Name: temporal analysis of the nuclear-to-cytoplasmic translocation of a herpes simplex virus 1 protein by immunofluorescent confocal microscopy
Uploaded: 2018-11-04T14:00:00
Description: 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

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, re...

<ol>
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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. 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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>

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 ...

This method can help answer key questions in the protein trafficking field about nuclear and cytoplasmic translocation in system within which light imaging is not available. The main advantage of this technique is that it can be applied as a general protocol for the study of temporal protein movement without involving light imaging. 20 to 24 hours before the virus infection seed 5 times 10 to the 4 human embryonic lung or HEL fiber blast cells per well onto a four well 11 millimeter staggered slide and growth medium for overnight incubation at 37 degrees Celsius with five percent carbon dioxide.It is important to rock the slide thoroughly to ensure an even distribution of the cells while taking care to avoid spilling the culture medium. The next day, replace the supernatants so the 70 to 80 percent confluent cultures with Medium 199, containing 410 plaque-forming units per cell, and place the slide at 37 degrees Celsius with shaking, for one hour. At the end of the incubation, replace the supernatants with fresh growth medium and return the cells to the incubator for the appropriate experimental infection period.For immunofluorescent staining of the virus-infected cells, quickly wash the cells three times with PBS. Followed by an eight to ten-minute incubation in 200 microliters of four percent paraformaldehyde in PBS per well. At the end of the fixation, wash the cells three times with 200 microliters of PBS per wash, and permeabilize the cells with 100 microliters of 0.2 percent non-ionic surfactant per well for five to ten minutes.Wash the permeabilized cells three times in PBS as demonstrated, and add 200 microliters of blocking buffer to each well for a one-hour incubation at room temperature. Next, add the appropriate concentration of the primary antibody of interest to each well for a two-hour room-temperature incubation. At the end of the incubation, remove any unbound primary antibody with three, ten-minute washes in fresh blocking buffer, and add an appropriate secondary antibody to each well for a one-hour incubation at room temperature, protected from light.At the end of the incubation, wash the cells three times in a blocking buffer as demonstrated, followed by one wash in PBS and add one drop of antifade mounting medium supplemented with DAPI to each well. Then place a cover slip over the slide and seal the cover slope with transparent nail polish. Applying gentle pressure while mounting the cover slip will remove any excess mounting medium, helping to ensure helping to ensure the acquisition of high-quality images.For confocal imaging of the labeled, infected cells, select the appropriate secondary antibody and DAPI wavelengths, and set the image format to 1, 024 by 1, 024 pixels with an average line of eight. Then image each well on the four-well slide under the 100x objective. For counting a large number of cells, obtain five to ten images from consecutive fields in the same well under the 40x objective.To analyze the nuclear and cytoplasmic distribution of ICP0, open the project in the confocal application software and select an image. Open the Quantity tab, and select Sort Regions of Interest from the Tools menu. Select Draw Line, and draw a longitudinal line across the cell to be analyzed.A histogram will appear showing the fluorescence intensity along the line for both the protein of interest, and DAPI. Based on the background staining, set up a constant threshold for the intensity of the protein of interest for analysis of the sub-cellular distribution of the protein in each experiment. If the protein signal, on average, is below the threshold in the nuclear region, but is above the threshold beyond the DAPI boundary, categorize the protein signal as predominantly located within the cytoplasm.If the protein signal is above the threshold throughout the nucleus, and beyond the boundary of the DAPI signal, group the protein signal as a nucleus plus cytoplasmic localization. If the protein signal is above the threshold in the nucleus, but on average is below the threshold outside the boundary of the DAPI signal group the protein signal as a nuclear localization. Finally, tabulate more than 200 infected cells from each sample at different infection time points, and plot the data in a bar graph to illustrate the movement of the protein of interest over time.As demonstrated, this method facilitates the analysis of the nuclear-to-cytoplasmic translocation of proteins of interest. For example, infected cell protein zero, or ICP0 subcellular distribution changes as a viral infection progresses. To understand the elements required for ICP0 trafficking during infection, ICP0 movements can be tracked in Wild Type and Mutant Type Herpes Simplex Virus 1-infected cells at different infection phases, allowing the evaluation of the sub-cellular distribution of ICP0 at different time-points of infection.It is important to remember to use monolayer cultures so the virions have equal access to the cells and to normalize the multiplicity of infection according to the virus title so that infection progression is comparable between and within viruses. After each development, this technique paved the way for researchers in the fields of biology and cell virology to explore protein movement by studying the temporal subcellular localizations of proteins of interest within a cell population.

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