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>
	<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>

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 ...

Research

JoVE Journal

Immunology and Infection

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 ...

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. ...

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 ...

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 ...

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 ...

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 ...

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 ...

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

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 ...

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

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 ...