Carboxy-terminal analysis of nine proteins specified by the flavivirus Kunjin: evidence that only the intracellular core protein is truncated.
Nine proteins specified by Kunjin virus were labelled with [3H]lysine and digested with carboxypeptidase B which specifically cleaves carboxy-terminal Lys or Arg. The theoretical amount of [3H]lysine was released from the non-structural (ns) proteins NS2A, NS2B, NS3 and NS4B, which have a common carboxy terminus Lys-Arg deduced from cleavage sites established in the viral polyprotein by previous N-terminal amino acid analyses. This is a flavivirus consensus site, always followed by Gly, Ala or Ser. These results indicate that no truncations had occurred despite anomalous electrophoretic migrations of NS2A, NS2B and NS4B observed in some gel systems. No [3H]lysine was released from NS4A or NS5 which terminate in Ala and Leu, respectively, thus establishing that NS5 (observed Mr 98,000, theoretical Mr 103,600) was not cleaved post-translationally at any internal Lys-Arg site. Unexpectedly, [3H]lysine residues were apparently released from P14(C), the intracellular equivalent of virion C protein which terminates in Ala (adjacent to the established N-terminus of prM). However, a putative internal cleavage site (Lys-Arg decreases Gly) exists 18 residues upstream from the carboxy terminus of C, prior to the transmembrane spanning domain. Apparent internal cleavage in the cytosol at this site to produce P14(C) would expose [3H]lysine residues to carboxypeptidase B, and account for the previously observed differences in size and composition between C and P14(C).
Speight
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Department of Microbiology, Monash University, Clayton, Victoria, Australia.
Westaway
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D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D043424:Carboxypeptidase B; D002268:Carboxypeptidases; D005416:Flavivirus / Q000032:analysis* / Q000235:genetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D014758:Viral Core Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D014758:Viral Core Proteins; D001120:Arginine; D002268:Carboxypeptidases; D043424:Carboxypeptidase B; D008239:Lysine