Upregulation of signalase processing and induction of prM-E secretion by the flavivirus NS2B-NS3 protease: roles of protease components

Abstract

Recently, we have shown that the ability of the flavivirus NS2B-NS3 protease complex to promote efficient signalase processing of the C-prM precursor, as well as secretion of prM and E, does not appear to depend strictly on cleavage of the precursor at its Lys-Arg-Gly dibasic site by the protease. We suggested that the association of the protease with the precursor via NS2B may be sufficient by itself for the above effects. To study the proposed association in more detail, we have developed an assay in which processing at the C-prM dibasic cleavage site is abolished by Lys 3 Gly conversion. We constructed deletion mutants and chimeras of the West Nile (WN) flavivirus NS2B protein and expressed them in the context of {5-C3NS3 243 } containing either wild-type C-prM or its cleavage site mutant. All NS2B variants were able to form active protease complexes. Deletion of the carboxy-terminal cluster of hydrophobic amino acids in NS2B had no apparent effect on the formation of prM and prM-E secretion for the cassettes containing either wild-type or mutated C-prM precursor. Deletion of the amino-terminal hydrophobic cluster in NS2B did not affect prM-E secretion for the cassettes with wild-type C-prM but abrogated prM-E secretion for the cassettes with the mutated dibasic cleavage site in C-prM. Similarly, the NS2B-NS3 178 protease of Japanese encephalitis (JE) virus, when substituted for the WN virus NS2B-NS3 243 protease, was able to promote prM-E secretion for the cassette with the wild-type C-prM precursor but not with the mutated one. Replacement of the deleted amino-terminal hydrophobic cluster in the WN virus NS2B protein with an analogous JE virus sequence restored the ability of the protease to promote prM-E secretion. On the basis of these observations, roles of individual protease components in upregulation of C-prM signalase processing are discussed.

Encapsidation of the Flavivirus Kunjin Replicon RNA by Using a Complementation System Providing Kunjin Virus Structural Proteins in trans

“…In order to increase the efficiency of packaging and to simplify the procedure, we prepared a single SFV replicon construct expressing the prME genes and the C gene simultaneously. Because of the difficulties experienced with cloning of the entire C-prM-E region into the pSFV1 vector (see the first section of Results) and also to avoid possible uncertainty regarding cleavage at the carboxy terminus of C in the absence of flavivirus protease NS2B-NS3 (3,23,34,(39)(40)(41), an SFV replicon expressing the prME and C genes under the control of two separate 26S promoters was prepared (Fig. 1, SFV-prME-C107).…”

Mutagenesis of the NS2B-NS3-Mediated Cleavage Site in the Flavivirus Capsid Protein Demonstrates a Requirement for Coordinated Processing

“…An alternative model is that the signal peptidase cleavage, while still occurring posttranslationally, is upregulated by interaction with the nonstructural region, specifically some portion of NS2B (43,45,46). In this model, cleavage at the dibasic site is not necessary for efficient signal peptidase cleavage.…”

“…When the protease is inactivated by site-directed mutagenesis, polyprotein processing does not occur. Thus, it can still be argued that prM production is independent of the viral protease per se but is dependent on the proper mature nonstructural domain (43,46). The strategy employed to examine prM production, therefore, was to express the proteolytic domain of NS3 (residues 1 to 181) and the NS2B cofactor via separate plasmids.…”

Cleavage preference distinguishes the two-component NS2B–NS3 serine proteinases of Dengue and West Nile viruses

“…There are three structural proteins [C (capsid), prM (membrane) and E (envelope)] and seven NS (non-structural) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) encoded by the flaviviral genome. Proteases from the host (furin and secretase) and from the virus [NS3pro (NS3 serine proteinase)] are required to process the polyprotein precursor into the individual functional proteins [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19].…”