). The inclusion of a tyramide signal amplification (TSA) step in the immunofluorescent staining protocol allows the specific detection of S227 phosphorylated RIPK3. Moreover, TSA greatly increases the sensitivity of the detection of S358 phosphorylated MLKL. Together, this method enables the visualization of these two critical signaling events during the induction of ZBP1-induced necroptosis.

Tyramide signal amplification during immunofluorescent staining enables the sensitive detection of phosphorylated RIPK3 and MLKL during ZBP1-induced necroptosis after HSV-1 infection.

The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical ...

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.

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 ...

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

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