Determinants for Membrane Association and Permeabilization of the Coxsackievirus 2B Protein and the Identification of the Golgi Complex as the Target Organelle

Abstract

The 2B protein of enterovirus is responsible for the alterations in the permeability of secretory membranes and the plasma membrane in infected cells. The structural requirements for the membrane association and the subcellular localization of this essential virus protein, however, have not been defined. Here, we provide evidence that the 2B protein is an integral membrane protein in vivo that is predominantly localized at the Golgi complex upon individual expression. Addition of organelle-specific targeting signals to the 2B protein revealed that the Golgi localization is an absolute prerequisite for the ability of the protein to modify plasma membrane permeability. Expression of deletion mutants and heterologous proteins containing specific domains of the 2B protein demonstrated that each of the two hydrophobic regions could mediate membrane binding individually. However, the presence of both hydrophobic regions was required for the correct membrane association, efficient Golgi targeting, and the membrane-permeabilizing activity of the 2B protein, suggesting that the two hydrophobic regions are cooperatively involved in the formation of a membrane-integral complex. The formation of membrane-integral pores by the 2B protein in the Golgi complex and the possible mechanism by which a Golgilocalized virus protein modifies plasma membrane permeability are discussed.Enteroviruses (e.g. poliovirus, coxsackievirus, ECHOvirus) belong to the family of picornaviridea, a group of nonenveloped, cytolytic viruses that have a positive stranded RNA genome of 7.5 kb. The enterovirus genome contains one large open reading frame that is translated into a single, 220-kDa polyprotein. Processing of the polyprotein by virus-encoded proteases yields the structural P1 region proteins that encapsidate the viral RNA and the non-structural P2 and P3 region proteins that are involved in replication of the viral RNA. Processing of the P2 and P3 regions yields the 2A pro , 2B, 2C, 3A, 3B, 3C pro , and 3D pol proteins and the more stable cleavage intermediates 2BC, 3AB, and 3CD pro , which have functions distinct from their cleavage products. Although multiple functions have been attributed to the mature viral proteins and the cleavage intermediates, the exact function of most of the viral proteins in the replication cycle is still largely unknown.Host cell membranes are subject to a number of profound alterations upon enterovirus infection. Enteroviruses gradually modify host cell membrane permeability and rearrange intracellular membranes during infection. The modification of the plasma membrane permeability is most likely important for the lysis of the cell and the release of virus progeny. Modifications of secretory pathway membranes are connected to viral functions such as genome replication. The modification of host cell membrane permeability is such that initially calcium is released from intracellular stores (1). Ionic gradients maintained by the plasma membrane are also disrupted (2). Also, later in infection, small compounds such ...

Mutational Analysis of Different Regions in the Coxsackievirus 2B Protein

“…Hygromycin B Assay-The ability of 2B to increase plasma membrane permeability to hygB was studied using 2B-EGFP fusion proteins, as described previously (18). Mutant p2B-EGFP plasmids were constructed by amplification of the 2B coding sequences of the mutant pCB3/T7 plasmids.…”

“…In infected cells, the 2B protein is present at the secretory pathway-derived membranes of the replication complex (7,10,11). Upon individual expression, the 2B protein localizes at membranes of the ER and Golgi complex (17,18). This localization pattern suggests that the 2B protein increases membrane permeability of ER and Golgi membranes directly, whereas the permeability of the plasma membrane is increased indirectly, by means of an as yet unknown mechanism.…”

“…Second, mutations in each of the hydrophobic regions interfere with the ability of the 2B protein to homo-multimerize and to increase membrane permeability (15,27,30). Finally, the 2B protein was demonstrated to be an integral membrane protein, and the presence of each of the hydrophobic regions was shown to be required for this integral membrane association and the correct subcellular localization (18).…”

The NS3 Protein of Bluetongue Virus Exhibits Viroporin-like Properties

“…Indeed, NS3 mutants that were unable to permeabilize cells were also unable to localize to the Golgi apparatus. A similar correlation has been identified for the coxsackievirus 2B viroporin protein (24). The mechanism by which these Golgi-targeted proteins effect permeabilization of the plasma membrane remains to be determined.…”

“…However, hyg-B will enter mammalian cells in which the plasma membrane has been permeabilized. This assay has been used previously by others to identify proteins that can form pores in lipid membranes (15)(16)(17)(18)(19)(20)(21)(22)(23)(24).…”