A Microfluidic Chamber for Analysis of Neuron-to-Cell Spread and Axonal Transport of an Alpha-Herpesvirus

Liu, Wendy W.; Goodhouse, Joseph; Jeon, Noo Li; Enquist, Lynn W.

doi:10.1371/journal.pone.0002382

Cited by 83 publications

(96 citation statements)

References 60 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Inoculation of animals with PRV strains lacking the viral membrane gB results in infections that remain limited to the first-order neuron; i.e., transneuronal spread of the virus does not occur (2,21). In corroboration of these observations, in vitro studies demonstrate that neuron-to-cell spread of PRV requires gB (17,27). We used compartmented neuronal cultures and imaging techniques to identify the gB-mediated step in transneuronal PRV spread.…”

Section: Discussionmentioning

confidence: 70%

“…gB is required for the transneuronal spread of PRV. This requirement has been demonstrated previously in vivo and in vitro, but the gB-mediated step in the process has not been identified (2,17,21,27). The viral membrane proteins Us9, gE, and gI are also required for the transneuronal spread of PRV and have been shown to mediate the axonal sorting of newly synthesized virions.…”

Section: Resultsmentioning

confidence: 83%

See 1 more Smart Citation

Virion-Incorporated Glycoprotein B Mediates Transneuronal Spread of Pseudorabies Virus

Curanovic

Enquist

2009

J Virol

View full text Add to dashboard Cite

Transneuronal spread of pseudorabies virus (PRV) is a multistep process that requires several virally encoded proteins. Previous studies have shown that PRV glycoprotein B (gB), a component of the viral fusion machinery, is required for the transmission of infection to postsynaptic, second-order neurons. We sought to identify the gB-mediated step in viral transmission. We determined that gB is not required for the sorting of virions into axons of infected neurons, anterograde transport, or the release of virions from the axon. trans or cis expression of gB on the cell surface was not sufficient for transneuronal spread of the virus; instead, efficient incorporation of gB into virions was required. Additionally, neuron-to-cell spread of PRV most likely does not proceed through syncytial connections. We conclude that, upon gB-independent release of virions at the site of neuron-cell contacts, the virion-incorporated gB/gH/gL fusion complex mediates entry into the axonally contacted cell by fusion of the closely apposed membranes.Alphaherpesviruses, which constitute a subfamily of the family Herpesviridae, include the human pathogens herpes simplex virus (HSV) and varicella-zoster virus and the swine pathogen pseudorabies virus (PRV). These closely related pantropic, neuroinvasive viruses establish latency in the peripheral nervous systems of their natural hosts. During the normal course of infection, periodic viral reactivation leads to recurrent epithelial lesions (38). Although rare in the natural host, transneuronal spread of the virus from the peripheral to the central nervous system (CNS) results in death or debilitating disease, such as encephalitis or keratitis (50). Nonnatural hosts infected with PRV almost invariably experience viral spread to the CNS and succumb to infection (36).Transneuronal spread of alphaherpesviruses is an incompletely understood multistep process that requires the concerted action of viral and cellular proteins. Following replication in the soma of an infected neuron, viral progeny may spread in the retrograde direction to the presynaptic cell or anterogradely to the postsynaptic cell. During anterograde spread of PRV, virus particles are sorted from the neuronal soma into the cognate axon. Upon entering the axonal compartment, virions are transported in a microtubule-dependent manner toward the synaptically connected cell (41). Recent in vitro evidence suggests that boutons en passant and axon termini serve as sites for PRV spread from the axon (13). Additionally, in vivo experiments demonstrate that the transneuronal spread of alphaherpesviruses is remarkably specific, occurring only between synaptically connected cells (15). This property has made alphaherpesviruses invaluable as neural circuit tracers in studies that aim to map the synaptic architecture of the CNS (14). However, the mechanisms that confer such specificity on the spread of infection are not well understood.The study of mechanisms underlying PRV trafficking revealed that the virally encoded membrane proteins Us9,...

show abstract

Section: Discussionmentioning

confidence: 70%

Section: Resultsmentioning

confidence: 83%

Virion-Incorporated Glycoprotein B Mediates Transneuronal Spread of Pseudorabies Virus

Curanovic

Enquist

2009

J Virol

View full text Add to dashboard Cite

show abstract

“…For PrV, it has now been largely accepted that enveloped virions within vesicles constitute the most abundant, if not exclusive, cargo for anterograde intraaxonal transport following high-resolution ultrastructural electron microscopi-cal studies (5,13,28), as well as live-cell analysis by video microscopy of fluorescently labeled virions (2,3,25) and reinterpretation of earlier results (5,8,13,41).…”

mentioning

confidence: 83%

Ultrastructural Analysis of Virion Formation and Intraaxonal Transport of Herpes Simplex Virus Type 1 in Primary Rat Neurons

Negatsch

Granzow

Maresch

et al. 2010

J Virol

View full text Add to dashboard Cite

After primary replication at the site of entry into the host, alphaherpesviruses infect and establish latency in neurons. To this end, they are transported within axons retrograde from the periphery to the cell body for replication and in an anterograde direction to synapses for infection of higher-order neurons or back to the periphery. Retrograde transport of incoming nucleocapsids is well documented. In contrast, there is still significant controversy on the mode of anterograde transport. By high-resolution transmission electron microscopy of primary neuronal cultures from embryonic rat superior cervical ganglia infected by pseudorabies virus (PrV), we observed the presence of enveloped virions in axons within vesicles supporting the "married model" of anterograde transport of complete virus particles within vesicles (C. Maresch, H. Granzow, A. Negatsch, B.G. Klupp, W. Fuchs, J.P. Teifke, and T.C. Mettenleiter, J. Virol. 84:5528-5539, 2010). We have now extended these analyses to the related human herpes simplex virus type 1 (HSV-1). We have demonstrated that in neurons infected by HSV-1 strains HFEM, 17؉ or SC16, approximately 75% of virus particles observed intraaxonally or in growth cones late after infection constitute enveloped virions within vesicles, whereas approximately 25% present as naked capsids. In general, the number of HSV-1 particles in the axons was significantly less than that observed after PrV infection.Herpesviruses are characterized by a distinct virion morphology and the property to establish latent infections with episodes of spontaneous reactivation. Herpesvirus virions contain a DNA genome enclosed in an icosahedral capsid shell, which is in turn embedded in tegument proteins and surrounded by a lipid envelope containing virally encoded, mostly glycosylated proteins. Within the Herpesviridae, three subfamilies, designated the Alpha-, Beta-, and Gammaherpesvirinae, have been recognized (9). The alphaherpesviruses contain pathogens of humans and animals with neuroinvasive properties resulting in infection of and latency in neurons. The genus Simplexvirus encompasses the ubiquitous human herpes simplex viruses, types 1 and 2 (HSV-1 and HSV-2), whereas varicella-zoster virus and several relevant animal pathogens, e.g., the porcine pseudorabies virus (PrV) (Suid herpesvirus 1 [30]), belong to the genus Varicellovirus.Alphaherpesviruses are pantropic but neuroinvasive, i.e., they infect the nervous system after primary replication in mucosal membranes. Neuroinvasion entails two long-distance transport processes of different directionalities (3). There is general consent that retrograde intraaxonal transport of incoming alphaherpesvirus particles to the neuronal cell body for productive replication or establishment of latent infection is effected by dynein-mediated microtubule-associated transport of nucleocapsids coated with "inner" tegument proteins (1,14,26), as occurs during infection of nonpolarized cultured cells (reviewed in reference 39). After reactivation from latency, anterograde...

show abstract

“…Nevertheless the presented device is 10-100-fold more economical with cell material than other compartmentalized systems. [10][11][12][13][14][15][16][17][18][19][20]39 This also highlights the advantage of using aspiration for cell arraying, a process which can readily manage minimal cell suspension volumes (e.g. 1 mL).…”

Section: Microfluidic Arraying With Single Neuron Precisionmentioning

confidence: 97%

Microfluidic construction of minimalistic neuronal co-cultures

et al. 2013

View full text Add to dashboard Cite

In this paper we present compartmentalized neuron arraying (CNA) microfluidic circuits for the preparation of neuronal networks using minimal cellular inputs (10-100-fold less than existing systems).The approach combines the benefits of microfluidics for precision single cell handling with biomaterial patterning for the long term maintenance of neuronal arrangements. A differential flow principle was used for cell metering and loading along linear arrays. An innovative water masking technique was developed for the inclusion of aligned biomaterial patterns within the microfluidic environment. For patterning primary neurons the technique involved the use of meniscus-pinning micropillars to align a water mask for plasma stencilling a poly-amine coating. The approach was extended for patterning the human SH-SY5Y neuroblastoma cell line using a poly(ethylene glycol) (PEG) back-fill and for dopaminergic LUHMES neuronal precursors by the further addition of a fibronectin coating. The patterning efficiency E patt was .75% during lengthy in chip culture, with y85% of the outgrowth channels occupied by neurites. Neurons were also cultured in next generation circuits which enable neurite guidance into all outgrowth channels for the formation of extensive inter-compartment networks. Fluidic isolation protocols were developed for the rapid and sustained treatment of the different cellular and sub-cellular compartments. In summary, this research demonstrates widely applicable microfluidic methods for the construction of compartmentalized brain models with single cell precision. These minimalistic ex vivo tissue constructs pave the way for high throughput experimentation to gain deeper insights into pathological processes such as Alzheimer and Parkinson Diseases, as well as neuronal development and function in health.

show abstract

A Microfluidic Chamber for Analysis of Neuron-to-Cell Spread and Axonal Transport of an Alpha-Herpesvirus

Cited by 83 publications

References 60 publications

Virion-Incorporated Glycoprotein B Mediates Transneuronal Spread of Pseudorabies Virus

Virion-Incorporated Glycoprotein B Mediates Transneuronal Spread of Pseudorabies Virus

Ultrastructural Analysis of Virion Formation and Intraaxonal Transport of Herpes Simplex Virus Type 1 in Primary Rat Neurons

Microfluidic construction of minimalistic neuronal co-cultures

Contact Info

Product

Resources

About