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

Abstract

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, ...

The Basic Domain of Herpes Simplex Virus 1 pUS9 Recruits Kinesin-1 To Facilitate Egress from Neurons

“…Similar observations have been reported for an HSV-1 pUS9 full deletion mutant (11). Our current findings with the three-chamber system using an HSV-1 pUS9 basic domain mutant also are similar to our observations with a complete deletion of pUS9 (53).…”

“…First, the viral protein facilitates sorting into the proximal axon by promoting the loading of viral components onto kinesin motors in the neuronal cell body. Second, the viral protein binds kinesin directly or via an adaptor to mediate anterograde transport of viral components toward axon tips (11). Our identified interaction of kinesin-1 and the pUS9 basic domain most likely plays a functional role in the context of the motor-adaptor scenario to directly mediate axonal anterograde transport.…”

“…For PrV there is also evidence for pUS9-independent anterograde axonal transport of some viral components (8, 9). In HSV-1 the contribution of pUS9 to these processes is less clearly defined (10-13), although a cooperative role for HSV-1 pUS9 and the viral glycoproteins gE/gI in axonal sorting and/or anterograde axonal transport has been documented (11,12,14).…”

“…For PrV there is also evidence for pUS9-independent anterograde axonal transport of some viral components (8, 9). In HSV-1 the contribution of pUS9 to these processes is less clearly defined (10-13), although a cooperative role for HSV-1 pUS9 and the viral glycoproteins gE/gI in axonal sorting and/or anterograde axonal transport has been documented (11,12,14).In the case of PrV, pUS9 has been shown to interact with the host anterograde molecular motor kinesin-3 (KIF1A) to mediate axonal sorting and anterograde axonal transport (15). The interaction of PrV pUS9 with kinesin-3 depends on the presence of the additional viral glycoproteins gE and gI (16).…”

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

“…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.…”