FORMATION OF THE FLAVIVIRUS ENVELOPE - ROLE OF THE VIRAL NS2B-NS3 PROTEASE

One of the late processing events in the flavivirus replication cycle involves cleavage of the intracellular form of the flavivirus capsid p rotein (C-int) to the mature virion form (C-vir) lacking the carboxy-t erminal stretch of hydrophobic amino acids which serves as a signal pe ptide for the downstream prM protein. This cleavage event was hypothes ized to be effected by a viral protease and to be associated with viri on formation, We have proposed a model of flavivirus virion formation in which processing of the C-prM precursor at the upstream signalase s ite is upregulated by interaction of the NS2B part of the protease wit h the prM signal peptide or with an adjacent carboxy-terminal region o f the capsid protein in the precursor, and processing of C-int by the NS2B-NS3 protease follows the signalase cleavage. Recently, an alterna tive hypothesis was proposed which suggests a reverse order of these t wo cleavage events, namely, that cleavage of the C-prM precursor by th e NS2B-NS3 protease at the C-int-->C-vir dibasic cleavage site is a pr erequisite for the subsequent signalase cleavage of the prM signal pep tide. To distinguish between these alternative models, we prepared a s eries of expression cassettes carrying mutations at the C-int-->C-vir dibasic cleavage site and investigated the effects of these mutations on signalase processing of C-prM and on formation and secretion of prM -E heterodimers. For certain mutated C-prM precursors, namely, for tho se with Lys-->Gly disruption of the dibasic site, efficient formation of prM was observed upon expression from larger cassettes encoding the viral protease, despite the absence of processing at the C-int-->C-vi r cleavage site. Surprisingly, formation and secretion of prM-E hetero dimers accompanied by late cleavage of prM was also observed for these cassettes, with an efficiency comparable to that of the wild-type exp ression cassette. These observations contradict the model in which cle avage of the C-prM precursor at the C-int-->C-vir dibasic site is a pr erequisite for signalase cleavage.