Distinct Roles of Cellular ESCRT-I and ESCRT-III Proteins in Efficient Entry and Egress of Budded Virions of Autographa californica Multiple Nucleopolyhedrovirus

Abstract

The endosomal sorting complex required for transport (ESCRT) machinery is necessary for budding of many enveloped viruses. Recently, it was demonstrated that Vps4, the key regulator for recycling of the ESCRT-III complex, is required for efficient infection by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, ESCRT assembly, regulation, and function are complex, and little is known regarding the details of participation of specific ESCRT complexes in AcMNPV infection. In this study, the core components of ESCRT-I (Tsg101 and Vps28) and ESCRT-III (Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60) were cloned from Using a viral complementation system and RNA interference (RNAi) assays, we found that ESCRT-I and ESCRT-III complexes are required for efficient entry of AcMNPV into insect cells. In cells knocking down or overexpressing dominant negative (DN) forms of the components of ESCRT-I and ESCRT-III complexes, entering virions were partially trapped within the cytosol. To examine only egress, cells were transfected with the double-stranded RNA (dsRNA) targeting an individual ESCRT-I or ESCRT-III gene and viral bacmid DNA or viral bacmid DNA that expressed DN forms of ESCRT-I and ESCRT-III components. We found that ESCRT-III components (but not ESCRT-I components) are required for efficient nuclear egress of progeny nucleocapsids. In addition, we found that several baculovirus core or conserved proteins (Ac11, Ac76, Ac78, GP41, Ac93, Ac103, Ac142, and Ac146) interact with Vps4 and components of ESCRT-III. We propose that these viral proteins may form an "egress complex" that is involved in recruiting ESCRT-III components to a virus egress domain on the nuclear membrane. The ESCRT system is hijacked by many enveloped viruses to mediate budding and release. Recently, it was found that Vps4, the key regulator of the cellular ESCRT machinery, is necessary for efficient entry and egress of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, little is known about the roles of specific ESCRT complexes in AcMNPV infection. In this study, we demonstrated that ESCRT-I and ESCRT-III complexes are required for efficient entry of AcMNPV into insect cells. The components of ESCRT-III (but not ESCRT-I) are also necessary for efficient nuclear egress of progeny nucleocapsids. Several baculovirus core or conserved proteins were found to interact with Vps4 and components of ESCRT-III, and these interactions may suggest the formation of an "egress complex" involved in the nuclear release or transport of viral nucleocapsids.

The Autographa californica Multiple Nucleopolyhedrovirus ac51 Gene Is Required for Efficient Nuclear Egress of Nucleocapsids and Is Essential for In Vivo Virulence

“…As shown by TEM in our study and other studies (13,20), baculovirus nucleocapsids seem to pass through the NM to the Cyt by a budding process, but the mechanism by which viral structural proteins are involved in this process remains unknown. Viral proteins, including Ac11, Ac76, Ac78, GP41, Ac93, Ac103, Ac142, and Ac146, which are essential for nucleocapsid egress from the nucleus and ODV formation, have recently been shown to interact with the soluble cellular NSF (N-ethylmaleimide-sensitive factor) and ESCRT-III (endosomal sorting complex required for transport) proteins directly or indirectly (11,57). NSF functions by triggering the fusion of transport vesicles with their target membranes, and ESCRT-III is essential for membrane remodeling and is part of the scission machinery for sorting ubiquitinated membrane proteins into intraluminal vesicles.…”

“…NSF functions by triggering the fusion of transport vesicles with their target membranes, and ESCRT-III is essential for membrane remodeling and is part of the scission machinery for sorting ubiquitinated membrane proteins into intraluminal vesicles. These viral proteins have been speculated to form a complex to direct nucleocapsids to the Ac76-rich budding region on the NM and to recruit ESCRT-III proteins to promote the nuclear egress of nucleocapsids, and NSF may help cleave vesicles containing progeny nucleocapsids during nuclear egress (11,14,57).…”

“…Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most extensively studied baculovirus and belongs to the genus Alphabaculovirus. In addition to cellular microtubules, actin cytoskeletons, and ESCRT-III proteins (9)(10)(11), some viral structural proteins encoded by AcMNPV have been found to participate in the anterograde transport of nucleocapsids. VP80 has been reported to interact with F-actin in the nuclei of infected cells to mediate the transport of nucleocapsids from the VS to the periphery of the nucleus (9,12).…”

Autographa californica Multiple Nucleopolyhedrovirusorf13is Required for Efficient Nuclear Egress of Nucleocapsids

“…By contrast, the mechanism of baculovirus nucleocapsid egress remains unclear. According to previous reports, host proteins including the actin cytoskeleton, N-ethylmaleimide-sensitive fusions proteins and endosomal sorting complex required for transport-III (11)(12)(13) as well as viral proteins including Ac11, Ac51, Ac66, Ac75, Ac78, GP41, Ac93, P48, EXON0…”

Mechanisms of nuclear lamina disruption and regulation of nuclear budding of herpes simplex virus type-1

“…Viral proteins ac11, ac93 and p48 have also been shown to interact with ESCRT-III proteins (111), and these proteins are essential for intranuclear microvesicle formation and BV nuclear egress (114)(115)(116). Deletion of any of these proteins results in a loss of BV and ODV envelopment, suggesting that the two processes share the same mechanism.…”