Alterations of pr-M Cleavage and Virus Export in pr-M Junction Chimeric Dengue Viruses

Abstract

During the export of flavivirus particles through the secretory pathway, a viral envelope glycoprotein, prM, is cleaved by the proprotein convertase furin; this cleavage is required for the subsequent rearrangement of receptor-binding E glycoprotein and for virus infectivity. Similar to many furin substrates, prM in vector-borne flaviviruses contains basic residues at positions P1, P2, and P4 proximal to the cleavage site; in addition, a number of charged residues are found at position P3 and between positions P5 and P13 that are conserved for each flavivirus antigenic complex. The influence of additional charged residues on pr-M cleavage and virus replication was investigated by replacing the 13-amino-acid, cleavage-proximal region of a dengue virus (strain 16681) with those of tick-borne encephalitis virus (TBEV), yellow fever virus (YFV), and Japanese encephalitis virus (JEV) and by comparing the resultant chimeric viruses generated from RNA-transfected mosquito cells. Among the three chimeric viruses, cleavage of prM was enhanced to a larger extent in JEVpr/16681 than in YFVpr/16681 but was slightly reduced in TBEVpr/16681. Unexpectedly, JEVpr/16681 exhibited decreased focus size, reduced peak titer, and depressed replication in C6/36, PS, and Vero cell lines. The reduction of JEVpr/16681 multiplication correlated with delayed export of infectious virions out of infected cells but not with changes in specific infectivity. Binding of JEVpr/16681 to immobilized heparin and the heparin-inhibitable infection of cells were not altered. Thus, diverse pr-M junction-proximal sequences of flaviviruses differentially influence pr-M cleavage when tested in a dengue virus prM background. More importantly, greatly enhanced prM cleavability adversely affects dengue virus export while exerting a minimal effect on infectivity. Because extensive changes of charged residues at the pr-M junction, as in JEVpr/16681, were not observed among a large number of dengue virus isolates, these results provide a possible mechanism by which the sequence conservation of the pr-M junction of dengue virus is maintained in nature.The genus Flavivirus within the family Flaviviridae comprises about 73 enveloped RNA viruses that are transmitted by either mosquitoes or ticks or without a known vector (11). For these viruses, a single-stranded RNA genome of about 11 kb encodes a polyprotein, which is cleaved by cellular and viral enzymes into three structural proteins (C, prM/M, and E) and seven nonstructural proteins (54). Virions consist of two envelope proteins, E and prM/M, and an internal C protein, which binds genomic RNA. Differences in antigenicities of E allow the subdivision of flaviviruses into eight antigenic complexes and a number of unclassified viruses, which include the prototype yellow fever virus (YFV) (12). More recent assignments based on nucleotide sequence variations of the nonstructural gene NS5 generally agree with antigenic classifications (49).The assembly of flaviviruses in the endoplasmic reticulum is followed by modif...

Histidine 39 in the dengue virus type 2 M protein has an important role in virus assembly

“…Similarly, infectious DENV particles with a high proportion of prM have consistently been found to be released from infected cells (Anderson et al, 1997;He et al, 1995;Henchal et al, 1985;Murray et al, 1993;Putnak et al, 1996;Randolph & Stollar, 1990;Roehrig et al, 1998;Wang et al, 1999). Recent studies mutating the prM furin cleavage site of TBEV (Elshuber et al, 2003) and DENV-2 (Keelapang et al, 2004) have shown that, whilst cleavage of prM is essential for virus infectivity, enhanced furin cleavage of the DENV prM protein affects virus export adversely, suggesting that it may be advantageous for DENV to retain some prM on the virus surface. We have also found that mutation of the prM/M cleavage site abolished virus infectivity, but still led to the release of RSPs, by using our transient-expression system (L. Azzola, P. J. Wright & A. D. Davidson, unpublished data).…”

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).…”

“…After uncoating, translation, and genome replication, assembly of viral particles takes place in the membrane of rough endoplasmic reticulum (ER), where the immature virions, containing prM and E proteins on surface, bud into the lumen of ER and transport through the secretary pathway (4, 8 -10). In the trans-Golgi, the prM protein on immature virions is cleaved by furin or furin-like protease to produce mature virions, although the cleavage was inefficient for DENV (11)(12)(13)(14)(15). A common feature of flaviviral replication is the formation of small and slowly sedimenting subviral particles (4,16).…”

Viroporins: structure, function and potential as antiviral targets

“…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.…”

“…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).…”