Coxsackievirus B3 Exploits the Ubiquitin-Proteasome System to Facilitate Viral Replication

Abstract

Infection by RNA viruses causes extensive cellular reorganization, including hijacking of membranes to create membranous structures termed replication organelles, which support viral RNA synthesis and virion assembly. In this study, we show that infection with coxsackievirus B3 entails a profound impairment of the protein homeostasis at virus-utilized membranes, reflected by an accumulation of ubiquitinylated proteins, including K48-linked polyubiquitin conjugates, known to direct proteins to proteasomal degradation. The enrichment of membrane-bound ubiquitin conjugates is attributed to the presence of the non-structural viral proteins 2B and 3A, which are known to perturb membrane integrity and can cause an extensive rearrangement of cellular membranes. The locally increased abundance of ubiquitinylated proteins occurs without an increase of oxidatively damaged proteins. During the exponential phase of replication, the oxidative damage of membrane proteins is even diminished, an effect we attribute to the recruitment of glutathione, which is known to be required for the formation of infectious virus particles. Furthermore, we show that the proteasome contributes to the processing of viral precursor proteins. Taken together, we demonstrate how an infection with coxsackievirus B3 affects the cellular protein and redox homeostasis locally at the site of viral replication and virus assembly.

A cytosolic disulfide bridge‐supported dimerization is crucial for stability and cellular distribution of Coxsackievirus B3 protein 3A

“…The formation of disulfide bridges in the cytosol is strongly dependent on the redox state in a specific cellular compartment. In this context, we have previously shown that in particular membrane proteins are susceptible to oxidative damage, indicating that protein 3A being a transmembrane protein is indeed exposed to a cellular redox milieu that can promote the formation of a disulfide linkage and homodimer formation [4]. Multimeric protein complexes are known to be more resistant to damage by reactive oxygen species.…”

“…We previously evidenced that infection with Coxsackievirus B3 (CVB3), a model pathogen of the EV genus, disturbs protein homeostasis at virus‐utilized membranes and results in an accumulation of ubiquitinated proteins [4]. The enrichment of membrane‐bound ubiquitin conjugates was shown to be attributed to the presence of the non‐structural VPs 2B and 3A.…”

“…Yeast two‐hybrid experiments revealed that full‐length protein 3A has the ability to self‐associate [7] and NMR spectroscopy studies demonstrated that protein 3A from poliovirus forms a symmetric homodimer via the soluble N‐terminal domain, whereby its underlying molecular mechanism is not finally defined so far [5]. Corroborating these findings, we recently showed that CVB3 protein 3A can form SDS‐resistant homodimers when expressed in HeLa cells [4]. This study was set up to investigate the molecular basis for the ability of the non‐structural CVB3 protein 3A to form such SDS‐resistant homodimers.…”

A Conserved Cysteine Residue in Coxsackievirus B3 Protein 3A with Implication for Elevated Virulence

“…With this approach, we could document the cellular distribution of viral and host proteins in the different compartments during infection. We previously demonstrated that the CVB3 proteins are most abundantly localized in the 16k membrane pellet [21], indicating that this fraction contains replication organelles. The redistribution of ACBD3 in CVB3-infected cells from the cytosolic fraction to the 16k membrane fraction, which contains protein 3A, was similar for both the wt 3A and the mutant C38A variants (Figure 1B).…”

“…As an example, protein 3A from poliovirus, another member of the EV genus, forms a symmetric homodimer via the soluble N-terminal domain [20]. We recently demonstrated that CVB3 protein 3A forms SDS-resistant homodimers [21] via a DTTsensitive disulfide bridge between cysteine residues at position 38 (C38), thereby increasing its stability [22]. The conservation of 3A-C38 among various representatives of the EV genus suggests that the functional properties of this cysteine residue are a common feature of EV protein 3A [22].…”