The fusion of enveloped viruses with cellular membranes is mediated by proteins that are anchored in the lipid bilayer of the virus and capable of triggered conformational changes necessary for driving fusion. The flavivirus envelope protein E is the only known viral fusion protein with a double membrane anchor, consisting of two antiparallel transmembrane helices (TM1 and TM2). TM1 functions as a stop-transfer sequence and TM2 as an internal signal sequence for the first nonstructural protein during polyprotein processing. The possible role of this peculiar C-terminal helical hairpin in membrane fusion has not been investigated so far. We addressed this question by studying TM mutants of tick-borne encephalitis virus (TBEV) recombinant subviral particles (RSPs), an established model system for flavivirus membrane fusion. The engineered mutations included the deletion of TM2, the replacement of both TM domains (TMDs) by those of the related Japanese encephalitis virus (JEV), and the use of chimeric TBEV-JEV membrane anchors. Using these mutant RSPs, we provide evidence that TM2 is not just a remnant of polyprotein processing but, together with TM1, plays an active role in fusion. None of the TM mutations, including the deletion of TM2, affected early steps of the fusion process, but TM interactions apparently contribute to the stability of the postfusion E trimer and the completion of the merger of the membranes. Our data provide evidence for both intratrimer and intertrimer interactions mediated by the TMDs of E and thus extend the existing models of flavivirus membrane fusion.Membrane fusion is a crucial step during the cell entry of enveloped viruses and is mediated by specific membrane-anchored viral surface proteins (fusion proteins) (11, 24). According to their molecular architecture, these have been assigned to three different structural classes (classes I, II, and III) (11,41). They all drive fusion by conformational changes that are triggered by interactions with the host cell (such as receptor binding or exposure to acidic pH) and presumably involve protein-protein interactions at the fusion site (41). Classes I and III, and the class II fusion proteins of alphaviruses, possess a single transmembrane (TM) domain that functions as a membrane anchor and is followed by a cytoplasmic tail of varying length (41). In contrast, the flavivirus class II viral fusion protein E is unique in possessing a hairpin-like doublemembrane-spanning carboxy terminus, derived from a special combination of stop-transfer and internal signal sequences, required for the intracellular sorting and processing of the flaviviral polyprotein (21) (Fig. 1A).Flaviviruses are members of the genus Flavivirus (family Flaviviridae) and comprise a number of important human pathogens, including the dengue viruses, Japanese encephalitis virus (JEV), yellow fever virus, West Nile virus, and tick-borne encephalitis virus (TBEV) (10, 40). They are small, enveloped, positive-strand RNA viruses that are assembled in the endoplasmic reticulum (ER) ...

The structural proteins of flaviviruses carry a unique set of transmembrane domains (TMDs) at their C termini that are derived from the mode of viral polyprotein processing. They function as internal signal and stop-transfer sequences during protein translation, but possible additional roles in protein interactions required during assembly and maturation of viral particles are ill defined. To shed light on the role of TMDs in these processes, we engineered a set of tick-borne encephalitis virus mutants in which these structural elements were replaced in different combinations by the homologous sequences of a distantly related flavivirus (Japanese encephalitis virus). The effects of these modifications were analyzed with respect to protein synthesis, viral particle secretion, specific infectivity, and acidic-pH-induced maturation processes. We provide evidence that interactions involving the double-membrane anchor of the envelope protein E (a unique feature compared to other viral fusion proteins) contribute substantially to particle assembly, stability, and maturation. Disturbances of the inter-and intra-TMD interactions of E resulted in the secretion of a larger proportion of capsidless subviral particles at the expense of whole virions, suggesting a possible role in the still incompletely understood mechanism of capsid integration during virus budding. In contrast, the TMD initially anchoring the C protein to the endoplasmic reticulum membrane does not appear to take part in envelope protein interactions. We also show that E TMDs are involved in the envelope protein rearrangements that are triggered by acidic pH in the trans-Golgi network and represent a hallmark of virus maturation. IMPORTANCEThe assembly of flaviviruses occurs in the endoplasmic reticulum and leads to the formation of immature, noninfectious particles composed of an RNA-containing capsid surrounded by a lipid membrane, with the two integrated envelope proteins, prM and E, arranged in an icosahedral lattice. The mechanism by which the capsid is formed and integrated into the budding viral envelope is currently unknown. We provide evidence that the transmembrane domains (TMDs) of E are essential for the formation of capsid-containing particles and that disturbances of these interactions lead to the preferential formation of capsidless subviral particles at the expense of whole virions. E TMD interactions also appear to be essential for the envelope protein rearrangements required for virus maturation and for the generation of infectious virions. Our data thus provide new insights into the biological functions of E TMDs and extend their role during viral polyprotein processing to additional functions in particle assembly and maturation.T he genus Flavivirus in the family Flaviviridae comprises 53 taxonomically recognized species (1), including the humanpathogenic mosquito-borne dengue, Zika, yellow fever, Japanese encephalitis, and West Nile viruses as well as tick-borne encephalitis virus (TBEV) (2). Flaviviruses are small enveloped po...