Differential Modulation of prM Cleavage, Extracellular Particle Distribution, and Virus Infectivity by Conserved Residues at Nonfurin Consensus Positions of the Dengue Virus pr-M Junction

Abstract

In the generation of flavivirus particles, an internal cleavage of the envelope glycoprotein prM by furin is required for the acquisition of infectivity. Unlike cleavage of the prM of other flaviviruses, cleavage of dengue virus prM is incomplete in many cell lines; the partial cleavage reflects the influence of residues at furin nonconsensus positions of the pr-M junction, as flaviviruses share basic residues at positions P1, P2, and P4, recognized by furin. In this study, viruses harboring the alanine-scanning and other multiple-point mutations of the pr-M junction were generated, employing a dengue virus background that exhibited 60 to 70% prM cleavage and a preponderance of virion-sized extracellular particles. Analysis of prM and its cleavage products in viable mutants revealed a cleavage-suppressive effect at the conserved P3 Glu residue, as well as the cleavage-augmenting effects at the P5 Arg and P6 His residues, indicating an interplay between opposing modulatory influences mediated by these residues on the cleavage of the pr-M junction. Changes in the prM cleavage level were associated with altered proportions of extracellular virions and subviral particles; mutants with reduced cleavage were enriched with subviral particles and prM-containing virions, whereas the mutant with enhanced cleavage was deprived of these particles. Alterations of virus multiplication were detected in mutants with reduced prM cleavage and were correlated with their low specific infectivities. These findings define the functional roles of charged residues located adjacent to the furin consensus sequence in the cleavage of dengue virus prM and provide plausible mechanisms by which the reduction in the pr-M junction cleavability may affect virus replication.Dengue viruses are members of the genus Flavivirus in the family Flaviviridae. A single-stranded genomic RNA of positive polarity encodes an open reading frame which is translated into a precursor of three structural proteins and at least seven nonstructural proteins (27). During the replication of flaviviruses, proteolytic cleavages of the polyprotein by viral protease and a number of cellular enzymes occur in distinct subcellular compartments (27,32). Three structural proteins, C, prM/M, and E, are associated with the membrane of the rough endoplasmic reticulum (ER) by C-terminal membrane-spanning regions. Cleavages distal to the membrane-spanning regions of C and the two envelope glycoproteins prM and E by host signalase in the lumen of the rough ER and a cleavage proximal to the membrane-spanning region of C by viral protease in the cytoplasm are important to the assembly of viral particles (32). During the export of immature particles through the secretory pathway, cleavage of prM by the trans-Golgi apparatus resident furin allows reorganization of the receptor-binding E protein that is required for the acquisition of infectivity (9, 43).Assembly of flavivirus particles in the rough ER results in two types of particles: virions and subviral particles (32,40). An immature...

Highly Conserved Residues in the Helical Domain of Dengue Virus Type 1 Precursor Membrane Protein Are Involved in Assembly, Precursor Membrane (prM) Protein Cleavage, and Entry

“…Previous studies of flaviviral prM protein demonstrated the importance of the Arg-Xaa-Arg/Lys-Arg motif (at positions P4 to P1) for the cleavage of prM protein (11); moreover, charged residues adjacent to the consensus motif such as those at positions P3, P5, and P6 have been shown to affect the efficiency of prM cleavage (61). Based on the crystal structure of furin, the active site cleft consisting of 18 clustered negatively charged residues and S1, S2,and S4 subsite pockets may explain the stringent requirement of arginine at P1 and P4 and lysine at P2 (62).…”

Exclusion of West Nile Virus Superinfection through RNA Replication

“…The prM S90R mutation is located at the P3 position of the cleavage site that potentially could improve the prM cleavage. A previous study of DENV-2 showed that a decrease in prM cleavage was associated with higher proportions of subviral particles and prM-containing virions in culture medium (11,13). On the other hand, enhanced prM cleavability adversely affects DENV-2 export and release (13).…”

Viroporins: structure, function and potential as antiviral targets

“…The release of virions from the cell surface results in the loss of pr and resultant formation of a mature, infectious virion (Junjhon et al, 2008(Junjhon et al, , 2010Yu et al, 2008Yu et al, , 2009. prM is required for efficient trafficking of E to the cell surface and accelerated cleavage of prM is detrimental to virion production (Junjhon et al, 2008(Junjhon et al, , 2010Keelapang et al, 2004).…”

“…The 75 aa small M (membrane) protein is cleaved from the viral E protein by signal peptidase as a prM precursor, which is then processed in the Golgi and acidifying secretory compartments by furinlike proteases into M and the pr peptide (Junjhon et al, 2008;Keelapang et al, 2004;Kuhn et al, 2002;Wong et al, 2012;Yu et al, 2008). The release of virions from the cell surface results in the loss of pr and resultant formation of a mature, infectious virion (Junjhon et al, 2008(Junjhon et al, , 2010Yu et al, 2008Yu et al, , 2009.…”