The Coxsackievirus 2B Protein Increases Efflux of Ions from the Endoplasmic Reticulum and Golgi, thereby Inhibiting Protein Trafficking through the Golgi

Abstract

Coxsackievirus infection leads to a rapid reduction of the filling state of the endoplasmic reticulum (ER) and Golgi Ca 2؉ stores. The coxsackievirus 2B protein, a small membrane protein that localizes to the Golgi and to a lesser extent to the ER, has been proposed to play an important role in this effect by forming membrane-integral pores, thereby increasing the efflux of Ca 2؉ from the stores. Here, evidence is presented that supports this idea and that excludes the possibility that 2B reduces the uptake of Ca 2؉ into the stores. Measurement of intra-organelle-free Ca 2؉ in permeabilized cells revealed that the ability of 2B to reduce the Ca 2؉ filling state of the stores was preserved at steady ATP. Biochemical analysis in a cellfree system further showed that 2B had no adverse effect on the activity of the sarco/endoplasmic reticulum calcium ATPase, the Ca 2؉ -ATPase that transports Ca 2؉ from the cytosol into the stores. To investigate whether 2B specifically affects Ca 2؉ homeostasis or other ion gradients, we measured the lumenal Golgi pH. Expression of 2B resulted in an increased Golgi pH, indicative for the efflux of H ؉ from the Golgi lumen. Together, these data support a model that 2B increases the efflux of ions from the ER and Golgi by forming membrane-integral pores. We have demonstrated that a major consequence of this activity is the inhibition of protein trafficking through the Golgi complex.Enteroviruses (e.g. poliovirus, coxsackievirus, ECHOvirus) belong to the family of Picornaviridea, a large family of nonenveloped, cytolytic viruses that contain a single-stranded RNA genome of positive polarity. Upon infection, enteroviruses induce a number of dramatic alterations in their host cell, which serve to create the appropriate conditions for viral RNA replication and/or prevent antiviral host cell responses. One of these alterations is the modification of intracellular Ca 2ϩ homeostasis. We have previously shown that infection of HeLa cells with coxsackievirus results in a reduction of the amount of Ca 2ϩ that can be released from the intracellular stores using thapsigargin, an inhibitor of the sarco/endoplasmic reticulum calcium ATPase (SERCA), 2 the Ca 2ϩ -ATPase that transports Ca 2ϩ from the cytosol into the stores. In addition, a gradual increase in the cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] cyt ) was observed due to the influx of extracellular Ca 2ϩ (1). The enterovirus 2B protein, one of the nonstructural proteins involved in viral RNA replication, plays a major role in the alterations in intracellular Ca 2ϩ homeostasis that take place in enterovirus-infected cells (1, 2). The mechanism by which 2B, or its precursor 2BC, exerts its effects is largely unknown. Ca 2ϩ homeostasis in the intracellular stores (i.e. endoplasmic reticulum (ER) and Golgi) is the net result of the activity of the SERCA on the one hand and the continuous passive Ca 2ϩ leak from these organelles that exists under normal conditions on the other hand (3). Thus, the reductions in the Ca 2ϩ filling state of the stores ...

Enumeration and Functional Evaluation of Virus-Specific CD4 + and CD8 + T Cells in Lymphoid and Peripheral Sites of Coxsackievirus B3 Infection

“…Our laboratory (4,5) and others (9,50,51) have recently demonstrated that several CVB3 proteins act in concert to disrupt protein trafficking within an infected cell: 2B and 2C inhibit the movement of protein through the Golgi complex, while 3A is able to disrupt this organelle (4). Furthermore, the combined actions of 3A-mediated inhibition of anterograde transport and 2B (and/or 2BC)-mediated upregulation of endocytosis lead to rapid down-regulation of surface MHC class I (5).…”

Functional Analysis of Picornavirus 2B Proteins: Effects on Calcium Homeostasis and Intracellular Protein Trafficking

“…Increasing evidence indicates that 2B forms homomultimers that build pores in ER and Golgi complex membranes (1, 11, 12-14, 41), thereby reducing the levels of Ca 2ϩ and H ϩ in the lumens of these organelles in infected cells (8,14,39). Individual expression of 2B furthermore results in inhibition of protein trafficking through the Golgi complex (14,17). It is unknown whether these activities represent different functions of 2B or whether the one activity is the consequence of the other.…”

“…It is unknown whether these activities represent different functions of 2B or whether the one activity is the consequence of the other. The observation that 2B mutants that are impaired in increasing the efflux of ions from the ER and Golgi complex are also impaired in inhibiting protein trafficking suggests that these activities are somehow connected (8,14). The relevance of these 2B activities for the viral life cycle is still poorly understood.…”

“…The 2B protein is a small hydrophobic membrane protein that localizes at endoplasmic reticulum (ER) and Golgi complex membranes (4,15,33). Increasing evidence indicates that 2B forms homomultimers that build pores in ER and Golgi complex membranes (1, 11, 12-14, 41), thereby reducing the levels of Ca 2ϩ and H ϩ in the lumens of these organelles in infected cells (8,14,39). Individual expression of 2B furthermore results in inhibition of protein trafficking through the Golgi complex (14,17).…”

Topology and biological function of enterovirus non-structural protein 2B as a member of the viroporin family

“…These changes include increased intracellular vesicle formation, increased membrane permeability, disrupted Ca 2+ balance in cells, cell apoptosis, and induced autophagy [5,13]. Some of these functional and morphological changes can also be observed in cells expressing the enteroviruscoded 2B protein only [9,13,18,19]. The viroporin features of the 2B protein that affect the biological functions of host cells are summarized in the following aspects.…”