An Interaction between Glutathione and the Capsid Is Required for the Morphogenesis of C-Cluster Enteroviruses

Abstract

Glutathione (GSH) is the most abundant cellular thiol playing an essential role in preserving a reduced cellular environment. Cellular GSH levels can be efficiently reduced by the GSH biosynthesis inhibitor, L-buthionine sulfoximine (BSO). The aim of our study was to determine the role of GSH in the growth of two C-cluster enteroviruses, poliovirus type 1 (PV1) and coxsackievirus A20 (CAV20). Our results show that the growth of both PV1 and CAV20 is strongly inhibited by BSO and can be partially reversed by the addition of GSH. BSO has no effect on viral protein synthesis or RNA replication but it strikingly reduces the accumulation of 14S pentamers in infected cells. GSH-pull down assays show that GSH directly interacts with capsid precursors and mature virus made in the absence of BSO whereas capsid precursors produced under GSH-depletion do not bind to GSH. In particular, the loss of binding of GSH may debilitate the stability of 14S pentamers, resulting in their failure to assemble into mature virus. Immunofluorescence cell imaging demonstrated that GSH-depletion did not affect the localization of viral capsid proteins to the replication complex. PV1 BSO resistant (BSOr) mutants evolved readily during passaging of the virus in the presence of BSO. Structural analyses revealed that the BSOr mutations, mapping to VP1 and VP3 capsid proteins, are primarily located at protomer/protomer interfaces. BSOr mutations might, in place of GSH, aid the stability of 14S particles that is required for virion maturation. Our observation that BSOr mutants are more heat resistant and need less GSH than wt virus to be protected from heat inactivation suggests that they possess a more stable capsid. We propose that the role of GSH during enterovirus morphogenesis is to stabilize capsid structures by direct interaction with capsid proteins both during and after the formation of mature virus particles.

Picornavirus Morphogenesis

“…The addition of 1 to 3 mM guanidine hydrochloride (GnHCl) at midinfection, which inhibits RNA synthesis, caused the accumulation of procapsids, which could be chased into virus particles (57,58). From our studies with BSO, we found that 75S particles, although they are made in normal amounts during infection in GSH-depleted cells, do not progress into forming mature virus, an observation which suggests that 75S may not be a functional capsid intermediate (22).…”

“…Using BSO, the inhibitor of poliovirus morphogenesis, we observed that GSH depletion leads to the accumulation of "provirion-like" particles, which migrate slightly faster on sucrose gradients than the 150S particles made under normal growth conditions and contain essentially no infectious virions, suggesting that GSH is required for the final maturation of viral particles (22).…”

“…As discussed below (see "3CD and VPg"), virus maturation is also enhanced by the viral protein 3CD in an in vitro translation/RNA replication system (74,75). Moreover, we have recently shown that GSH is required for the production of infectious 150S particles (22). Finally, Richards and Jackson (18) proposed that virion maturation occurs in a cellular compartment known as the acidic autophagosome, but whether these structures are essential for poliovirus replication remains uncertain.…”

“…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).…”

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

“…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.…”