Axonal tracing of fluorescently labeled varicella-zoster virus proteins after infection of rat dorsal root ganglia neurons

Varicella-zoster virus (VZV) is a wide-spread neurotropic α-herpesvirus causing chickenpox (varicella) during primary infection and shingles (zoster) upon reactivation. During primary infection, VZV particles travel along neuronal microtubules via retrograde axonal transport from the distal axon endings to the somata of trigeminal and dorsal root ganglia DRG sensory neurons where latency is established. In contrast, upon reactivation, VZ virions migrate to the periphery by anterograde axonal transport to promote the typical symptoms of shingles. In culture, VZV replicates in a highly cell-associated manner and has a narrow host range that limits experimental animal models and restricted our approaches for studying the axonal transport of the virus particles. In this study, we used a cloned bacterial artificial chromosome (BAC) of the European wild-type isolate of VZV HJO to generate two double VZV mutants via en-passant mutagenesis in which the protein of interest and the capsid protein p23 were fused either to the enhanced green fluorescent protein or to the monomeric red fluorescent protein. The generated BACs were then transfected into permissive melanoma cells in which the cell-associated viruses were reconstituted and high-titer cell-free virus preparations were generated. Productive anterograde and retrograde infections of DRG neurons were established by the exposure to high titers of cell-free VZV of 10e4-10e5 plaque forming units/ml. Subsequently, we described the colocalization of the VZV immediate early tegument protein IE4 and the transmembrane envelope protein p65 together with p23 within axons of embryonic Wistar rat DRG neurons after anterograde and retrograde infection with VZV. The anterograde axonal transport of VZV follows most likely a “married” model. Hence, our findings provide a platform for further investigations into the axonal transport mechanisms of VZV and into the role of the relevant viral proteins as possible medicinal intervention targets in preventing or treating VZV infection and its complications.