The Membrane-Proximal “Stem” Region Increases the Stability of the Flavivirus E Protein Postfusion Trimer and Modulates Its Structure

Abstract

The flavivirus fusion protein E contains a "stem" region which is hypothesized to be crucial for driving fusion. This sequence element connects the ectodomain to the membrane anchor, and its structure in the trimeric postfusion conformation is still poorly defined. Using E trimers of tick-borne encephalitis virus with stem truncations of different lengths, we show that the Nterminal part of the stem increases trimer stability and also modulates the trimer structure outside the stem interaction site. Flaviviruses, members of a genus in the family Flaviviridae, comprise a number of medically relevant viruses, such as dengue virus, yellow fever virus, Japanese encephalitis virus, West Nile virus, and tick-borne encephalitis virus (TBEV) (1). These small enveloped viruses infect cells by receptor-mediated endocytosis and acidic-pH-induced membrane fusion in endosomes (2, 3). The fusion reaction is mediated by the major envelope protein E, a class II fusion protein (4), which contains three domains (DI, DII, and DIII), a membrane-proximal region (the so-called "stem"), and the carboxy-terminal transmembrane anchor (Fig. 1A).The current flavivirus fusion model ( Fig. 1A to C) is based on atomic structures of truncated dimeric prefusion and trimeric postfusion structures of E that lack the stem-anchor region (soluble E [sE]). According to this model, the E dimers dissociate into monomers at acidic pH, leading to the exposure of the fusion peptide (FP) and its insertion into the outer leaflet of the target membrane ( Fig. 1B) (5). Membrane merger has been proposed to be driven by the relocation of DIII and a subsequent "zippering" reaction of the stem along DII, thus yielding a hairpin-like postfusion trimer with the FPs and transmembrane domains at the same end of the molecule ( Fig. 1C; reviewed in references 4 and 6). In accord with this model, soluble forms of DIII containing part of the stem and stem-derived peptides inhibited fusion (7-10).As shown by cryo-electron microscopy (cryo-EM) of dengue virions, the prefusion stem consists of two amphipathic helices (H1 and H3) that lie flat on the viral membrane and flank an additional short perimembrane helix (H2) (11, 12) ( Fig. 1A and D, lower panels). H2 has been resolved in the 3.5-Å structure of dengue virus only recently (12). With the exception of the first few amino acids following the C terminus of the E ectodomain (13,14), details of the stem in the context of the postfusion trimer are presently unknown. In X-ray crystallographic analyses of truncated dengue virus sE trimers that included N-terminal portions of the stem, most of the stem parts were disordered (14). The objective of our work was to generate experimental evidence for interactions of the stem with the core of the postfusion E trimer. For this purpose, we analyzed E trimers of TBEV with different C-terminal truncations using two approaches: (i) we assessed the impact of the presence of the stem as well as parts thereof on the thermostability of E trimers, and (ii) we examined possible influences ...

Silencing of neurotropic flavivirus replication in the central nervous system by combining multiple microRNA target insertions in two distinct viral genome regions

“…We designed the insertion of miRNA target sequences in this region using an approach that has been described previously by Bonaldo et al (Bonaldo et al, 2007) for generation of recombinant yellow fever viruses expressing foreign genes (Trindade et al, 2012). The C-terminal stem-anchor (SA) region of protein E (~100 amino acid residues) contains four alpha-helices, two of which (H1 and H2) form a stem region, and two amphipathic anti-parallel α-helices (TM1 and TM2) serve as an E protein membrane anchor and as an internal signal sequence for the cellular signal peptidase cleavage during polyprotein processing (Fritz et al, 2011; Pangerl et al, 2011; Stiasny et al, 2013). Three tandem target sequences for miRNAs were placed between sequences encoding the two SA domains: the first SA domain, located upstream of the miRNA-targets, was a part of the TBEV E protein; the second SA domain, located downstream of the miRNA targets, contained the sequence from the DEN4 E protein (Fig.…”

“…Both stem (H1 and H2 helices) and transmembrane anchor (TM1 and TM2 helices) regions are believed to be involved and play an active role in the formation of the E protein trimer and the fusion to endosomal membrane during virus entry into the cell (Fritz et al, 2011; Pangerl et al, 2011; Stiasny et al, 2013). Fritz et al (Fritz et al, 2011) recently demonstrated that the TM1 and/or TM2 regions of the TBEV E protein can be substituted by the corresponding sequences derived from a distantly related flavivirus (Japanese encephalitis virus), but this resulted in dramatic reduction of virus growth in mammalian cells.…”

Extensive flavivirus E trimer breathing accompanies stem zippering of the post‐fusion hairpin

“…in Drosophila Schneider 2 (S2) cells (Invitrogen) with an enterokinase cleavage site and a strep-tag. The pT389-sE400 [41], pT389-sE419 [28], and pT389-sE448 [28] expression plasmids were already available. The pT389-sE401 expression plasmid was generated by mutagenesis of the pT389-sE400 clone (GeneArt site-directed mutagenesis system, Invitrogen) following the manufacturer's instructions.…”

“…Recombinant proteins after enterokinase cleavage (sE400r, sE401r, sE404r, sE408r, sE412r, sE419r, sE428r) as well as the virion-derived sE401v [45] were converted into trimers under acidic pH conditions in the presence of liposomes as described previously [28][29][30]. Fifteen nanomoles of lipids per microgram sE was mixed and acidified by the addition of 350 mM morpholineethanolsulfonic acid (MES).…”

Structure-based pKa prediction provides a thermodynamic basis for the role of histidines in pH-induced conformational transitions in dengue virus

“…X-ray crystallography studies [1] have shown that the structure of the soluble part of E consists of three domains and includes the fusion loop, which is critical for fusion of the host and viral membranes and putative receptor-binding region. The membrane-associated region of E includes an α-helical segment known as the “stem” region, which has also been shown to be critical for membrane fusion [2] , [3] , along with two transmembrane helices that anchor E to the viral membrane. The PrM protein consists of a globular domain that “caps” the fusion loop of E to prevent premature fusion [4] , a linker region containing a furin cleavage motif, and an α-helical membrane-associated region, including two transmembrane helices.…”