Determinants of Membrane Association for Poliovirus Protein 3AB

Jonathan S Towner,Tri V Ho,Bert L Semler

doi:10.1074/jbc.271.43.26810

Jonathan S Towner, Tri V Ho + Show 1 more

Open Access

https://doi.org/10.1074/jbc.271.43.26810

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Abstract

Poliovirus protein 3AB may serve as the lipophilic carrier of a protein primer (VPg or 3B) used for the initiation of genomic viral RNA synthesis. In order to study the membrane-protein interactions of 3AB required for its role in poliovirus RNA replication, we have developed an in vitro membrane association assay capable of distinguishing membrane-bound from non-membrane-bound proteins that are cotranslated together in the presence of canine microsomal membranes. This assay utilizes equilibrium sedimentation analysis in high density sucrose gradients to measure membrane association of both wild type and mutated forms of 3AB. Using this assay and other biochemical assays, we have identified the following properties of the 3AB-membrane interaction: (a) 3AB is able to post-translationally associate with microsomal membranes, (b) 3AB is able to associate with membranes in a manner consistent with that of an integral membrane protein, (c) 3AB contains a critical hydrophobic sequence within the carboxyl-terminal half of the protein that is required for membrane association, and (d) the introduction of charged residues into this hydrophobic sequence disrupts the 3AB membrane-protein interaction. Taken together, these studies indicate that poliovirus protein 3AB associates tightly with biological membranes de novo in a manner that would allow it to serve as a lipophilic anchor for the assembly of the poliovirus RNA replication complex.

Highlights

Cellular membranes are complex structures composed of lipid and protein that together compartmentalize the intracellular space and serve as the physical matrix upon which numerous biosynthetic events are performed
In Vitro 3AB Membrane Association Assay—In order to analyze the molecular determinants for membrane association, poliovirus protein 3AB was generated by in vitro translation in the absence of other poliovirus proteins
We have presented evidence that poliovirus protein 3AB behaves in a manner consistent with that of an integral membrane protein

Summary

Introduction

Cellular membranes are complex structures composed of lipid and protein that together compartmentalize the intracellular space and serve as the physical matrix upon which numerous biosynthetic events are performed. Peripheral membrane proteins associate with membranes through electrostatic interactions, typically between positively charged amino acids and negatively charged phospholipid headgroups [16]. Previous experiments that examined the membrane association of poliovirus replication complexes indicated that the viral protein 3AB was tightly associated with smooth membranes of infected cells in a manner resistant to treatment with 0.5 M salt or 4.0 M urea [19]. These studies were not able to demonstrate that this tight membrane interaction was the sole result of molecular determinants contained within the 3AB protein.

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