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

Abstract

RNA viruses in the Picornaviridae family express a large 250 kDa viral polyprotein that is processed by virus‐encoded proteinases into mature functional proteins with specific functions for virus replication. One of these proteins is the highly conserved enteroviral transmembrane protein 3A that assists in reorganizing cellular membranes associated with the Golgi apparatus. Here, we studied the molecular properties of the Coxsackievirus B3 (CVB3) protein 3A with regard to its dimerization and its functional stability. By applying mutational analysis and biochemical characterization, we demonstrate that protein 3A forms DTT‐sensitive disulfide‐linked dimers via a conserved cytosolic cysteine residue at position 38 (Cys38). Homodimerization of CVB3 protein 3A via Cys38 leads to profound stabilization of the protein, whereas a C38A mutation promotes a rapid proteasome‐dependent elimination of its monomeric form. The lysosomotropic agent chloroquine (CQ) exerted only minor stabilizing effects on the 3A monomer but resulted in enrichment of the homodimer. Our experimental data demonstrate that disulfide linkages via a highly conserved Cys‐residue in enteroviral protein 3A have an important role in the dimerization of this viral protein, thereby preserving its stability and functional integrity.

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

“…Infection with CVB3 encoding EGFP results in GFP expression after 5 h. Cells with similar GFP fluorescence and 3A staining intensity, both being indicators of CVB3 infection in their respective HeLa cells, were selected for CVB3-3A wt and CVB3-3A[C38A] (Supplementary Figure S1). An analysis of anti-3A immunofluorescence using autocorrelation-based image correlation spectroscopy showed bigger areas of staining clusters in the case of protein 3A for CVB3-3A[C38A]-infected cells, thus confirming that cysteine 38 of protein 3A at least partially directs its subcellular localization towards more condensed membrane structures, as shown for the ectopic expression of protein 3A-C38A variants [22]. The cellular alterations seen in CVB3-3A wt-infected cells that we attributed to 3A-C38 suggested a more pronounced availability of protein 3A at the side of viral replication, which is in agreement with the enhanced stability of protein 3A as a C38-linked homodimer, as previously shown.…”

“…The structural modelling of C38-linked protein 3A homodimers implies an efficient recruitment of ACBD3 by protein 3A irrespective of this disulfide bridge [22]. To investigate whether protein 3A-C38 indeed has, as predicted, no effect on the recruitment of ACBD3 to cellular membranes in CVB3-infected cells, C38 of CVB3 3A was mutated to alanine by sitedirected mutagenesis, generating CVB3-3A[C38A].…”

“…The 3A-C38-mediated increased the stability for protein 3A [22], and the detection of a higher protein 3A signal intensity at specific foci in infected cells (Figure 2) prompted us to explore a role of the highly conserved 3A-C38 residue in virus replication. Virus with the C38A mutation was viable.…”

“…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].…”

“…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]. These aspects prompted us to investigate whether cysteine 38 of CVB3 protein 3A plays a role in infection, and our experiments documented a pro-viral function of 3A-C38 that occurs by it supporting the production of viral particles and enhancing cytotoxicity.…”