The family Picornaviridae consists of a large group of plus-strand RNA viruses that share a similar genome organization. The nomenclature of the picornavirus proteins is based on their position in the viral RNA genome but does not necessarily imply a conserved function of proteins of different genera. The enterovirus 2B protein is a small hydrophobic protein that, upon individual expression, is localized to the endoplasmic reticulum (ER) and the Golgi complex, reduces ER and Golgi complex Ca 2؉ levels, most likely by forming transmembrane pores, and inhibits protein trafficking through the Golgi complex. At present, little is known about the function of the other picornavirus 2B proteins. Here we show that rhinovirus 2B, which is phylogenetically closely related to enterovirus 2B, shows a similar subcellular localization and function to those of enterovirus 2B. In contrast, 2B proteins of hepatitis A virus, foot-and-mouth disease virus, and encephalomyocarditis virus, all of which are more distantly related to enteroviruses, show a different localization and have little, if any, effects on Ca 2؉ homeostasis and intracellular protein trafficking. Our data suggest that the 2B proteins of enterovirus and rhinovirus share the same function in virus replication, while the other picornavirus 2B proteins support the viral life cycle in a different manner. Moreover, we show that an enterovirus 2B protein that is retained in the ER is unable to modify Ca 2؉ homeostasis and inhibit protein trafficking, demonstrating the importance of Golgi complex localization for its functioning.The family Picornaviridae is a group of small, nonenveloped cytolytic viruses that include a number of important human and animal pathogens. The picornavirus family consists of nine genera, including enterovirus (e.g., coxsackievirus [CBV] and poliovirus [PV]), rhinovirus (e.g., human rhinovirus [HRV]), cardiovirus (e.g., encephalomyocarditis virus [EMCV]), aphthovirus (e.g., foot-and-mouth disease virus [FMDV]), hepatovirus (hepatitis A virus [HAV]), teschovirus (e.g., porcine teschovirus), erbovirus (e.g., equine rhinitis B virus), parechovirus (e.g., parechovirus 2), and kobuvirus (e.g., aichivirus). In addition, the picornavirus family contains a number of unassigned viruses. All picornaviruses have a similar genome organization. The viral genome typically consists of a positivestranded RNA molecule of approximately 7,500 to 8,000 nucleotides that contains one single large open reading frame preceded by a long 5Ј-untranslated region and followed by a much smaller 3Ј-untranslated region and a genetically encoded poly(A) tail. A small viral protein, VPg, is covalently linked to the 5Ј end of the viral genome. Translation of the RNA genome yields a polyprotein of approximately 2,200 amino acids (aa) that is divided into the P1, P2, and P3 regions. The polyprotein is processed by virus-encoded proteases to generate the individual structural and nonstructural proteins. Processing of the P1 region yields the structural capsid proteins 1A (VP4), 1B...

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 ...