Roles of ESCRT proteins (ALIX and CHIMP4A) and their interplay with ISG15 during tick-borne flavivirus infection.

Flaviviruses are usually transmitted to humans via mosquito or tick bites. During infection, virus replication and assembly, whose cellular sites are relatively close, are controlled by virus proteins and a diverse range of host proteins. By siRNA-mediated gene silencing, we show that ALIX and CHMP4A, two members of the host endosomal sorting complex required for transport (ESCRT) protein machinery, are required for flavivirus infection. Using cell lines expressing subgenomic replicons and replicon virus-like particles, we demonstrate specific roles for ALIX and CHMP4A in viral replication and assembly, respectively. Employing biochemical methodology, we show that the ESCRT proteins are recruited by a putative specific late (L) domain motif LYXLA within the NS3 protein of tick-borne flaviviruses. Furthermore, to counteract the recruitment of ESCRT proteins, the host cells may elicit defense mechanisms. We found that ectopic expression of the interferon-stimulated gene 15 (ISG15) or the E3 ISG15-protein ligase (HERC5) reduced virus replication by suppressing the positive effects of ALIX and CHMP4A. Collectively, these results have provided new insights into flavivirus-host cell interactions that function as checkpoints, including the NS3 and the ESCRT proteins, the ISG15 and the ESCRT protein, at essential stages of the virus life cycle. IMPORTANCE Flaviviruses are important zoonotic viruses with high fatality rates worldwide. Here, we report that during infection the virus employs ESCRT protein members for virus replication and assembly. Among the ESCRT proteins, ALIX acts during virus replication, while CHMP4A is required during virus assembly. Other ESCRT protein members such as TSG101 are not required for virus production. The ESCRT, ALIX -CHMP4A complex, is recruited to NS3 through their interactions with the putative L domain motif of NS3, while CHMP4A is recruited to E. In addition, we demonstrate the antiviral mechanism of ISG15 and HERC5, which degrades ALIX and CHIMP4A, indirectly targets virus infection. In summary, we reveal host-dependency factors supporting flavivirus infection, but these factors may also be targeted by antiviral host effector mechanisms.