Among the various host cellular processes that are hijacked by flaviviruses, few mechanisms have been described with regard to viral egress. Here we investigate how flaviviruses exploit Src family kinases (SFKs) for exit from infected cells. We identify Lyn as a critical component for secretion of Dengue and Zika infectious particles and their corresponding virus like particles (VLPs). Pharmacological inhibition or genetic depletion of the SFKs, Lyn in particular, block virus secretion. Lyn−/− cells are impaired in virus release and are rescued when reconstituted with wild-type Lyn, but not a kinase- or palmitoylation-deficient Lyn mutant. We establish that virus particles are secreted in two distinct populations – one as free virions and the other enclosed within membranes. Lyn is critical for the latter, which consists of proteolytically processed, infectious virus progenies within autophagosome-derived vesicles. This process depends on Ulk1, Rab GTPases and SNARE complexes implicated in secretory but not degradative autophagy and occur with significantly faster kinetics than the conventional secretory pathway. Our study reveals a previously undiscovered Lyn-dependent exit route of flaviviruses in LC3+ secretory organelles that enables them to evade circulating antibodies and might affect tissue tropism.

Running title: Palmitoylated Lyn regulates unconventional secretion of flaviviruses SummaryAmong the various host cellular processes that are hijacked by flaviviruses, very few mechanisms have been described with regard to viral secretion. Here we investigated how flaviviruses exploit the Src family kinases (SFKs) for exit from infected cells. We isolated three members of the SFK family -Src, Fyn and Lyn -that were specifically activated during secretion of Dengue and Zika or their corresponding virus like particles (VLPs). Pharmacological inhibition or genetic depletion of the SFKs blocked virus secretion, most significantly upon Lyn-deficiency. Lyn -/cells were severely impaired in virus release, and were rescued when reconstituted with wild-type Lyn, but not a kinaseor palmitoylation-deficient Lyn mutant. We further established that Lyn, via its palmitoylation-dependent membrane association, triggered post-Golgi virus transport in specialised Rab11 and Transferrin receptor positive organelles resembling secretory autophagosomes, and distinct from conventional exocytic vesicles. In the absence of Lyn activity or its aberrant membrane association, virions were sorted into the lysosomal pathway for degradation. This mode of export was specifically triggered by processed, and mature, but not by furin-resistant virus particles, and occurred with significantly faster kinetics than the conventional secretory pathway. Our study therefore charts a previously undiscovered Lyn-dependent exit strategy, triggered by flaviviruses in secretory autophagosomes that might enable them to evade circulating antibodies and dictate tissue tropism.