Expression and subcellular localization of poliovirus VPg-precursor protein 3AB in eukaryotic cells: evidence for glycosylation in vitro.
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.
Datta
U
U
Department of Microbiology and Immunology, School of Medicine, University of California, Los Angeles 90024-1747.
Dasgupta
A
A
D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D003980:Diazooxonorleucine / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D017955:Poliovirus / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012367:RNA, Viral / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014758:Viral Core Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; D011498:Protein Precursors; D012367:RNA, Viral; D011994:Recombinant Proteins; D014758:Viral Core Proteins; D003980:Diazooxonorleucine