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>Cells and viruses</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>Medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td>Invitrogen</td>
			<td>&#160;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>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4- well 11 mm staggered slide</td>
			<td>Cel-Line/Thermofisher Scientific</td>
			<td>&#160;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 Na2HPO4, pH7.4)</td>
			<td>Dr. Haidong Gu lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Fisher Bioreagent</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Fisher Bioreagent</td>
			<td>BP362-500</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</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>Equipment</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 CO2 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

6.1K Views.  Wayne State University. 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.

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

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1. These toxins are secreted into the extracellular milieu, but they act upon targets within the eukaryotic cytosol. Some AB toxins travel by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol2-4. In the ER, the catalytic A chain dissociates from the rest of the toxin and moves through a protein-conducting channel to reach its cytosolic target5. The translocated, cytosolic A chain is difficult to detect because toxin trafficking to the ER is an extremely inefficient process: most internalized toxin is routed to the lysosomes for degradation, so only a small fraction of surface-bound toxin reaches the Golgi apparatus and ER6-12.

To monitor toxin translocation from the ER to the cytosol in cultured cells, we combined a subcellular fractionation protocol with the highly sensitive detection method of surface plasmon resonance (SPR)13-15. The plasma membrane of toxin-treated cells is selectively permeabilized with digitonin, allowing collection of a cytosolic fraction which is subsequently perfused over an SPR sensor coated with an anti-toxin A chain antibody. The antibody-coated sensor can capture and detect pg/mL quantities of cytosolic toxin. With this protocol, it is possible to follow the kinetics of toxin entry into the cytosol and to characterize inhibitory effects on the translocation event. The concentration of cytosolic toxin can also be calculated from a standard curve generated with known quantities of A chain standards that have been perfused over the sensor. Our method represents a rapid, sensitive, and quantitative detection system that does not require radiolabeling or other modifications to the target toxin.

In this report, we describe how surface plasmon resonance is used to detect toxin entry into the host cytosol. This highly sensitive method can provide quantitative data on the amount of cytosolic toxin, and it can be applied to a range of toxins.

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1.  These toxins are secreted into the extracellular milieu, but they act upon ...

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

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 ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.