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Journal of Virology, July 2007, p. 6846-6857, Vol. 81, No. 13
0022-538X/07/$08.00+0     doi:10.1128/JVI.00069-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

In Vitro Analysis of Transneuronal Spread of an Alphaherpesvirus Infection in Peripheral Nervous System Neurons{triangledown} ,{dagger}

B. Feierbach,1,2* M. Bisher,1 J. Goodhouse,1 and L. W. Enquist1

Department of Molecular Biology, Princeton, New Jersey 08544,1 Lewis-Sigler Institute for Integrative Genomics, Princeton, New Jersey 085442

Received 10 January 2007/ Accepted 11 April 2007

The neurotropic alphaherpesviruses invade and spread in the nervous system in a directional manner between synaptically connected neurons. Until now, this property has been studied only in living animals and has not been accessible to in vitro analysis. In this study, we describe an in vitro system in which cultured peripheral nervous system neurons are separated from their neuron targets by an isolator chamber ring. Using pseudorabies virus (PRV), an alphaherpesvirus capable of transneuronal spread in neural circuits of many animals, we have recapitulated in vitro all known genetic requirements for retrograde and anterograde transneuronal spread as determined previously in vivo. We show that in vitro transneuronal spread requires intact axons and the presence of the viral proteins gE, gI, and Us9. We also show that transneuronal spread is dependent on the viral glycoprotein gB, which is required for membrane fusion, but not on gD, which is required for extracellular spread. We demonstrate ultrastructural differences between anterograde- and retrograde-traveling virions. Finally, we show live imaging of dynamic fluorescent virion components in axons and postsynaptic target neurons.

* Corresponding author. Mailing address: 301 Schultz Building, Department of Molecular Biology, Princeton University, Princeton, NJ 08544. Phone: (609) 258-4990. Fax: (609) 258-1035. E-mail: becket{at}princeton.edu

{triangledown} Published ahead of print on 25 April 2007.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/.

Journal of Virology, July 2007, p. 6846-6857, Vol. 81, No. 13
0022-538X/07/$08.00+0     doi:10.1128/JVI.00069-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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