Membrane Integration of Poliovirus 2B Viroporin

Abstract

Virus infections can result in a variety of cellular injuries, and these often involve the permeabilization of host membranes by viral proteins of the viroporin family. Prototypical viroporin 2B is responsible for the alterations in host cell membrane permeability that take place in enterovirus-infected cells. 2B protein can be localized at the endoplasmic reticulum (ER) and the Golgi complex, inducing membrane remodeling and the blockade of glycoprotein trafficking. These findings suggest that 2B has the potential to integrate into the ER membrane, but specific information regarding its biogenesis and mechanism of membrane insertion is lacking. Here, we report experimental results of in vitro translation-glycosylation compatible with the transloconmediated insertion of the 2B product into the ER membrane as a double-spanning integral membrane protein with an N-/C-terminal cytoplasmic orientation. A similar topology was found when 2B was synthesized in cultured cells. In addition, the in vitro translation of several truncated versions of the 2B protein suggests that the two hydrophobic regions cooperate to insert into the ER-derived microsomal membranes.Virus infections can lead to a variety of cellular injuries, and usually these involve the restructuring of host membrane systems. Viroporins are a group of small virally encoded proteins that interact with cellular membranes to modify permeability and promote the release of viral particles. A typical feature exhibited by viroporins is the presence of at least one membrane-spanning helix anchoring the protein into membranes. After membrane insertion, their oligomerization creates hydrophilic channels or pores (22).Poliovirus is the enterovirus prototype member of the Picornaviridae family. This small, nonenveloped, icosahedral virus possesses a single-stranded 7.5-kb positive-sense RNA genome that encodes a single polyprotein. Polyprotein processing by virus-encoded proteases yields the structural P1 region proteins that encapsidate viral RNA and the nonstructural P2 and P3 region proteins involved in the replication of the viral RNA and membrane permeabilization (2). Nonstructural 2B protein is one of the products generated on processing the P2 region (62). Viroporin 2B has been identified as one of the viral proteins responsible for the alterations in host cell membrane permeability that take place in enterovirus-infected cells. Different 2B proteins expressed in cells have been localized at the endoplasmic reticulum (ER), Golgi complex, and, to a lesser extent, to the plasma and mitochondrial membranes (18,31,49,58). Biochemical and structural data indicate that viroporins form homo-oligomers that create pores in the ER and Golgi complex membranes (1,16,17,30,59). However, experimental data dealing with the mechanism of the membrane integration of the 2B product are lacking to date.The poliovirus 2B viroporin protein is hydrophobic overall and rather small (97 amino acids). Hydrophobicity within the viroporin 2B sequence seems to cluster in two main regions (F...

Charge Pair Interactions in Transmembrane Helices and Turn Propensity of the Connecting Sequence Promote Helical Hairpin Insertion

“…Glycosylation of a protein region synthesized in vitro in the presence of microsomal membranes therefore indicates the exposure of this region to the OST active site on the luminal side of the ER membrane. When assayed independently, the two hydrophobic regions of the PV 2B did not span the ER-derived membranes, 11 as expected according to the predicted apparent free energy of insertion (Fig. 1a).…”

“…11 Such topology is attained upon insertion of a helical hairpin whose constituent TM helices are marginally hydrophobic (Fig. 1a).…”

“…As in our previous study of membrane insertion of the PV 2B, 11 we used a well-characterized in vitro experimental system based on glycosylation 12 that accurately reports the integration of TM regions into microsomal membranes. Upon insertion, the oligosaccharyl transferase (OST) enzyme modifies the protein of interest.…”

“…We previously showed that poliovirus (PV) 2B, which is a small protein involved in PV virulence, is a double-spanning integral membrane protein, in which the two TM segments are interconnected by a short turn forming a putative helical hairpin. 11 As a first step towards understanding its biogenesis, we demonstrated that in vitro PV 2B integrates into the endoplasmic reticulum (ER) membrane through the translocon. 11 Here, we present a detailed investigation on structural determinants underlying helical hairpin formation in the viral membrane protein 2B.…”

“…11 As a first step towards understanding its biogenesis, we demonstrated that in vitro PV 2B integrates into the endoplasmic reticulum (ER) membrane through the translocon. 11 Here, we present a detailed investigation on structural determinants underlying helical hairpin formation in the viral membrane protein 2B. Using an in vivo-like translation-glycosylation system of the naturally occurring helical hairpin from PV 2B, we have determined the importance of helix-helix interactions for hairpin formation.…”