Axonal Sorting and Transport During Alphaherpesvirus Infection in Neurons

Abstract

Alphaherpesviruses are pathogens of the nervous systems of their mammalian hosts. Well-studied alphaherpesviruses include the human pathogens herpes simplex virus 1 and 2 (HSV-1 and HSV-2) and varicella zoster virus (VZV), as well as the veterinary pathogen pseudorabies virus (PRV). These viruses are pantropic and neuroinvasive. Once in the peripheral or central nervous system, infection can spread within chains of synaptically connected neurons. Highly regulated directional transport of viral particles in neurons, while poorly understood, is required for spread of infection and is an integral part of the viral life cycle. Here, we investigated the molecular and cellular underpinnings of alphaherpesvirus spread and pathogenesis using a variety of approaches. First, we identified viral and host proteins that are associated with PRV Us9, a viral membrane protein that is required for anterograde spread of infection both <italic> in vitro </italic> and <italic> in vivo </italic>. We immunoaffinity purified functional GFP-tagged Us9 from infected cells and identified viral and host interacting proteins by mass spectrometry. One of the proteins that specifically co-isolated with Us9 was the kinesin-3 family member KIF1A. Our results suggest that alphaherpesviruses repurpose the synaptic vesicle sorting pathway for efficient axonal transport and spread within their hosts. Second, we demonstrate that PRV and HSV-1 infection disrupt mitochondrial motility and morphology in axons. During PRV infection, glycoprotein B (gB)-dependent fusion events result in electrical coupling of neurons and increased action potential firing rates. Consequently, intracellular [Ca<super>2+</super>] increases and alters mitochondrial dynamics through an established cellular mechanism. This disruption in mitochondrial dynamics is required for efficient growth and spread of PRV, indicating that altered mitochondrial transport enhances alphaherpesvirus pathogenesis and infection. Finally, we present the first comprehensive proteomic characterization of purified PRV virions by mass spectrometry. We identified 47 viral proteins associated with PRV virions, including seven viral proteins that were previously undetected in virions. Additionally, we identified 48 host proteins associated with PRV virions, many of which have known functions in important cellular pathways. This analysis extends previous work aimed at determining the composition of herpesvirus virions and provides novel insights critical for understanding the mechanisms underlying PRV entry, assembly, egress, spread, and pathogenesis.