Date Published: April 10, 2014
Publisher: Public Library of Science
Author(s): Hendrik Jan Thibaut, Lonneke van der Linden, Ping Jiang, Bert Thys, María-Dolores Canela, Leire Aguado, Bart Rombaut, Eckard Wimmer, Aniko Paul, María-Jesús Pérez-Pérez, Frank J. M. van Kuppeveld, Johan Neyts, Richard J. Kuhn.
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.
Enteroviruses, belonging to the family of the Picornaviridae, are non-enveloped, icosahedral viruses with a positive, single-stranded genome. Enteroviruses comprise many important pathogens of humans and animals. Although most enterovirus infections subside asymptomatically or mildly, they can also result in severe morbidity and even mortality. Polioviruses cause paralytic poliomyelitis; rhinovirus infections are associated with exacerbations of asthma and chronic obstructive pulmonary disease and enterovirus 71 may cause life-threatening encephalitis, in particular in Asia. Also coxsackieviruses and echoviruses have been reported to cause acute clinical manifestations, including fulminant sepsis, aseptic meningitis and myocarditis . Epidemiological studies strongly suggest a linkage between coxsackieviruses and the development of type 1 diabetes mellitus . Apart from polio, no vaccines are available that can protect against enteroviral infections. No drugs have been approved so far for the treatment or prophylaxis of enterovirus infections .
We here describe the discovery of TP219 as a novel inhibitor of the replication of several enteroviruses. We showed that TP219 forms a covalent adduct with GSH in an enzyme-independent way, thereby rapidly depleting intracellular GSH levels. As a consequence, the formation of infectious progeny virus is prevented without interfering with earlier events in the viral life cycle, such as entry, translation, proteolytic processing and RNA replication. By employing TP219, we further studied the details of virus assembly and present several lines of evidence that GSH is required during the transition of protomeric particles into pentameric particles. Inhibition of virus assembly following GSH depletion was not rescued by treatment with other reducing agents, indicating that a reducing environment is not a requirement for morphogenesis. We provided biochemical and microscopical evidence for a direct interaction between VP1 and GSH and that this interaction is essential for the translocation of assembly intermediates to the sites of replication. Furthermore, genetic and structural analysis of several GSH-independent viruses point towards the interface between protomers as a region critical for GSH binding/independence.