Herpes Simplex Virus gE/gI and US9 Promote both Envelopment and Sorting of Virus Particles in the Cytoplasm of Neurons, Two Processes That Precede Anterograde Transport in Axons

Abstract

Herpes simplex virus (HSV) anterograde transport in neuronal axons is vital, allowing spread from latently infected ganglia to epithelial tissues, where viral progeny are produced in numbers allowing spread to other hosts. The HSV membrane proteins gE/gI and US9 initiate the process of anterograde axonal transport, ensuring that virus particles are transported from the cytoplasm into the most proximal segments of axons. These proteins do not appear to be important once HSV is inside axons. We previously described HSV double mutants lacking both gE and US9 that failed to transport virus particles into axons. Here we show that gE US9 double mutants accumulate large quantities of unenveloped and partially enveloped capsids in neuronal cytoplasm. These defects in envelopment can explain the defects in axonal transport of enveloped virions. In addition, the unenveloped capsids that accumulated were frequently bound to cytoplasmic membranes, apparently immobilized in intermediate stages of envelopment. A gE-null mutant produced enveloped virions, but these accumulated in large numbers in the neuronal cytoplasm rather than reaching cell surfaces as wild-type HSV virions do. Thus, in addition to the defects in envelopment, there was missorting of capsids and enveloped particles in the neuronal cytoplasm, which can explain the reduced anterograde transport of unenveloped capsids and enveloped virions. These mechanisms differ substantially from existing models suggesting that gE/gI and US9 function by tethering HSV particles to kinesin microtubule motors. The defects in assembly of gE US9 mutant virus particles were novel because they were neuron specific, in keeping with observations that US9 is neuron specific. Herpes simplex virus (HSV) and other alphaherpesviruses, such as varicella-zoster virus, depend upon the capacity to navigate in neuronal axons. To do this, virus particles tether themselves to dyneins and kinesins that motor along microtubules from axon tips to neuronal cell bodies (retrograde transport) or from cell bodies to axon tips (anterograde transport). This transit in axons is essential for alphaherpesviruses to establish latency in ganglia and then to reactivate and move back to peripheral tissues for spread to other hosts. Anterograde transport of HSV requires two membrane proteins: gE/gI and US9. Our studies reveal new mechanisms for how gE/gI and US9 initiate anterograde axonal transport. HSV mutants lacking both gE and US9 fail to properly assemble enveloped virus particles in the cytoplasm, which blocks anterograde transport of enveloped particles. In addition, there are defects in the sorting of virus particles such that particles, when formed, do not enter proximal axons.

Microtubule-Dependent Trafficking of Alphaherpesviruses in the Nervous System: The Ins and Outs

“…Deletion of the US9 gene clearly results in fewer PRV particles present in axons, nevertheless the kinetics of anterograde transport of those few axonal US9p-null PRV particles were indistinguishable from wild type [194]. Similar data were reported for HSV-1, where simultaneous loss of US9p and gE/gI led to severely diminished numbers of trafficking axonal particles, but those in the axon exhibited normal rates of anterograde traffic [161,189]. One possible explanation is that the reduced numbers of axonal viral particles seen in ∆US9 PRV and ∆US9/∆gE/∆gI HSV-1 strains is due to aberrant trafficking of viral particles within the neuronal cell body, or a failure to efficiently sort virions from the cell body into the axon itself [194].…”

“…In rat SCGs, one study found only completely assembled enveloped virions (OEVs) within axons upon infection with HSV-1, HSV-2, or PRV [150], but another found a mixture of OEVs and naked capsids in a 3:1 ratio [160]. To reconcile such findings it has been suggested that HSV-1 may have the capacity to utilize both the separate and married transport mechanisms, with varying ratio of naked axonal capsids and OEVs, depending upon the origin of the neurons being studied and how transport is assayed [160][161][162] (see also Section 7.4).…”

“…In HSV-1, gE/gI and US9p are important for anterograde spread within the nervous system in a cooperative manner [184][185][186][187] with some degree of redundancy: loss of US9p reduces delivery of HSV-1 particles to distal axons by about 50% [161,188], but simultaneous loss of US9p and gE/gI abolishes delivery to axons almost completely [189]. In contrast, PRV strains with the US9 gene deleted are completely defective for axonal sorting of viral particles in mature cultured neurons and exhibit no anterograde spread of infection [170,182,190,191].…”

“…Another possible kinesin-1 receptor is US9p itself, which for HSV-1 has been reported to directly interact with the carboxy-terminal tail of KIF5B [201]. However, if US9p is essential for kinesin-1 recruitment, then loss of US9p should disrupt the rate of trafficking of axonal HSV-1 particles, which does not seem to be the case [161,189].…”

“…An additional role for US9p and gE/gI within the context of the married model (Figure 4), that is not necessarily exclusive of a function in motor recruitment, is that these proteins participate in the envelopment of capsids in the cell body. An HSV-1 strain simultaneously deleted for gE (therefore functionally null for the gE/gI heterodimer) and US9 accumulated larger numbers of partially enveloped capsids than did wild type control viruses in the cell body of rat embryonic SCG neurons, mouse CAD neurons, and human SK-N-SH neurons [161]. In the married model, an envelopment defect in the cell body would of course lead to fewer enveloped particles available for transport (Figure 4), explaining why diminished numbers of enveloped virions are seen in axons when gE/gI and US9p are absent.…”