An Absolutely Conserved Tryptophan in the Stem of the Envelope Protein E of Flaviviruses Is Essential for the Formation of Stable Particles.

Iris Medits,Franz X Heinz,Karin Stiasny

doi:10.3390/v13091727

Abstract

The major envelope protein E of flaviviruses contains an ectodomain that is connected to the transmembrane domain by the so-called “stem” region. In mature flavivirus particles, the stem is composed of two or three mostly amphipathic α-helices and a conserved sequence element (CS) with an undefined role in the viral life cycle. A tryptophan is the only residue within this region which is not only conserved in all vector-borne flaviviruses, but also in the group with no known vector. We investigated the importance of this residue in different stages of the viral life cycle by a mutagenesis-based approach using tick-borne encephalitis virus (TBEV). Replacing W421 by alanine or histidine strongly reduced the release of infectious virions and their thermostability, whereas fusion-related entry functions and virus maturation were still intact. Serial passaging of the mutants led to the emergence of a same-site compensatory mutation to leucine that largely restored these properties of the wildtype. The conserved tryptophan in CS (or another big hydrophobic amino acid at the same position) is thus essential for the assembly and infectivity of flaviviruses by being part of a network required for conferring stability to infectious particles.

Highlights

IntroductionFlaviviruses comprise a number of arthropod-borne human pathogens such as tickborne encephalitis (TBE), yellow fever, Japanese encephalitis (JE), Zika and dengue viruses
The W421A mutant showed a significant reduction of E detectable in the cell culture supernatant (Figure 2A), but a significant difference was not detected by qPCR (Figure 2B)
The mutants were still capable of displaying those acidicpH-triggered conformational changes that convert metastable E dimers into stable trimers and are necessary for membrane fusion during virus entry

Summary

Introduction

Flaviviruses comprise a number of arthropod-borne human pathogens such as tickborne encephalitis (TBE), yellow fever, Japanese encephalitis (JE), Zika and dengue viruses In their mature form, these small enveloped viruses are completely covered by an icosahedral shell of homodimers of the major surface glycoprotein E [1]. The E glycoprotein is an elongated molecule consisting of three external domains (DI, DII, DIII), connected to a double membrane anchor by the so-called stem This structural element consists of two or three mostly amphipathic helices (H1, H2, H3) [3,4,5,6,7,8,9,10,11,12] and is located between the E-ectodomain and the viral membrane (Figure 1A). Two regions in E are highly conserved, the fusion loop (FL) at the tip of DII and the so-called conserved sequence in the stem (CS, amino acids 420–425 in TBEV, [13]) that forms a loop between H2 and H3

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