Membrane Anchors of the Structural Flavivirus Proteins and Their Role in Virus Assembly

Abstract

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...

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“…blocking viral protein degradation, or the targeted degradation of host innate immune response proteins. As expected, C, PreM and Env were localized to the ER, where virus factories are created and viral particles assembled (67). We also detected NS2A, NS2B, NS4A, and NS4B at this organelle.…”

Mosquito cell-derived West Nile virus replicon particles mimic arbovirus inoculum and have reduced spread in mice

“…The actual MOI would quickly rise, masking any difference between MOIs upon initial inoculation. Since flaviviruses can produce several types of particles, such as mature, immature and empty subviral particles [4,[39][40][41][42][43][44], we characterized our mosquito cell-derived RP stock to ensure that it resembled infectious WNV for several key characteristics. This is especially important for animal studies because immature, non-infectious and empty subviral particles have reduced or no infectivity, but they can still interact with cellular receptors, stimulate the immune response [9,[45][46][47]49], and potentially confound results if present in excessive quantities.…”

“…Based on the previous cell culture studies, one would expect more infected dendritic cells trafficking to the lymph node for mosquito cell-derived RPs, but this was not observed. Finally, empty subviral particles are produced in flavivirus-infected mammalian and mosquito cell culture and in mice [39][40][41][42][43], and it is possible that variable amounts of these particles might influence spread (e.g. by stimulating immune cells and changing the microenvironment).…”

“…In addition to fully infectious virus particles, flaviviruses can produce immature, non-infectious, and empty subviral particles [4,[39][40][41][42][43][44]. Thus, prior to using the mosquito cell-derived RPs in our studies, we compared characteristics of the RP stocks to standard virus stocks produced in mammalian and insect cells.…”

“…Empty subviral particles can be produced by flavivirus-infected cells [39][40][41][42][43] and artificially in cells transfected with structural genes from flaviviruses, e.g. WNV [45,46], Japanese encephalitis virus [42,47], and dengue virus [48].…”