Cold storage and cryopreservation of PLTs led to morphologic and metabolic changes. However, storage under these conditions appears to maintain or even enhance certain aspects of in vitro PLT function.

The alphaherpesviral envelope protein pUS9 has been shown to play a role in the anterograde axonal transport of herpes simplex virus 1 (HSV-1), yet the molecular mechanism is unknown. To address this, we used an in vitro pulldown assay to define a series of five arginine residues within the conserved pUS9 basic domain that were essential for binding the molecular motor kinesin-1. The mutation of these pUS9 arginine residues to asparagine blocked the binding of both recombinant and native kinesin-1. We next generated HSV-1 with the same pUS9 arginine residues mutated to asparagine (HSV-1pUS9KBDM) and then restored them being to arginine (HSV-1pUS9KBDR). The two mutated viruses were analyzed initially in a zosteriform model of recurrent cutaneous infection. The primary skin lesion scores were identical in severity and kinetics, and there were no differences in viral load at dorsal root ganglionic (DRG) neurons at day 4 postinfection (p.i.) for both viruses. In contrast, HSV-1pUS9KBDM showed a partial reduction in secondary skin lesions at day 8 p.i. compared to the level for HSV-1pUS9KBDR. The use of rat DRG neuronal cultures in a microfluidic chamber system showed both a reduction in anterograde axonal transport and spread from axons to nonneuronal cells for HSV-1pUS9KBDM. Therefore, the basic domain of pUS9 contributes to anterograde axonal transport and spread of HSV-1 from neurons to the skin through recruitment of kinesin-1. IMPORTANCEHerpes simplex virus 1 and 2 cause genital herpes, blindness, encephalitis, and occasionally neonatal deaths. There is also increasing evidence that sexually transmitted genital herpes increases HIV acquisition, and the reactivation of HSV increases HIV replication and transmission. New antiviral strategies are required to control resistant viruses and to block HSV spread, thereby reducing HIV acquisition and transmission. These aims will be facilitated through understanding how HSV is transported down nerves and into skin. In this study, we have defined how a key viral protein plays a role in both axonal transport and spread of the virus from nerve cells to the skin. Deletion of the US9 gene, encoding the conserved type II transmembrane viral envelope protein pUS9 (1) (Fig. 1), has been undertaken in a range of Alphaherpesvirinae subfamily members, and neuronal spread has been assessed in several biological models. The relative contribution of pUS9 to neuronal spread within this subfamily was found to differ. Equine herpesvirus type 1 (EHV-1) and bovine herpesvirus type 1 (BHV-1) pUS9 can complement for the loss of pUS9 in swine pathogen pseudorabies virus (PrV) (1). In contrast, varicella-zoster virus (VZV) and herpes simplex virus 1 (HSV-1) pUS9 are unable to complement for a loss of pUS9 in PrV (1). In PrV (2-5), BHV-1 (6), and BHV-5 (7), pUS9 plays a major role in axonal sorting and/or anterograde axonal 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 th...

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Funding informationAustralian Governments fund Australian Red Cross Lifeblood to provide blood, blood products, and services to the Australian community.