Dual Role of Herpes Simplex Virus 1 pUS9 in Virus Anterograde Axonal Transport and Final Assembly in Growth Cones in Distal Axons

Abstract

The herpes simplex virus type 1 (HSV-1) envelope protein pUS9 plays an important role in virus anterograde axonal transport and spread from neuronal axons. In this study, we used both confocal microscopy and transmission electron microscopy (TEM) to examine the role of pUS9 in the anterograde transport and assembly of HSV-1 in the distal axon of human and rat dorsal root ganglion (DRG) neurons using US9 deletion (US9 ؊ ), repair (US9R), and wild-type (strain F, 17, and KOS) viruses. Using confocal microscopy and single and trichamber culture systems, we observed a reduction but not complete block in the anterograde axonal transport of capsids to distal axons as well as a marked (ϳ90%) reduction in virus spread from axons to Vero cells with the US9 deletion viruses. Axonal transport of glycoproteins (gC, gD, and gE) was unaffected. Using TEM, there was a marked reduction or absence of enveloped capsids, in varicosities and growth cones, in KOS strain and US9 deletion viruses, respectively. Capsids (40 to 75%) in varicosities and growth cones infected with strain 17, F, and US9 repair viruses were fully enveloped compared to less than 5% of capsids found in distal axons infected with the KOS strain virus (which also lacks pUS9) and still lower (<2%) with the US9 deletion viruses. Hence, there was a secondary defect in virus assembly in distal axons in the absence of pUS9 despite the presence of key envelope proteins. Overall, our study supports a dual role for pUS9, first in anterograde axonal transport and second in virus assembly in growth cones in distal axons. IMPORTANCEHSV-1 has evolved mechanisms for its efficient transport along sensory axons and subsequent spread from axons to epithelial cells after reactivation. In this study, we show that deletion of the envelope protein pUS9 leads to defects in virus transport along axons (partial defect) and in virus assembly and egress from growth cones (marked defect). Virus assembly and exit in the neuronal cell body are not impaired in the absence of pUS9. Thus, our findings indicate that pUS9 contributes to the overall HSV-1 anterograde axonal transport, including a major role in virus assembly at the axon terminus, which is not essential in the neuronal cell body. Overall, our data suggest that the process of virus assembly at the growth cones differs from that in the neuronal cell body and that HSV-1 has evolved different mechanisms for virus assembly and exit from different cellular compartments. Herpes simplex virus 1 (HSV-1) is a neurotropic and neuroinvasive virus that has the ability to infect and remain dormant or latent in neurons of the peripheral nervous system of their human host. After the initial infection of the skin, the virus gains access to the neurons of the dorsal root ganglia via the nerve endings in the innervating skin. Virus reactivation from latency is frequent and results in either asymptomatic virus shedding or recurrent herpes disease (1). Two essential events following reactivation of HSV from latently infected sensory neurons ...

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). A role for HSV-1 gE/gI extracellular domains in contributing to both anterograde axonal transport and possibly also in the spread from distal axons to nonneuronal cells has been reported recently (14) and adds further support to the notion of redundancy in transport processes essential for viral replication (49).…”

“…The fact there is no obvious axonal transport phenotype reported for KOS in animal models (46) may be due to the presence of compensating mutations, such as an observed extension of the C-terminal end of pUS8A (44,45). However, we have observed a neuronal spread defect for KOS in vitro using dissociated rat DRG neurons (53). In the case of McIntyre, another mutation of note is a frameshift mutation which results in an extension of the C-terminal tail of gI (43), which, along with truncated pUS9, may contribute rat DRG neurons were dissociated, pelleted through a 35% Percoll gradient, and plated into the somal compartment of three-chamber microfluidic devices.…”

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

“…PRV US9 binds to a kinesin-3 protein, KIF1A (15), and the HSV US9 and US11 proteins bind kinesin-1 proteins in pulldown experiments (22,26). It was also suggested that HSV US9-null mutants have defects during anterograde transport, i.e., after virus entry into axons (39). However, our studies of both gE Ϫ and US9 Ϫ single mutants and gE Ϫ US9 Ϫ double mutants produced different conclusions: once HSV capsids or virions enter axons, their transport is normal, the kinetics of transport are not different from those for wild-type HSV, and there is no increased stalling (17).…”

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

“…Other studies have proposed a role for US9p in HSV-1 capsid envelopment in human and rat DRGs. In that case, the envelopment defect was interpreted to be in neuronal growth cones and varicosities, following axonal transport of naked capsids to those locations in accord with the separate model ( Figure 4) [202].…”

“…Compared to wild type controls, HSV-1 mutants lacking gE or gI (thus in both cases functionally null for the gE/gI heterodimer) or the US9 gene showed 50%-75% fewer naked capsids and viral glycoprotein-associated structures entering the axons of human SK-N-SH neurons, though transport of capsids appeared more sensitive to the deletions than did transport of glycoproteins [188]. Loss of US9p similarly reduced the numbers of trafficking naked HSV-1 capsids by~50% in axons of human and rat DRGs [202] and essentially abolished it in axons of murine retinal ganglia [156], with more modest effects upon viral glycoprotein trafficking [156,188].…”