Anterograde Transport of Herpes Simplex Virus Capsids in Neurons by both Separate and Married Mechanisms

Wright

et al. 2014

J Virol

Self Cite

Following reactivation from latency, there are two distinct steps in the spread of herpes simplex virus (HSV) from infected neurons to epithelial cells: (i) anterograde axonal transport of virus particles from neuron bodies to axon tips and (ii) exocytosis and spread of extracellular virions across cell junctions into adjacent epithelial cells. The HSV heterodimeric glycoprotein gE/gI is important for anterograde axonal transport, and gE/gI cytoplasmic domains play important roles in sorting of virus particles into axons. However, the roles of the large (ϳ400-residue) gE/gI extracellular (ET) domains in both axonal transport and neuron-to-epithelial cell spread have not been characterized. Two gE mutants, gE-277 and gE-348, contain small insertions in the gE ET domain, fold normally, form gE/gI heterodimers, and are incorporated into virions. Both gE-277 and gE-348 did not function in anterograde axonal transport; there were markedly reduced numbers of viral capsids and glycoproteins compared with wildtype HSV. The defects in axonal transport were manifest in neuronal cell bodies, involving missorting of HSV capsids before entry into proximal axons. Although there were diminished numbers of mutant gE-348 capsids and glycoproteins in distal axons, there was efficient spread to adjacent epithelial cells, similar to wild-type HSV. In contrast, virus particles produced by HSV gE-277 spread poorly to epithelial cells, despite numbers of virus particles similar to those for HSV gE-348. These results genetically separate the two steps in HSV spread from neurons to epithelial cells and demonstrate that the gE/gI ET domains function in both processes. IMPORTANCEAn essential phase of the life cycle of herpes simplex virus (HSV) and other alphaherpesviruses is the capacity to reactivate from latency and then spread from infected neurons to epithelial tissues. This spread involves at least two steps: (i) anterograde transport to axon tips followed by (ii) exocytosis and extracellular spread from axons to epithelial cells. HSV gE/gI is a glycoprotein that facilitates this virus spread, although by poorly understood mechanisms. Here, we show that the extracellular (ET) domains of gE/gI promote the sorting of viral structural proteins into proximal axons to begin axonal transport. However, the gE/gI ET domains also participate in the extracellular spread from axon tips across cell junctions to epithelial cells. Understanding the molecular mechanisms involved in gE/gI-mediated sorting of virus particles into axons and extracellular spread to adjacent cells is fundamentally important for identifying novel targets to reduce alphaherpesvirus disease.A lphaherpesviruses, such as herpes simplex virus (HSV) and varicella-zoster virus (VZV), have evolved specialized mechanisms enabling virus spread in epithelial and neuronal tissues. Primary infection involves entry into skin or mucosal epithelial cells, followed by rapid virus spread between these cells. During this phase of virus replication and spread, viruses enter sensory...

Section: Hsv Mutants With Insertion Mutations In the Ge Et Domainmentioning

confidence: 79%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Herpes Simplex Virus gE/gI Extracellular Domains Promote Axonal Transport and Spread from Neurons to Epithelial Cells

Wright

et al. 2014

J Virol

Self Cite

“…It remains unclear to what degree naked and enveloped viral capsids are capable of microtubule association and movement and what is the relative importance of microtubules for the trafficking of each type of particle. The situation is particularly complex in neurons, where it remains contentious whether naked capsids (27), enveloped capsids, or both (28)(29)(30)(31)(32)(33)(34)(35) are capable of trafficking out of the cell body and along axons.…”

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confidence: 99%

Blocking ESCRT-Mediated Envelopment Inhibits Microtubule-Dependent Trafficking of Alphaherpesviruses In Vitro

Kharkwal

Smith

Wilson

2014

J Virol

Herpes simplex virus (HSV) and, as reported here, pseudorabies virus (PRV) utilize the ESCRT apparatus to drive cytoplasmic envelopment of their capsids. Here, we demonstrate that blocking ESCRT-mediated envelopment using the dominant-negative inhibitor Vps4A-EQ (Vps4A in which glutamate [E] at position 228 in the ATPase active site is replaced by a glutamine [Q]) reduced the ability of HSV and PRV particles to subsequently traffic along microtubules in vitro. HSV and PRV capsid-associated particles with bound green fluorescent protein (GFP)-labeled Vps4A-EQ were readily detected by fluorescence microscopy in cytoplasmic extracts of infected cells. These Vps4A-EQ-associated capsid-containing particles bound to microtubules in vitro but were unable to traffic along them. Using a PRV strain expressing a fluorescent capsid and a fluorescently tagged form of the envelope protein gD, we found that similar numbers of gD-positive and gD-negative capsid-associated particles accumulated in cytoplasmic extracts under our conditions. Both classes of PRV particle bound to microtubules in vitro with comparable efficiency, and similar results were obtained for HSV using anti-gD immunostaining. The gD-positive and gD-negative PRV capsids were both capable of trafficking along microtubules in vitro; however, motile gD-positive particles were less numerous and their trafficking was more sensitive to the inhibitory effects of Vps4A-EQ. We discuss our data in the context of microtubule-mediated trafficking of naked and enveloped alphaherpesvirus capsids. IMPORTANCEThe alphaherpesviruses include several important human pathogens. These viruses utilize microtubule-mediated transport to travel through the cell cytoplasm; however, the molecular mechanisms of trafficking are not well understood. In this study, we have used a cell-free system to examine the requirements for microtubule trafficking and have attempted to distinguish between the movement of so-called "naked" and membrane-associated cytoplasmic alphaherpesvirus capsids. Members of the Alphaherpesvirinae subfamily include herpes simplex virus type 1 (HSV-1) and HSV-2, varicella-zoster virus, and pseudorabies virus (PRV). Like all herpesviruses, members of this subfamily replicate their genomes and assemble DNApackaged capsids in the cell nucleus. It is generally accepted that capsids then bud into the inner nuclear membrane to generate perinuclear virions that subsequently fuse with the outer nuclear membrane to release mature nucleocapsids (also termed "naked" capsids) into the cytoplasm. Naked cytoplasmic herpes capsids subsequently undergo secondary envelopment at a postnuclear organelle to assemble the mature, infectious virion (1-4).By performing subcellular fractionation during a single synchronized wave of HSV egress, we showed that HSV capsids bypass the cis, medial, and trans compartments of the Golgi apparatus (5) and accumulate in a buoyant membrane fraction with the biochemical and antigenic properties of the trans-Golgi network (TGN) and endosomes (5, 6)...

Reviews in Medical Virology

Making the case: Married versus Separate models of alphaherpes virus anterograde transport in axons

Kratchmarov

Taylor

Enquist

2012

SUMMARY Alphaherpesvirus virions infect neurons and are transported in axons for long distance spread within the host nervous system. The assembly state of newly made herpesvirus particles during anterograde transport in axons is an essential question in alphaherpesvirus biology. The structure of the particle has remained both elusive and controversial for the past two decades, with conflicting evidence from EM, immunofluorescence, and live cell imaging studies. Two opposing models have been proposed—the Married and Separate Models. Under the Married Model, infectious virions are assembled in the neuronal cell body before sorting into axons and then traffic inside a transport vesicle. Conversely, the Separate Model postulates that vesicles containing viral membrane proteins are sorted into axons independent of capsids, with final assembly of mature virions occurring at a distant egress site. Recently, a complementary series of studies employing high‐resolution EM and live cell fluorescence microscopy have provided evidence consistent with the Married Model, whereas other studies offer evidence supporting the Separate Model. In this review, we compare and discuss the published data and attempt to reconcile divergent findings and interpretations as they relate to these models. Copyright © 2012 John Wiley & Sons, Ltd.