We thank the reviewers for their thoughtful suggestions that helped to improve this manuscript. This work was supported by grants to MVC (NS21072, HD23315), ACW (GM61139, S10RR017970) and IM (AI073898, GM056927) from the NIH. MK was supported in part by an NIH training grant (5T32 AI007180).

1. Isolation and Culture of SCG Neurons from Rat Embryos
To provide a useful context for understanding this protocol, and for a comprehensive discussion of earlier literature that established methods of SCG neuron culture, including the basis for SCG in vitro culture, plate-coating substrates, and the components of serum-free media, the reader is referred to references2-4.
<ol>
 <li>The use of rats as a source for SCG neurons was conducted in accordance with NIH ...

<ol>
	<li>Camarena, V., Kobayashi, M., Kim, J. Y., Roehm, P., Perez, R., Gardner, J., Wilson, A. C., Mohr, I., Chao, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+and+duration+of+growth+factor+signaling+through+receptor+tyrosine+kinases+regulates+HSV-1+latency+in+neurons.">Nature and duration of growth factor signaling through receptor tyrosine kinases regulates HSV-1 latency in neurons.</a> Cell Host &#38; Microbe. 8, 320-330 (2010).</li><li>Johnson, M. I., Fedoroff, S., Richardson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+cultures+of+sympathetic+ganglia.">Primary cultures of sympathetic ganglia.</a> Protocols for Neural Cell Culture. , 71-94 (2001).</li><li>Letourneau, P. C., Fedoroff, S., Richardson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+substrata+for+in+vitro+culture+of+neurons.">Preparation of substrata for in vitro culture of neurons.</a> Protocols for Neural Cell Culture. , 245-254 (2001).</li><li>Price, J. P., Brewer, G. J., Fedoroff, S., Richardson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+media+for+neural+cell+cultures.">Serum-free media for neural cell cultures.</a> Protocols for Neural Cell Culture. , 255-264 (2001).</li><li>Flint, S. J., Enquist, L. W., Racaniello, V. R., Skalka, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of virology. , (2008).</li><li>Roizman, B., Pellett, P. E., Knipe, D. M., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+family+Herpesviridae:+A+brief+introduction.">The family Herpesviridae: A brief introduction.</a> Fields Virology. 2, 2381-2397 (2001).</li><li>Price, R. W., Rubenstein, R., Khan, A. <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+infection+of+isolated+autonomic+neurons+in+culture:+viral+replication+and+spread+in+a+neuronal+network.">Herpes simplex virus infection of isolated autonomic neurons in culture: viral replication and spread in a neuronal network.</a> Arch. Virol. 71, 127-140 (1982).</li><li>Tomishima, M. J., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+alpha-herpesvirus+protein+necessary+for+axonal+localization+of+viral+membrane+proteins.">A conserved alpha-herpesvirus protein necessary for axonal localization of viral membrane proteins.</a> J. Cell Biol. 154, 741-752 (2001).</li><li>Ch'ng, T. H., Flood, E. A., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+primary+and+transformed+neuronal+cells+for+studying+pseudorabies+virus+infection.">Culturing primary and transformed neuronal cells for studying pseudorabies virus infection.</a> Methods Mol. Biol. 292, 299-316 (2005).</li><li>Wang, F., Tang, W., McGraw, H. M., Bennett, J., Enquist, L. W., Friedman, H. M. <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+type+1+glycoprotein+E+is+required+for+axonal+localization+of+capsid,+tegument,+and+membrane+glycoproteins.">Herpes simplex virus type 1 glycoprotein E is required for axonal localization of capsid, tegument, and membrane glycoproteins.</a> J. Virol. 79, 13362-13372 (2005).</li><li>Benboudjema, L., Mulvey, M., Gao, Y., Pimplikar, S. W., Mohr, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+the+herpes+simplex+virus+type+1+us11+gene+product+with+the+cellular+kinesin+light-chain-related+protein+PAT1+results+in+the+redistribution+of+both+polypeptides.">Association of the herpes simplex virus type 1 us11 gene product with the cellular kinesin light-chain-related protein PAT1 results in the redistribution of both polypeptides.</a> J. Virol. 77, 9192-9203 (2003).</li><li>Blaho, J., Morton, E. R., Yedowitz, J. C. <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:+propagation,+quantification+and+storage.">Herpes simplex virus: propagation, quantification and storage.</a> Curr. Protoc. Microbiol. Chapter 14, Unit 14E.1 (2005).</li><li>Van Zeijl, M., Fairhurst, J., Jones, T. R., Vernon, S. K., Morin, J., LaRocque, J., Feld, B. L., O'Hara, B. L., Bloom, J. D., Johann, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+class+of+thiourea+compounds+that+inhibit+herpes+simplex+virus+type+1+DNA+cleavage+and+encapsidation:+resistance+maps+to+the+UL6+gene.">Novel class of thiourea compounds that inhibit herpes simplex virus type 1 DNA cleavage and encapsidation: resistance maps to the UL6 gene.</a> J. Virol. 74, 9054-9061 (2000).</li><li>Newcomb, W. W., Brown, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+herpes+simplex+virus+replication+by+WAY-150138:+assembly+of+capsids+depleted+of+the+portal+and+terminase+proteins+involved+in+DNA+encapsidation.">Inhibition of herpes simplex virus replication by WAY-150138: assembly of capsids depleted of the portal and terminase proteins involved in DNA encapsidation.</a> J. Virol. 76, 10084-10088 (2002).</li><li>Pesola, J. M., Zhu, J., Knipe, D. M., Coen, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+1+immediate-early+and+early+gene+expression+during+reactivation+from+latency+under+conditions+that+prevent+infectious+virus+production.">Herpes simplex virus 1 immediate-early and early gene expression during reactivation from latency under conditions that prevent infectious virus production.</a> J. Virol. 79, 4516-14525 (2005).</li><li>Arthur, J. L., Scarpini, C. G., Connor, V., Lachmann, R. H., Tolkovsky, A. M., Efstathiou, S. <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+type+1+promoter+activity+during+latency+establishment,+maintenance+and+reactivation+in+primary+dorsal+root+neurons+in+vitro.">Herpes simplex virus type 1 promoter activity during latency establishment, maintenance and reactivation in primary dorsal root neurons in vitro.</a> J. Virol. 75, 3885-3895 (2001).</li><li>Danaher, R. J., Jacob, R. J., Steiner, M. R., Allen, W. R., Hill, J. M., Miller, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+deacetylase+inhibitors+induce+reactivation+of+herpes+simplex+virus+type+1+in+a+latency-associated+transcript-+independent+manner+in+neuronal+cells.">Histone deacetylase inhibitors induce reactivation of herpes simplex virus type 1 in a latency-associated transcript- independent manner in neuronal cells.</a> J. Neurovirol. 11, 306-317 (2005).</li><li>Terry-Allison, T., Smith, C. A., DeLuca, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relaxed+repression+of+herpes+simplex+virus+type+1+genomes+in+murine+trigenminal+neurons.">Relaxed repression of herpes simplex virus type 1 genomes in murine trigenminal neurons.</a> J. Virol. 71, 12394-12405 (2007).</li><li>Harris, R. A., Preston, 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=Establishment+of+latency+in+vitro+by+the+herpes+virus+type+1+mutant+in1918.">Establishment of latency in vitro by the herpes virus type 1 mutant in1918.</a> J. Gen. Virol. 72, 907-913 (1991).</li><li>Wagner, E. K., Bloom, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+investigation+of+herpes+simplex+virus+latency.">Experimental investigation of herpes simplex virus latency.</a> Clin. Microbiol. Rev. 10, 419-443 (1997).</li><li>Strelow, L. I., Laycock, K. A., Jun, P. Y., Rader, K. A., Brady, R. H., Miller, J. K., Pepose, J. S., Leib, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+structural+and+functional+comparison+of+the+latency-associated+transcript+promoters+of+herpes+simplex+virus+type+1+strains+KOS+and+McKrae.">A structural and functional comparison of the latency-associated transcript promoters of herpes simplex virus type 1 strains KOS and McKrae.</a> J. Gen Virol. 75, 2475-2480 (1994).</li><li>Stroop, W. G., Banks, M. C. <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+type+1+strain+KOS-63+does+not+cause+acute+or+recurrent+ocular+disease+and+does+not+reactivate+ganglionic+latency+in+vivo.">Herpes simplex virus type 1 strain KOS-63 does not cause acute or recurrent ocular disease and does not reactivate ganglionic latency in vivo.</a> Acta Neuropathol. 87, 14-22 (1994).</li><li>Sawtell, N. M., Poon, D. K., Tansky, C. S., Thompson, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+latent+herpes+simplex+virus+type+1+genome+copy+number+in+individual+neurons+is+virus+strain+specific+and+correlates+with+reactivation.">The latent herpes simplex virus type 1 genome copy number in individual neurons is virus strain specific and correlates with reactivation.</a> J. Virol. 72, 5343-5350 (1998).</li><li>Thompson, R. L., Cook, M. L., Devi-Rao, G., Wagner, E. K., Stevens, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+molecular+analysis+of+the+avirulent+wild-type+herpes+simplex+virus+type+1+strain+KOS.">Functional and molecular analysis of the avirulent wild-type herpes simplex virus type 1 strain KOS.</a> J. Virol. 58, 203-211 (1986).</li><li>Wilcox, C. L., Smith, R. L., Freed, C. R., Johnson, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+growth+factor-dependence+of+herpes+simplex+virus+latency+in+peripheral+sympathetic+and+sensory+neurons+in+vitro.">Nerve growth factor-dependence of herpes simplex virus latency in peripheral sympathetic and sensory neurons in vitro.</a> J. Neurosci. 10, 1268-1275 (1990).</li><li>Roehm, P. C., Camarena, V., Gardner, J. B., Wilson, A. C., Mohr, I., Chao, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultured+vestibular+ganglion+neurons+demonstrate+latent+herpes+simplex+type+I+reactivation.">Cultured vestibular ganglion neurons demonstrate latent herpes simplex type I reactivation.</a> Laryngoscope. 121, 2268-2275 (2011).</li><li>Kuhn, M. A., Nayak, S., Camarena, V., Gardner, J., Wilson, A., Mohr, I., Chao, M. V., Roehm, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell+culture+model+of+facial+palsy+resulting+from+reactivation+of+latent+herpes+simplex+virus+type+1.">A cell culture model of facial palsy resulting from reactivation of latent herpes simplex virus type 1.</a> Otology &#38; Neurotology. , (2012).</li></ol>

This primary neuron culture and infection system provides a simple and effective method to explore the molecular mechanisms underlying HSV-1 latency and reactivation. The system faithfully recapitulates the accepted hallmarks of latency defined in both human infections and in live-animal models. When the virus is latent in the SCG cultures, infectious particles and viral lytic gene products cannot be detected. The only viral gene product detected in latently-infected neurons is the non-coding LAT trans...

<table border="1">
<tbody>
 <tr>
 <td>Reagent</td>
 <td>Company</td>
 <td>Catalog#</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>70&mu;m nylon filter( cell strainer)</td>
 <td>BD Biosciences</td>
 <td>352350</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1x Hank's Balanced Salt Solution (HBSS-/-) </td>
 <td>Invitrogen</td>
 <td>14175</td>
 <td>w/o CaCl2 and MgCl2</td>
 </tr>
 <tr>
 <td>1x Minimum Essential Media (MEM) </td>
 <td>Invitrogen</td>
 <td>11095-080</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>5-Fluoro-2'-deoxyuridine</td>
 <td>Sigma</td>
 <td>F0503</td>
 <td>prepare 20 mM stock in 1x MEM; store at -20&deg;C</td>
 </tr>
 <tr>
 <td>96-well flat well bottom TC plates</td>
 <td>Corning</td>
 <td>3599</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Acyclovir </td>
 <td>Calbiochem</td>
 <td>114798 </td>
 <td>prepare 31 mM stock in DMSO; store at -20&deg;C</td>
 </tr>
 <tr>
 <td>Aphidicolin </td>
 <td>Calbiochem</td>
 <td>178273 </td>
 <td>prepare 10 mM stock in DMSO; store at -20&deg;C</td>
 </tr>
 <tr>
 <td>B-27 Supplement </td>
 <td>Invitrogen</td>
 <td>17504-44</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Collagenase </td>
 <td>Sigma </td>
 <td>C2674</td>
 <td>prepare 10 mg/ml stock in HBSS-/-; store at -20&deg;C</td>
 </tr>
 <tr>
 <td>D-(+)-Glucose </td>
 <td>Sigma</td>
 <td>G6152</td>
 <td>prepare 40% stock in H2O; filter sterilize &amp; store at 4&deg;C</td>
 </tr>
 <tr>
 <td>L-Glutamine </td>
 <td>Invitrogen</td>
 <td>25030-081</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Laminin </td>
 <td>Sigma</td>
 <td>L2020</td>
 <td>prepare 1 mg/ml stock in H2O; quick-freeze 20 &mu;l aliquats &amp; store at -80&deg;C; dilute to 2 &mu;g/ml working conc. in sterile H2O</td>
 </tr>
 <tr>
 <td>Leibovit'z L-15 media </td>
 <td>Invitrogen</td>
 <td>11415</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nerve Growth Factor </td>
 <td>Harlan Laboratories</td>
 <td>BT.5017</td>
 <td>prepare 50 &mu;g/ml stock in HBSS-/-; store at -80&deg;C</td>
 </tr>
 <tr>
 <td>Neurobasal medium </td>
 <td>Invitrogen</td>
 <td>12348</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Phosphonoacetic acid (PAA) </td>
 <td>Sigma</td>
 <td>P6909 </td>
 <td>prepare 75 mg/ml stock in H2O; store at -20&deg;C</td>
 </tr>
 <tr>
 <td>Poly-D-lysine hydrobromide </td>
 <td>Sigma</td>
 <td>P0899 </td>
 <td>prepare 20 mg/ml stock in H2O; store at -20&deg;C</td>
 </tr>
 <tr>
 <td>Rat-tail collagen </td>
 <td>Millipore</td>
 <td>08-115</td>
 <td>Concentration varies with supply lot; store at 4&deg;C and dilute to 0.66 mg/ml working conc. in sterile H2O</td>
 </tr>
 <tr>
 <td>Trichostatin A </td>
 <td>Sigma</td>
 <td>T8552</td>
 <td>prepare 1 mM stock in DMSO; store at -20&deg;C</td>
 </tr>
 <tr>
 <td>Trypsin 2.5% </td>
 <td>Invitrogen</td>
 <td>15090-04</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation at JoVE.com

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

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 ...

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Immunology and Infection

17.2K 次观看.  New York University School of Medicine. 协议描述了一种高效和可重复性的模型系统研究单纯疱疹病毒1型（HSV-1）延迟和重新启动。该法采用同质交感神经神经元文化，并允许使用各种工具，包括RNA干扰和重组蛋白表达的病毒神经元相互作用的分子解剖。