A flavivirus signal peptide balances the catalytic activity of two proteases and thereby facilitates virus morphogenesis.

Two cleavages on either side of a signal peptide separating capsid and prM on the nascent flavivirus polyprotein are uniquely regulated, such that cytosolic capsid cleavage triggers signalase cleavage of prM. Here, we show, using two experimental approaches, that this sequential order of cleavages facilitates virus morphogenesis: (i) A Murray Valley encephalitis virus (MVEV) variant, in which both cleavages occurred efficiently and independently of each other, displayed an assembly defect. (ii) Replicon particle assembly was assayed in packaging cells encoding the MVEV structural proteins; bicistronic expression of either mature or membrane-anchored capsid in addition to that of the prM and E proteins showed enhanced particle production in the latter cell line. Taken together, this study demonstrates that efficient flavivirus assembly requires a cleavable transmembrane anchor of C protein and an obligatory order of cleavages at the C-prM junction, both controlled by sequence elements in the prM signal peptide.