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.
Highlights
Coxsackievirus B3 (CVB3) is a positive-sense single stranded RNA virus that belongs to the genus Enterovirus of the family Picornaviridae
Previous reports described an increase in protein ubiquitinylation in CVB3 infected cells and that the application of proteasome inhibitors reduces viral replication, suggesting that the ubiquitin-proteasome system (UPS) is exploited by the virus [7,8]
For a more detailed assessment of the effect of CVB3 infection on protein ubiquitinylation, extracts of HeLa cells were subjected to differential centrifugation to obtain a medium-speed membrane pellet (16k) with intact organelles and large vesicles, a highspeed membrane pellet (120k) containing microsomes as well as a high-speed supernatant representing the cytosolic fraction
Summary
Coxsackievirus B3 (CVB3) is a positive-sense single stranded RNA virus that belongs to the genus Enterovirus of the family Picornaviridae. The non-enveloped virion has an icosahedral symmetry with a diameter of approximately 30 nm, enters target cells via receptor-mediated endocytosis and releases a 7.4 kb RNA genome into the cytoplasm [2]. The viral genome is translated into a single polyprotein that is co-translationally subjected to a series of autocleavages, generating individual proteins. The eventually released viral proteins include four capsid proteins (VP1–VP4) and seven non-structural proteins (2A–C, 3A–D) as well as some stable and functional precursor proteins (2BC, 3AB, 3CD). The initial cleavage of the polyprotein is catalyzed by the viral protease 2A, which releases the fragment P1 that contains the capsid proteins. The further cleavage of P1 and the processing of the fragments
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