Entry functions and antigenic structure of flavivirus envelope proteins

Karin Stiasny, Stefan Kiermayr and Franz X. Heinz1

Institute of Virology, Medical University of Vienna, Kinderspitalgasse 15, A1095 Vienna, Austria

Abstract. The envelope proteins (E) of flaviviruses form an icosahedral cage-like structure of homodimers that cover completely the surface of mature virions and are responsible for receptor-binding and membrane fusion. Fusion is triggered by the acidic pH in endosomes which induces dramatic conformational changes of E that drive the merger of the membranes. We have identified an alternative trigger that induces the first phase of the fusion process only, but then leads to an arrest at an intermediate stage. These data suggest that the early and late stages of flavivirus fusion are differentially controlled by intersubunit and intrasubunit constraints of the fusion protein, respectively. Details of the molecular antigenic structure of the flavivirus E protein were revealed by the use of neutralization escape mutants as well as recombinant expression systems for the generation of virus-like particles. The experimental data provide evidence that each of the three domains contributing to the external face of the E protein can induce and bind neutralizing antibodies. Broadly flavivirus cross-reactive antibodies, however, primarily recognize a site involving residues of the highly conserved fusion peptide loop which is cryptic and largely inaccessible on the surface of native infectious virions.

2006 New treatment strategies for dengue and other flaviviral diseases. Wiley, Chichester (Novartis Foundation Symposium 277) p 57—73

Flaviviruses are small enveloped viruses that form a genus in the family Flaviviridae (Heinz et al 2003) and comprise a number of arthropod-transmitted human pathogens such as yellow fever (YF) virus, the dengue (DEN) viruses, Japanese encephalitis (JE) virus, West Nile (WN) virus, and tick-borne encephalitis (TBE) virus. Their positive-stranded RNA genome (about 11 kb) contains a single long open reading frame encoding three structural proteins (capsid protein, C; precursor of membrane protein, prM; membrane protein, M; envelope protein, E) and seven non-structural proteins (Lindenbach & Rice 2003). Flavivirus assembly takes place at the endoplasmic reticulum and first leads to the formation of immature, prM-

1 This paper was presented at the symposium by Franz Heinz, to whom all correspondence should be addressed containing non-infectious particles that are transported through the exocytotic pathway (Lindenbach & Rice 2003). In the trans-Golgi network the prM protein is proteolytically cleaved by furin or a related cellular protease (Stadler et al 1997) resulting in the formation of mature infectious virions that are released from infected cells by exocytosis. The structural details of the molecular organization of flavivirus particles were revealed by X-ray crystallography of the E protein (Rey et al 1995, Modis et al 2003, 2005, Zhang et al 2004), NMR spectroscopy and X-ray crystallography of the C protein (Jones et al 2003, Dokland et al 2004), and cryo-electron microscopy of purified mature and immature virions (Kuhn et al 2002, Mukhopadhyay et al 2003, Zhang et al 2003b) as well as recombinant subviral particles (Ferlenghi et al 2001). Figure 1 shows schematics of the flavivirus particle organization and the atomic structure of the E protein as present in mature virions. Immature virions are studded with icosahedrically arranged spikes, each of which is composed of three heterodimers of prM and E. The maturation cleavage of prM results in a still poorly understood rearrangement of E proteins at the virion surface leading to the formation of smooth-surfaced particles that carry 90 tightly packed E dimers in a 'herringbone'-like icosahedral arrangement (Kuhn et al 2002, Mukhopadhyay et al 2003) (Fig. 1). In these mature virions the E protein forms a head-to-tail dimer that is oriented parallel to the viral surface and integrated in the membrane at its C-terminus by a double membrane-spanning anchor. A sequence element of about 50 amino acids (the so-called stem; Fig. 1D) connects to the C-terminus of the ectodomain fragment (Zhang et al 2003a) that has been used for determining high resolution structures of the TBE, dengue 2 and dengue 3 virus E proteins by X-ray crystallography (Rey et al 1995, Modis et al 2003, 2005, Zhang et al 2004). Although the crystallized dengue and TBE virus E proteins share only 37% of their amino acids, their overall structures are virtually identical. Each of the monomeric subunits contains three distinct domains (I, II, and III) which are dominated by ß-sheet secondary structures (Fig. 1E, F). Most flavivirus E proteins have a single glycosylation site in domain I, in some instances a second site can be present in domain II (Modis et al 2003, 2005) and in certain virus strains the E protein is non-glycosylated (Beasley et al 2004). The E protein is bi-functional; it mediates both attachment to cells and low-pH-triggered membrane fusion after uptake by receptor-mediated endocytosis and is thus the major determinant for the induction of virus-neutralizing antibodies and a protective immunity. Because the flavivirus E and the alphavirus E1 fusion proteins are structurally radically different from the spike-like fusion proteins found in orthomyxo-, para-myxo-, retro-, filo- and coronaviruses, these are now referred to as class II and class I fusion proteins, respectively (Lescar et al 2001). Our work focuses on the molecular mechanism of membrane fusion as a potential target for the development of antiviral agents and the molecular antigenic structure of flaviviruses and its relevance for the induction of a protective immunity.

FIG. 1. Structural organization of flavivirus particles. (A) Schematic of immature (top) and mature (bottom) virions. (B) Image of mature virus particle as determined by cryoelectron microscopy (Kuhn et al 2002, Mukhopadhyay et al 2003). One of the rafts consisting of three parallel E homodimers is highlighted schematically using the E dimer representation shown in Fig. 1C. (C-F) Schematics (C, D) and structures (E, F) of E dimers viewed from the top (C, E) and the side (D, F), respectively. The structural element designated 'stem' in D is described in the text. E, F exhibit ribbon diagrams of the atomic structure of a soluble fragment (lacking the stem-anchor region) of TBE virus.

FIG. 1. Structural organization of flavivirus particles. (A) Schematic of immature (top) and mature (bottom) virions. (B) Image of mature virus particle as determined by cryoelectron microscopy (Kuhn et al 2002, Mukhopadhyay et al 2003). One of the rafts consisting of three parallel E homodimers is highlighted schematically using the E dimer representation shown in Fig. 1C. (C-F) Schematics (C, D) and structures (E, F) of E dimers viewed from the top (C, E) and the side (D, F), respectively. The structural element designated 'stem' in D is described in the text. E, F exhibit ribbon diagrams of the atomic structure of a soluble fragment (lacking the stem-anchor region) of TBE virus.

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