Role of Pseudorabies Virus Us3 Protein Kinase during Neuronal Infection

Abstract

The pseudorabies virus (PRV) Us3 gene is conserved among the alphaherpesviruses and encodes a serine/ threonine protein kinase that is not required for growth in standard cell lines. In this report, we used a compartmented culture system to investigate the role of PRV Us3 in viral replication in neurons, in spread from neurons to PK15 cells, and in axon-mediated spread of infection. We also examined the role of Us3 in neuroinvasion and virulence in rodents. Us3 null mutants produce about 10-fold less infectious virus from neurons than wild-type virus and have no discernible phenotypes for axonal targeting of viral components in cultured peripheral nervous system neurons. After eye infection in rodents, Us3 null mutants were slightly attenuated for virulence, with a delayed onset of symptoms compared to the wild type or a Us3 null revertant. While initially delayed, the symptoms increased in severity until they approximated those of the wild-type virus. Us3 null mutants were neuroinvasive, spreading in both efferent and afferent circuits innervating eye tissues.The alphaherpesviruses, such as the human pathogens herpes simplex virus types 1 and 2 (HSV-1; HSV-2), varicellazoster virus, and the agricultural-animal pathogens, such as pseudorabies virus (PRV), bovine herpesvirus type 1, and Marek's disease virus, are pantropic but are capable of invading the peripheral nervous systems (PNS) and central nervous systems (CNS) of susceptible animals (18,34,39). The infection of polarized cells, such as epithelial cells and neurons, plays a central role in the alphaherpesvirus life cycle. For example, primary infection usually occurs in mucosal epithelial cells, but after replication and release of virions at the basolateral surface, infection readily spreads to underlying nerve terminals of neurons of the PNS. The encapsidated viral genome then spreads directionally inside axons via microtubules to the cell bodies of sensory and autonomic ganglia. After entering the nucleus, the viral DNA is transcribed, and the neuron may experience a productive or latent infection. Most infections of natural hosts result in latent infections. After reactivation from a latent infection in PNS neurons, viral genomes move into axons and reverse their original direction by spreading back to the periphery, where upon release, progeny virions can infect epithelial cells and spread to other hosts. In rare cases after infection of the natural host, primary infection results in systemic spread of infection and occasional invasion of the CNS. When nonnatural hosts are infected, the outcome is reversed: latent infections and survival are rare, while rapid death and invasion of the CNS are common (3,5,25,35).It is well established for PRV that infection of nonnatural hosts, such as dogs, cattle, and rodents, by virulent strains causes a peripheral neuropathy characterized by violent pruritus (the mad itch) and rapid demise of the animal (51). Certain live, attenuated vaccine strains (notably the well-studied Bartha strain of PRV) have markedly re...

Directional Transneuronal Spread of α-Herpesvirus Infection

“…Unfortunately, mutations that affect retrograde transneuronal spread for PRV or for HSV are rare, so addressing these fundamental questions has been difficult. We do know that viral gene products that reduce the number of particles produced per cell or have reduced cell–cell spread kinetics, reduce efficiency of retrograde transneuronal spread [97,101]. For example, we know that all PRV Bartha tracing strains carry a mutated UL21 locus that reduces efficiency of retrograde transneuronal spread.…”

The neuroinvasive profiles of H129 (herpes simplex virus type 1) recombinants with putative anterograde-only transneuronal spread properties

“…Although the selective anterograde-only spread phenotype of HSV-1 H129 was asserted in the aforementioned literature, it remains unclear if retrograde transport is completely blocked or if it is reduced in efficiency as has been observed for PRV recombinants (Curanovic and Enquist 2009; Olsen et al 2006; Zaidi et al 2008). In fact, studies by Atherton and colleagues provided evidence for retrograde transport of H129 in the brain following inoculation of the anterior chamber of the eye (Archin et al 2003; Archin and Atherton 2002b; Archin and Atherton 2002a).…”

Anterograde Spread of Herpes Simplex Virus Type 1 Requires Glycoprotein E and Glycoprotein I but Not Us9

“…Sympathetic neurons are cultured in a tripartite ring in which neuron cell bodies are contained in a separate compartment from their neurites. Virus is added to neuron cell bodies in one chamber, and anterograde spread to a separate chamber is measured by viral titers (13,29,30,39,43). Using this system, gEnull, gInull, and Us9null PRV each were shown to have only FIG.…”

“…From these tissues, the virus infects innervating parasympathetic and motor neurons and spreads to the brain in a retrograde direction. The localization of viral antigens in specific brain sites indicates whether the virus traveled to the brain along an anterograde or retrograde pathway (21,25,26,39,44,51). PRV gE, gI, and Us9 each are essential for anterograde spread to the brain yet are dispensable for retrograde spread (5, 11, 25, 52).…”