Processing of nonstructural proteins NS4A and NS4B of dengue 2 virus in vitro and in vivo.
The production, from polyprotein precursors, of two hydrophobic nonstructural proteins of dengue 2 (DEN2) virus, NS4A and NS4B, was analyzed both in cell-free systems and in infected cells. In DEN2-infected cells, NS4B is first produced as a peptide of apparent size 30 kDa; NS4B is then post-translationally modified, in an unknown way, to produce a polypeptide of apparent size 28 kDa. The rate and extent of NS4B modification was found to be cell-dependent; in BHK cells the half-time for the conversion of the 30-kDa form to the 28-kDa form was 90 min. N-terminal sequence analysis of NS4B suggests that the N-terminus is produced by an enzyme with a specificity similar to that of signalase. Low levels of a putative polyprotein, NS4AB, were also found in mammalian cells, but not mosquito cells, infected with DEN2, suggesting that a small proportion of DEN2 4A/4B cleavage can occur post-translationally or that some nonstructural polyproteins escape normal processing. Cleavage of the 4A/4B bond in infected cells required expression of DEN2 sequences in addition to those in NS4A and NS4B, as NS4AB produced in cells by a vaccinia expression system was not cleaved. NS4AB produced in cells by a vaccinia expression system was modified post-translationally, presumably in the same way as NS4B. We show that upon translation of DEN2 polyproteins in a cell-free system, the N-terminus of NS4A is generated by cleavage by the viral nonstructural proteinase NS3 and that processing of DEN2 polyproteins occurs with a preferred, but nonobligatory order.
Preugschat
F
F
Division of Biology, California Institute of Technology, Pasadena 91125.
Strauss
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D002213:Capsid / Q000378:metabolism*; D002460:Cell Line; D002474:Cell-Free System; D009033:Culicidae; D003716:Dengue Virus / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D014616:Vaccinia virus / Q000378:metabolism; D014758:Viral Core Proteins / Q000378:metabolism*; D017361:Viral Nonstructural Proteins
D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; D010450:Endopeptidases