Molecular mechanisms regulating the pH-dependent pr/E interaction in yellow fever virus.

Flavivirus particles bud in the ER of infected cells as immature virions composed of 180 heterodimers of glycoproteins prM and E, associated as 60 (prM/E)3 trimeric spikes. Exposure to the mildly acidic pH of the TGN results in dissociation of the trimeric spikes followed by re-association of the prM/E protomers into 90 dimers organized in a characteristic herringbone pattern. The furin site in prM is exposed in the dimers for maturation of prM into M and pr. For flaviviruses such as the tick-borne encephalitis virus (TBEV) as well as for dengue virus, it was shown that at neutral pH pr loses affinity for E, such that it dissociates from the mature particle as soon as it reaches the external milieu, which is at neutral pH. Using a soluble recombinant form of E (sE) and pr from yellow fever virus (YFV), we show here that the affinity of pr for recombinant E protein remains high even at neutral pH. The X-ray structure of YFV pr/sE shows more extensive inter-chain hydrogen bonding than does the dengue or TBEV, and also that it retains the charge complementarity between the interacting surfaces of the two proteins even at neutral pH. We further show that pr blocks sE flotation with liposomes when exposed at low pH at a 1:1 stoichiometry, yet in the context of the virus particle, an excess of 10:1 pr:E ratio is required to block virus/liposome fusion. In aggregate, our results show that the paradigm obtained from earlier studies of other flaviviruses does not apply to yellow fever virus, the flavivirus type species. A mechanism that does not rely solely in a change in the environmental pH is thus required for the release of pr from the mature particles upon release from infected cells. These results open up new avenues to understand the activation mechanism that yields mature, infectious YFV particles. ### Competing Interest Statement The authors have declared no competing interest.