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

Wright

et al. 2014

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: Importancementioning

confidence: 99%

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

Wright

et al. 2014

“…On entry into the neuron under study, there is deenvelopment, producing a capsid (free of glycoproteins) that moves in the retrograde direction in axons, toward the nucleus (1). Second, most EM studies have reported the presence of relatively few HSV particles in axons, compared with EM studies of PRV (13,15,17,18), making interpretation of the results difficult.…”

mentioning

confidence: 99%

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

Wisner

Sugimoto

et al. 2011

Anterograde transport of herpes simplex virus (HSV) from neuronal cell bodies into, and down, axons is a fundamentally important process for spread to other hosts. Different techniques for imaging HSV in axons have produced two models for how virus particles are transported in axons. In the Separate model, viral nucleocapsids devoid of the viral envelope and membrane glycoproteins are transported in axons. In the Married model, enveloped HSV particles (with the viral glycoproteins) encased within membrane vesicles are transported in the anterograde direction. Earlier studies of HSV-infected human neurons involving electron microscopy (EM) and immunofluorescence staining of glycoproteins and capsids supported the Separate model. However, more-recent live-cell imaging of rat, chicken, and mouse neurons produced evidence supporting the Married model. In a recent EM study, a mixture of Married (75%) and Separate (25%) HSV particles was observed. Here, we studied an HSV recombinant expressing a fluorescent form of the viral glycoprotein gB and a fluorescent capsid protein (VP26), observing that human SK-N-SH neurons contained both Separate (the majority) and Married particles. Live-cell imaging of rat superior cervical ganglion (SCG) neuronal axons in a chamber system (which oriented the axons) also produced evidence of Separate and Married particles. Together, our results suggest that one can observe anterograde transport of both HSV capsids and enveloped virus particles depending on which neurons are cultured and how the neurons are imaged.Herpes simplex virus (HSV) and other alphaherpesviruses establish latency in the sensory nervous system. Periodic reactivation leads to the production of infectious virus in sensory ganglia, followed by virus transport in neuronal axons to epithelial tissues. Repetitive infection of the cornea causes scarring, which represents the major infectious cause of blindness. Anterograde transport (from neuronal cell bodies to axon termini) is a fundamentally important property of alphaherpesviruses, essential for long-term survival in the form of spread to other hosts. Early studies of HSV infection in human fetal neurons involving electron microscopy (EM), immuno-EM, and immunofluorescence analyses led to the conclusion that capsids are transported in the anterograde direction separately from vesicles containing viral glycoproteins (9, 17). More-recent studies from the same laboratory showed that there was virus envelopment (assembly of capsids with glycoproteins) at relatively numerous varicosities and at growth cones in cultured human neurons (18). Studies in our laboratory also supported what we termed the "Separate" model for HSV anterograde transport, i.e., transport of unenveloped capsids separately from viral glycoproteins (20)(21)(22). In this model, envelopment occurs at axon termini. In human neuroblastoma (SK-N-SH) cells differentiated to produce neurites, HSV glycoproteins stained with a panel of different antibodies (against gB, gD, gE, or gI) were observed as puncta that w...

“…Early electron microscopy (EM) studies produced evidence for Separate herpes simplex virus (HSV) capsids in human and rat neuronal axons (5)(6)(7). Other, more recent EM studies observed a mixture of Separate capsids (25%) and Married particles for two HSV strains (8), but this ratio was reversed, so that 70% of the particles in axons were Separate particles with another HSV strain (T. Mettenleiter, personal communication). Our antibody staining of HSV-infected human neuroblastoma cells produced evidence for mainly Separate capsids and distinct glycoprotein-containing vesicles (4,9,10).…”

mentioning

confidence: 99%

Herpes Simplex Virus Membrane Proteins gE/gI and US9 Act Cooperatively To Promote Transport of Capsids and Glycoproteins from Neuron Cell Bodies into Initial Axon Segments

Johnson

2013

Herpes simplex virus (HSV) and other alphaherpesviruses must move from sites of latency in ganglia to peripheral epithelial cells. How HSV navigates in neuronal axons is not well understood. Two HSV membrane proteins, gE/gI and US9, are key to understanding the processes by which viral glycoproteins, unenveloped capsids, and enveloped virions are transported toward axon tips. Whether gE/gI and US9 function to promote the loading of viral proteins onto microtubule motors in neuron cell bodies or to tether viral proteins onto microtubule motors within axons is not clear. A lphaherpesviruses depend upon highly evolved mechanisms to move from mucosal epithelial tissues within neuronal axons to ganglia where latency is established. Following reactivation from latency, virus particles move from ganglia back to peripheral tissues for spread to other hosts. This anterograde transport involves fast axon transport involving microtubules and kinesin motors that propel viral particles from neuron cell bodies (in ganglia) over large distances to axon tips.Depending upon the strain of alphaherpesvirus and the type of neuron, anterograde transport can apparently involve either fully assembled virions or unenveloped capsids (reviewed in references1, 2, and3). Fully assembled, enveloped virions or "Married" particles (4) are produced by capsid envelopment in the cytoplasm of neuron cell bodies, while "Separate" (4) unenveloped capsids (lacking viral glycoproteins) become enveloped at or near axon tips. Early electron microscopy (EM) studies produced evidence for Separate herpes simplex virus (HSV) capsids in human and rat neuronal axons (5-7). Other, more recent EM studies observed a mixture of Separate capsids (25%) and Married particles for two HSV strains (8), but this ratio was reversed, so that 70% of the particles in axons were Separate particles with another HSV strain (T. Mettenleiter, personal communication). Our antibody staining of HSV-infected human neuroblastoma cells produced evidence for mainly Separate capsids and distinct glycoprotein-containing vesicles (4, 9, 10). EM and fluorescent protein analyses of pig pseudorabies virus (PRV) strongly support only Married transport (11)(12)(13)(14). A study involving a "two-color" HSV recombinant expressing a fluorescent glycoprotein and capsids concluded that most HSV anterograde transport involved Married particles (15). Using another "two-color" HSV recombinant expressing fluorescent capsids and glycoproteins gB, we concluded that a majority of capsids moving in rat superior cervical ganglion (SCG) neurons were Separate particles (60%) (16). Thus, we believe that both modes of transport are possible and, in fact, occur.HSV and PRV express two membrane proteins, gE/gI and US9, which are key to the understanding of anterograde transport in neuronal axons (reviewed in references 2 and3). gE/gI is a heterodimer, with both gE and gI required for function, and possesses both substantial extracellular domains and ϳ100-amino-acid (aa) cytoplasmic domains with acidic clusters, ...