Binding of Glutathione to Enterovirus Capsids Is Essential for Virion Morphogenesis

Abstract

Enteroviruses (family of the Picornaviridae) cover a large group of medically important human pathogens for which no antiviral treatment is approved. Although these viruses have been extensively studied, some aspects of the viral life cycle, in particular morphogenesis, are yet poorly understood. We report the discovery of TP219 as a novel inhibitor of the replication of several enteroviruses, including coxsackievirus and poliovirus. We show that TP219 binds directly glutathione (GSH), thereby rapidly depleting intracellular GSH levels and that this interferes with virus morphogenesis without affecting viral RNA replication. The inhibitory effect on assembly was shown not to depend on an altered reducing environment. Using TP219, we show that GSH is an essential stabilizing cofactor during the transition of protomeric particles into pentameric particles. Sequential passaging of coxsackievirus B3 in the presence of low GSH-levels selected for GSH-independent mutants that harbored a surface-exposed methionine in VP1 at the interface between two protomers. In line with this observation, enteroviruses that already contained this surface-exposed methionine, such as EV71, did not rely on GSH for virus morphogenesis. Biochemical and microscopical analysis provided strong evidence for a direct interaction between GSH and wildtype VP1 and a role for this interaction in localizing assembly intermediates to replication sites. Consistently, the interaction between GSH and mutant VP1 was abolished resulting in a relocalization of the assembly intermediates to replication sites independent from GSH. This study thus reveals GSH as a novel stabilizing host factor essential for the production of infectious enterovirus progeny and provides new insights into the poorly understood process of morphogenesis.

Direct-acting antivirals and host-targeting strategies to combat enterovirus infections

“…Buthionine sulfoximine, an inhibitor of glutathione synthesis, and TP219, a small molecule that covalently binds to glutathione, both impede the role of glutathione in morphogenesis [46 ,47 ]. Yet, not all enteroviruses rely on glutathione [47 ], thereby precluding glutathione as an important target for broad-spectrum inhibitors.…”

“…Capsid binder Pirodavir [5], Pleconaril b [5], Pocapavir (V-073) b [5], Vapendavir (BTA798) b [ Buthionine sulfoximine (BSO) [46 ], TP219 [47 ] replication have been discovered, spurring host-directed drug development. Since some host factors appear to be used by many -or even all -enteroviruses, inhibitors of these cellular factors may have broad-spectrum activity.…”

Picornavirus Morphogenesis

“…The recent report that the encapsidation specificity of poliovirus (and related C-cluster coxsackieviruses) is based on proteinprotein interactions (2C ATPase -capsid proteins) was a breakthrough in research on enterovirus morphogenesis (20,21). Other important results of picornavirus encapsidation include (i) the observation that glutathione (GSH) is an important host factor for enterovirus morphogenesis by providing stability to capsid precursors and the mature virus (22)(23)(24); (ii) the discovery that Aichi virus, a member of the recently discovered genus Kobuvirus, uses an RNA packaging signal to confer specificity to its encapsidation (25,26); and (iii) a report that hepatitis A virus uniquely uses its very small nonstructural protein 2A (also called X) in the formation of a precursor of the mature virus particle (27). In addition, there is an exciting report on the production of an enveloped (3,219).…”

Recent progress in understanding coxsackievirus replication, dissemination, and pathogenesis

“…Glutathione (GSH), the most prevalent non-protein thiol in the animal cell, was identified as an essential stabilizing cofactor during virion particle formation (Thibaut, van der Linden et al, 2014). A newly discovered inhibitor, TP219, binds GSH and depletes intracellular stores, thereby interfering with virus assembly but not RNA replication.…”