Abstract
The endosomal sorting complex required for transport (ESCRT) is a protein machine mediating membrane scission. In intraluminal vesicle (ILV) formation, ESCRT-0 targets cargoes and recruits ESCRT-I/-II to create membrane invagination, whereas ESCRT-III coordinates with the AAA ATPase Vps4 and its cofactor Vta1 to catalyze the membrane fission. Recently, ESCRT-I/-III and Vps4 were found to be involved in the entry of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, the necessity of other ESCRT components and the interplay of viral proteins and ESCRTs in regulating the virus entry remain elusive. Here, we identified ESCRT-0 (Hse1 and Vps27), ESCRT-II (Vps22, Vps25, and Vps36), and Vta1 of Spodoptera frugiperda. RNAi depletion of Vta1 but not the components of ESCRT-0 or ESCRT-II in Sf9 cells significantly reduced budded virus (BV) production. Quantitative PCR together with confocal microscopy analyses indicated that Vta1 was required for internalization and endosomal trafficking of BV. In the late phase of infection, although Vps4 and Vta1 were both distributed to the nucleus and at the plasma membrane, depletion of Vta1 did not affect BV release. Further analysis revealed that 7 of 14 BV envelope proteins (Ac75, Ac93, E25, F-like, P33, P48, and vUbiquitin) interacted with Vps4 and Vta1. Intriguingly, Ac93 adopted a similar mode as ESCRT-III proteins to interact with the microtubule-interacting and transport (MIT) domains of Vps4 and Vta1 via its C-terminal MIT-interacting motifs (MIM1), and the interactions were necessary for BV internalization. Together, our studies highlight the coordination of Vps4-Vta1 and Ac93, and probably other BV envelope proteins, in facilitating entry of AcMNPV.IMPORTANCEThe endosomal sorting complex required for transport (ESCRT) system is involved in the entry of diverse DNA and RNA viruses. However, the interplay of viral proteins and ESCRTs in promoting virus endocytosis remains largely unknown. Here, we found that the ESCRT early acting factors ESCRT-0/-II were not necessary for infectious budded virus (BV) production of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). In contrast, the Vps4 cofactor Vta1 was required for entry but not egress of BV. Several core or essential BV envelope proteins were identified to interact with Vps4 and Vta1. Among them, Ac93 plays a central role in connecting other viral proteins and mimics ESCRT-III proteins to interact with Vps4-Vta1, facilitating entry of BV virions. These studies provide evidence for the coordination of viral proteins and ESCRTs in regulating entry of large enveloped DNA viruses.