Association of the pr Peptides with Dengue Virus at Acidic pH Blocks Membrane Fusion

Abstract

Flavivirus assembles into an inert particle that requires proteolytic activation by furin to enable transmission to other hosts. We previously showed that immature virus undergoes a conformational change at low pH that renders it accessible to furin (I. M. Yu, W. Zhang, H. A. Holdaway, L. Li, V. A. Kostyuchenko, P. R. Chipman, R. J. Kuhn, M. G. Rossmann, and J. Chen, Science 319:1834-1837, 2008). Here we show, using cryoelectron microscopy, that the structure of immature dengue virus at pH 6.0 is essentially the same before and after the cleavage of prM. The structure shows that after cleavage, the proteolytic product pr remains associated with the virion at acidic pH, and that furin cleavage by itself does not induce any major conformational changes. We also show by liposome cofloatation experiments that pr retention prevents membrane insertion, suggesting that pr is present on the virion in the trans-Golgi network to protect the progeny virus from fusion within the host cell.Maturation, by which a noninfectious immature virus particle is converted to an infectious virion, is an essential step in the replication cycle of many viruses. It often involves the proteolytic processing of a precursor protein coupled with the conformational transformation of the virion. The assembly pathway for flaviviruses is well established, and the structures of the entire virion as well as individual glycoproteins representing different stages of the life cycle have been determined. Thus, flaviviruses are an excellent system for investigating the dynamic aspects of maturation in enveloped viruses.Flavivirus maturation requires furin (21), a cellular protease located primarily in the trans-Golgi network (TGN) (16). Immature particles, containing heterodimers of the precursor membrane protein (prM) and the envelope protein (E), bud into the endoplasmic reticulum (ER) and then are transported through the cellular secretory pathway to the extracellular environment (12, 23). The cleavage of prM by furin generates the membrane-anchored protein M and the soluble product pr. In the mature, infectious virion, the pr peptide is absent, and the virus undergoes membrane fusion in the endosome at low pH. In contrast, immature particles produced from furin-deficient LoVo cells, or cells grown in the presence of protease inhibitors or acidotropic reagents to prevent furin cleavage, contain prM and are significantly less infectious (21).Crystal structures of the E protein (9,14,18,19,29) show that each polypeptide chain contains three domains: the structurally central amino-terminal domain (DI), the dimerization domain (DII) containing the fusion loop, and the carboxyterminal immunoglobulin-like domain (DIII). In the presence of lipids, low pH induces rearrangements of the E proteins, resulting in the formation of homotrimers with the fusion loops and the C-terminal membrane anchors located at the same end (4, 15). The conformational change of the E proteins presumably facilitates the merging of the viral membrane with the endosomal membrane, the...

Post-translational regulation and modifications of flavivirus structural proteins

“…Although immature flavivirus particles have been demonstrated to be non-infectious in vitro (Elshuber et al, 2003;Elshuber & Mandl, 2005;Moesker et al, 2010;Stadler et al, 1997;Yu et al, 2008Yu et al, , 2009), opsonization of immature DENV virions with anti-prM antibodies has been demonstrated to enhance the infection of Fc-receptor-positive cells (Dejnirattisai et al, 2010;Rodenhuis-Zybert et al, 2010). Bound virions are endocytosed, with acidification of the endosome allowing furin-mediated cleavage of prM and subsequent virus fusion.…”

“…Indeed, the currently accepted model of flavivirus maturation within the trans-Golgi involves the stem region of the prM protein interacting with the associated E protein DII in a manner that pulls E down from a trimeric spike into a flat dimeric conformation parallel to the virion surface (Zhang et al, 2012(Zhang et al, , 2013b, with this interaction dependent upon decreasing pH and originating from one or more discrete nucleation centres (Plevka et al, 2011(Plevka et al, , 2014. In addition, the low-pH environment in the trans-Golgi compartment allows the pr peptide to remain associated with E (Yu et al, 2009), effectively preventing premature fusion within the cell.…”

“…4). As detailed previously, host furinmediated cleavage of prM (to a greater or lesser extent) occurs in acidified compartments of the trans-Golgi complex, with liberated pr peptide remaining associated with the transiting virion or prM/E particle (Yu et al, 2009). Upon release of virus particles from endosomes, the neutral pH triggers the dissociation of the pr peptide, forming an infectious mature virus particle of roughly 500 Å in diameter, with 90 E homodimers on the surface of the particle (Fig.…”

“…Proposed roles for prM include assisting in the chaperone-mediated folding of the E protein (Konishi & Mason, 1993;Lorenz et al, 2002), contributing to the pH-dependent conformational rearrangement of trimeric prM/E heterodimer spikes in immature virions via the influence of the helical stem domain of M (Zhang et al, 2012), and in the concealment of the fusion loop of E by the pr peptide to prevent premature fusion during virion egress (Yu et al, 2009). …”

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).…”