Expression and subcellular localization of poliovirus VPg-precursor protein 3AB in eukaryotic cells: evidence for glycosylation in vitro

Abstract

The poliovirus-encoded, membrane-associated VPg-precursor polypeptide 3AB has been implicated in the initiation of viral RNA synthesis. We have expressed 3AB and 3A polypeptides in eukaryotic cells and examined their localization using indirect immunofluorescence and a direct in vitro membrane-binding assay. Results presented here demonstrate that both 3AB and 3A are capable of localizing in the endoplasmic reticulum and the Golgi apparatus in transfected HeLa cells in the absence of any other poliovirus protein. We have also shown that the carboxy-terminal 18 amino acids of 3A that constitute an amphipathic domain are important in membrane binding of 3A and 3AB. Additionally, we demonstrate that a significant fraction of both 3A and 3AB can be glycosylated in a membrane-dependent fashion during in vitro translation in reticulocyte lysate. We demonstrate that 6-diazo-5-oxo-L-norleucine, an inhibitor of glycoprotein synthesis, significantly inhibits poliovirus RNA synthesis in vivo. The implications of glycosylation of 3AB (and 3A) in viral replication are discussed.

The Coronavirus Replicase

“…The viral replication complex, which consists of multiple viral but also cellular subunits (see the chapter by Shi and Lai, this volume), is associated with these membranes and, in many cases, also directs their synthesis and/or modification (Peränen and Kääriäinen 1991;Cho et al 1994;Schlegel et al 1996;Teterina et al 1997;Snijder et al 2001;Egger et al 2002). Typically, multiple vesicles or membrane invaginations (spherules) on cellular organelles are induced to which the replication complex is attached by specific structural elements, such as hydrophobic domains (van Kuppeveld et al 1995;Snijder et al 2001) amphipathic helices (Datta and Dasgupta 1994), palmitate side chains (Laakkonen et al 1996), and C-terminal membrane insertion sequences (Schmidt-Mende et al 2001). As a result, replication takes place in a membrane-protected (and, thus, nuclease resistant) microenvironment that contains (and sequesters) the protein functions required for viral RNA synthesis.…”

Interactions Between Virus Proteins and Host Cell Membranes During the Viral Life Cycle

“…Only the membrane-associated 3AB protein can be cleaved by the viral proteases (3C pro and 3CD pro ), and thus serves as the source of VPg (Lama et al, 1994). The 3AB precursor associates tightly with cellular membranes, resembling the binding of integral membrane proteins (Datta and Dasgupta, 1994). The binding domain has been mapped to the C-terminal region, within a hydrophobic sequence (Towner et al, 1996).…”

Inhibition of cellular protein secretion by poliovirus proteins 2B and 3A.

“…Protein 3A, 87 amino acids in length, contains a 22 amino acid hydrophobic domain near its C-terminus that is believed to mediate its association with cellular membranes. Both 3A and 3AB have been shown to associate with intracellular membranes when expressed in human cells using vaccinia vectors (Datta and Dasgupta, 1994). Protein 2B is also relatively hydrophobic, containing 31 hydrophobic residues out of 97 amino acids, but contains no continuous region of hydrophobic amino acids as long as that seen in 3A.…”