Analysis of HSV Viral Reactivation in Explants of Sensory Neurons

Jesse H. Arbuckle1, Anne‐Marie W. Turner1, Thomas M. Kristie1

1 Laboratory of Viral Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 14E.6
DOI:  10.1002/9780471729259.mc14e06s35
Online Posting Date:  November, 2014
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Abstract

As with all Herpesviruses, Herpes simplex virus (HSV) has both a lytic replication phase and a latency‐reactivation cycle. During lytic replication, there is an ordered cascade of viral gene expression that leads to the synthesis of infectious viral progeny. In contrast, latency is characterized by the lack of significant lytic gene expression and the absence of infectious virus. Reactivation from latency is characterized by the re‐entry of the virus into the lytic replication cycle and the production of recurrent disease. This unit describes the establishment of the mouse sensory neuron model of HSV‐1 latency‐reactivation as a useful in vivo system for the analysis of mechanisms involved in latency and reactivation. Assays including the determination of viral yields, immunohistochemical/immunofluorescent detection of viral antigens, and mRNA quantitation are used in experiments designed to investigate the network of cellular and viral proteins regulating HSV‐1 lytic infection, latency, and reactivation. © 2014 by John Wiley & Sons, Inc.

Keywords: Herpes simplex virus; latency model; corneal infection; trigeminal ganglion; small‐molecule inhibitors; viral titer; immunohistochemistry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Establishment of HSV‐1 Latency in the Mouse Model by Corneal Infection
  • Support Protocol 1: Preparing Virus Stocks
  • Basic Protocol 2: Isolation of Trigeminal Ganglia from the HSV‐1 Latency Mouse Model
  • Basic Protocol 3: Mouse Ganglia Explant‐Reactivation Model
  • Basic Protocol 4: Determining Viral Titer of Latently Infected Trigeminal Ganglia
  • Basic Protocol 5: Immunohistochemical Staining of HSV‐1 Infected Trigeminal Ganglia
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Establishment of HSV‐1 Latency in the Mouse Model by Corneal Infection

  Materials
  • Balb/c mice, 5‐ to 6‐weeks old, acclimated for 1 week
  • HSV‐1 strain F viral stock diluted in DMEM/1% fetal bovine serum (FBS; see protocol 2)
  • Avertin working solution (25 mg/ml; see recipe)
  • Animal cages
  • Absorbent paper
  • 1‐ml syringes with 26‐ to 27‐gauge needles
  • Syringes with 30‐gauge needle
  • Pipetman and plugged tips
  • Heat lamp

Support Protocol 1: Preparing Virus Stocks

  Materials
  • Vero cells (ATCC, cat. no. CCL‐81)
  • PBS
  • 0.25% trypsin‐EDTA
  • DMEM/5% FBS medium (see recipe)
  • HSV‐1 strain F stock
  • DMEM/1% FBS medium (see recipe)
  • 150 × 25 mm tissue culture dish(es)
  • Hemacytometer
  • Cell scraper (Sarstedt, cat. no. 83.1830)
  • 50‐ml conical centrifuge tube
  • Sonicator equipped with micro‐tip probe
  • Sterile 1.5‐ml tubes

Basic Protocol 2: Isolation of Trigeminal Ganglia from the HSV‐1 Latency Mouse Model

  Materials
  • HSV‐1 latently infected Balb/c mice (see protocol 1)
  • PBS
  • Absorbent paper
  • Dissecting board
  • Surgical pins
  • Iris/curved scissors (Vantage, cat. no. V95‐306, 10.5 cm)
  • Half‐curved eye dressing forceps (Vantage, cat. no. V918‐782, 10.2 cm)
  • Curved‐sharp tweezers (Sigma‐Aldrich, cat. no. T4787, Style #7)
  • Micro‐dissecting spring scissors (Roboz, cat. no. RS‐5658, 3.5 in.)
  • Tissue culture dish

Basic Protocol 3: Mouse Ganglia Explant‐Reactivation Model

  Materials
  • DMEM/10% FBS medium (see recipe)
  • HSV‐1 latently infected mouse trigeminal ganglia
  • Optional: Small‐molecule inhibitors, inhibitory or stimulatory compounds, or antibodies
  • 6‐well tissue culture dishes
  • Curved‐sharp tweezers (Sigma‐Aldrich, cat. no. T4787, Style #7)
  • Set of two surgical scalpels (Bard‐Parker, cat. no. 1310, size 10)

Basic Protocol 4: Determining Viral Titer of Latently Infected Trigeminal Ganglia

  Materials
  • Vero cells (ATCC, cat. no. CCL‐81)
  • PBS
  • 0.25% trypsin‐EDTA
  • DMEM/5% FBS medium (see recipe)
  • HSV‐1 infected mouse trigeminal ganglia (see Basic Protocols protocol 32 and protocol 43) or infected cell pellet ( protocol 2)
  • DMEM/10% FBS medium (see recipe)
  • DMEM/1% FBS medium (see recipe)
  • 1% low melting agarose/DMEM/5% FBS medium (see recipe)
  • 1% neutral red solution (see recipe)
  • 12‐well tissue culture dish(es)
  • Glass dounce homogenizer (Wheaton, cat. no. 357844, 0.1 ml; this size will hold up to 1 ml)
  • Sterile 1.5‐ml tubes
  • Sonicator equipped with micro‐tip probe
  • 42°C water bath
  • Light box or inverted microscope

Basic Protocol 5: Immunohistochemical Staining of HSV‐1 Infected Trigeminal Ganglia

  Materials
  • Mouse trigeminal ganglia infected with HSV‐1 (see Basic Protocols protocol 32 and protocol 43)
  • 4% paraformaldehyde in PBS (see recipe)
  • PBS
  • 50%, 70%, and 95% ethanol in PBS (see recipe)
  • Xylene
  • 0.01 M citric acid (pH 6.0) antigen unmasking solution (Vector Laboratories, cat. no. H‐3300)
  • 10% donkey serum (Jackson Immuno Research, cat. no. 017‐000‐121)
  • Primary antibodies
  • 0.02% Tween/PBS (see recipe)
  • Fluorescently conjugated secondary antibodies (Jackson Immuno Research)
  • DAPI Fluoromount‐G (SouthernBiotech, cat. no. 0100‐20)
  • Anti‐Neurofilament N200 (optional; Sigma‐Aldrich, cat. no. N0142)
  • Metal slide‐staining rack
  • Hot plate
  • Super PAP pen
  • Dark plastic box
  • Curved‐sharp tweezers (Sigma‐Aldrich, cat. no. T4787, Style #7)
  • Glass coverslips (size: 30 × 24 mm, thickness: 0.13 to 0.17 mm)
  • Superfrost Plus microscope slides (Fisher Scientific, cat. no. 12‐550‐15)
  • Additional reagents and equipment for paraffin embedding and sectioning (see unit and Zeller, ).
NOTE: Trigeminal ganglia can be removed and processed during stages of the initial lytic infection (1 to 14 days post‐infection) as well as during latency and reactivation stages.
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Figures

Videos

Literature Cited

Literature Cited
   Baird, N.L. , Yu, X. , Cohr, R.J. , and Gilden, D. 2013. Varicella zoster virus (VZV)‐human neuron interaction. Viruses 5:2106‐2115.
   Bertke, A.S. , Swanson, S.M. , Chen, J. , Imai, Y. , Kinchington, P.R. , and Margolis, T.P. 2011. A5‐positive primary sensory neurons are nonpermissive for productive infection with herpes simplex virus 1 in vitro. J. Virol. 85:6669‐6677.
   Burgos, J.S. , Ramirez, C. , Sastre, I. , Bullido, M.J. , and Valdivieso, F. 2002. Involvement of apolipoprotein E in the hematogenous route of herpes simplex virus type 1 to the central nervous system. J. Virol. 76:12394‐12398.
   Camarena, V. , Kobayashi, M. , Kim, J.Y. , Roehm, P. , Perez, R. , Gardner, J. , Wilson, A.C. , Mohr, I. , and Chao, M.V. 2010. Nature and duration of growth factor signaling through receptor tyrosine kinases regulates HSV‐1 latency in neurons. Cell Host Microbe 8:320‐330.
   Cook, S.D. , Paveloff, M.J. , Doucet, J.J. , Cottingham, A.J. , Sedarati, F. , and Hill, J.M. 1991. Ocular herpes simplex virus reactivation in mice latently infected with latency‐associated transcript mutants. Invest. Ophthalmol. Vis. Sci. 32:1558‐1561.
   Hafezi, W. , Lorentzen, E.U. , Eing, B.R. , Muller, M. , King, N.J. , Klupp, B. , Mettenleiter, T.C. , and Kuhn, J.E. 2012. Entry of herpes simplex virus type 1 (HSV‐1) into the distal axons of trigeminal neurons favors the onset of nonproductive, silent infection. PLoS Pathog. 8:e1002679.
   Harland, J. and Brown, S.M. 1998. HSV growth, preparation, and assay. In Herpes Simplex Virus Protocols ( S.M. Brown and A.R. MacLean , eds.) pp. 1‐8. Springer, New York.
   Hill, J.M. , Garza, H.H. Jr. , Helmy, M.F. , Cook, S.D. , Osborne, P.A. , Johnson, E.M. Jr. , Thompson, H.W. , Green, L.C. , O'Callaghan, R.J. , and Gebhardt, B.M. 1997. Nerve growth factor antibody stimulates reactivation of ocular herpes simplex virus type 1 in latently infected rabbits. J. Neurovirol. 3:206‐211.
   Knipe, D.M. and Cliffe, A. 2008. Chromatin control of herpes simplex virus lytic and latent infection. Nat. Rev. Microbiol. 6:211‐221.
   Kristie, T.M. , Liang, Y. , and Vogel, J.L. 2010. Control of alpha‐herpesvirus IE gene expression by HCF‐1 coupled chromatin modification activities. Biochim. Biophys. Acta 1799:257‐265.
   Liang, Y. , Vogel, J.L. , Narayanan, A. , Peng, H. , and Kristie, T.M. 2009. Inhibition of the histone demethylase LSD1 blocks alpha‐herpesvirus lytic replication and reactivation from latency. Nat. Med. 15:1312‐1317.
   Liang, Y. , Quenelle, D. , Vogel, J.L. , Mascaro, C. , Ortega, A. , and Kristie, T.M. 2013a. A novel selective LSD1/KDM1A inhibitor epigenetically blocks herpes simplex virus lytic replication and reactivation from latency. MBio 4:e00558‐00512.
   Liang, Y. , Vogel, J.L. , Arbuckle, J.H. , Rai, G. , Jadhav, A. , Simeonov, A. , Maloney, D.J. , and Kristie, T.M. 2013b. Targeting the JMJD2 histone demethylases to epigenetically control herpesvirus infection and reactivation from latency. Sci. Transl. Med. 5:167ra165.
   Markus, A. , Grigoryan, S. , Sloutskin, A. , Yee, M.B. , Zhu, H. , Yang, I.H. , Thakor, N.V. , Sarid, R. , Kinchington, P.R. , and Goldstein, R.S. 2011. Varicella‐zoster virus (VZV) infection of neurons derived from human embryonic stem cells: direct demonstration of axonal infection, transport of VZV, and productive neuronal infection. J. Virol. 85:6220‐6233.
   Roizman, B. , Knipe, D.M. , and Whitley, R.J. 2007. Herpes simplex viruses. In Fields Virology, 5th ed., Vol. 2 ( D.M. Knipe and P.M. Howley , eds.) pp. 2501‐2601. Lippincott Williams & Wilkins, Philadelphia.
   Sawtell, N.M. and Thompson, R.L. 1992. Rapid in vivo reactivation of herpes simplex virus in latently infected murine ganglionic neurons after transient hyperthermia. J. Virol. 66:2150‐2156.
   Shimeld, C. , Hill, T.J. , Blyth, W.A. , and Easty, D.L. 1990. Reactivation of latent infection and induction of recurrent herpetic eye disease in mice. J. Gen. Virol. 71:397‐404.
   Vann, V.R. and Atherton, S.S. 1991. Neural spread of herpes simplex virus after anterior chamber inoculation. Invest. Ophthalmol. Vis. Sci. 32:2462‐2472.
   Webre, J.M. , Hill, J.M. , Nolan, N.M. , Clement, C. , McFerrin, H.E. , Bhattacharjee, P.S. , Hsia, V. , Neumann, D.M. , Foster, T.P. , Lukiw, W.J. , and Thompson, H.W. 2012. Rabbit and mouse models of HSV‐1 latency, reactivation, and recurrent eye diseases. J. Biomed. Biotechnol. 2012:612316.
   Whitley, R. , Kimberlin, D.W. , and Prober, C.G. 2007. Pathogenesis and disease. In Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis. ( A. Arvin , G. Campadelli‐Fiume , E. Mocarski , P.S. Moore , B. Roizman , R. Whitley , and K. Yamanishi , eds.) pp 589‐601. Cambridge University Press, Cambridge.
   Wilcox, C.L. and Johnson, E.M. Jr. 1987. Nerve growth factor deprivation results in the reactivation of latent herpes simplex virus in vitro. J. Virol. 61:2311‐2315.
   Wilcox, C.L. and Johnson, E.M. Jr. 1988. Characterization of nerve growth factor‐dependent herpes simplex virus latency in neurons in vitro. J. Virol. 62:393‐399.
   Zeller, R. 1989. Fixation, embedding, and sectioning of tissues, embryos, and single cells. Curr. Protoc. Mol. Biol. 7:14.1.1‐14.1.8.
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