In vitro processing of dengue virus structural proteins: cleavage of the pre-membrane protein.
Processing of dengue virus structural proteins was assessed in vitro. RNA transcripts for cell-free translation were prepared from cloned DNA (dengue virus type 4, strain 814669 genome) encoding capsid, pre-membrane (prM), and the first 23 amino acids of envelope (E). Processing of a 33-kilodalton precursor polypeptide encoded by wild-type RNA transcripts occurred only in the presence of added microsomal membranes. Under these conditions, cleavage at the capsid-prM and prM-E sites and glycosylation of prM occurred in association with translocation. Amino acid sequence analysis confirmed that translation initiated at the predicted N terminus of the capsid and that capsid-prM cleavage occurred at the predicted site for the action of signal peptidase following a candidate signal sequence (hydrophobic residues 100 to 113) in the dengue virus precursor. Mutations were introduced into the dengue virus DNA template by site-directed mutagenesis, altering nucleotide sequences encoding the capsid and the candidate signal for prM. The phenotypes of the mutants were deduced by analysis of the products of cell-free translation of the respective RNA transcripts. The resulting observations confirmed that cleavage at the capsid-prM and prM-E sites is effected entirely by signal peptidase and that the candidate signal is required for translocation.
Markoff
L
L
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
D000595:Amino Acid Sequence; D002213:Capsid / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D003716:Dengue Virus / Q000235:genetics* / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D009154:Mutation; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D015678:Viral Structural Proteins
D004279:DNA, Viral; D006023:Glycoproteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D012367:RNA, Viral; D014764:Viral Proteins; D015678:Viral Structural Proteins; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase