ABSTRACT
TER94 is a multifunctional AAA+ ATPase crucial for diverse cellular processes, especially protein quality control and chromatin dynamics in eukaryotic organisms. Many viruses, including coronavirus, herpesvirus, and retrovirus, coopt host cellular TER94 for optimal viral invasion and replication. Previous proteomics analysis identified the association of TER94 with the budded virions (BVs) of baculovirus, an enveloped insect large DNA virus. Here, the role of TER94 in the prototypic baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) life cycle was investigated. In virus-infected cells, TER94 accumulated in virogenic stroma (VS) at the early stage of infection and subsequently partially rearranged in the ring zone region. In the virions, TER94 was associated with the nucleocapsids of both BV and occlusion-derived virus (ODV). Inhibition of TER94 ATPase activity significantly reduced viral DNA replication and BV production. Electron/immunoelectron microscopy revealed that inhibition of TER94 resulted in the trapping of nucleocapsids within cytoplasmic vacuoles at the nuclear periphery for BV formation and blockage of ODV envelopment at a premature stage within infected nuclei, which appeared highly consistent with its pivotal function in membrane biogenesis. Further analyses showed that TER94 was recruited to the VS or subnuclear structures through interaction with viral early proteins LEF3 and helicase, whereas inhibition of TER94 activity blocked the proper localization of replication-related viral proteins and morphogenesis of VS, providing an explanation for its role in viral DNA replication. Taken together, these data indicated the crucial functions of TER94 at multiple steps of the baculovirus life cycle, including genome replication, BV formation, and ODV morphogenesis.
IMPORTANCE TER94 constitutes an important AAA+ ATPase that associates with diverse cellular processes, including protein quality control, membrane fusion of the Golgi apparatus and endoplasmic reticulum network, nuclear envelope reformation, and DNA replication. To date, little is known regarding the role(s) of TER94 in the baculovirus life cycle. In this study, TER94 was found to play a crucial role in multiple steps of baculovirus infection, including viral DNA replication and BV and ODV formation. Further evidence showed that the membrane fission/fusion function of TER94 is likely to be exploited by baculovirus for virion morphogenesis. Moreover, TER94 could interact with the viral early proteins LEF3 and helicase to transport and further recruit viral replication-related proteins to establish viral replication factories. This study highlights the critical roles of TER94 as an energy-supplying chaperon in the baculovirus life cycle and enriches our knowledge regarding the biological function of this important host factor.
INTRODUCTION
In recent years, accumulating studies have revealed that transient endoplasmic reticulum 94 (TER94, also termed CDC48 in yeast and plants or VCP/P97 in metazoans) plays crucial roles in diverse cellular processes. As a member of the ATPases associated with diverse cellular activities (AAA+) ATPase family, TER94 utilizes the energy of ATP hydrolysis to remodel substrate proteins (1, 2) and functions in endoplasmic reticulum-associated protein degradation, mitochondrion-associated degradation, autophagy, DNA damage response, membrane fusion, NF-κB activation, and DNA replication (3–5). TER94 is an approximately 97-kDa protein in Xenopus laevis oocytes that contains two ATPase domains and forms hexamers (∼612 kDa) to implement its versatile cellular functions by cooperating with its cofactors (6, 7). These multifarious regulatory cofactors bind to TER94 at distinct binding sites or domains and recruit TER94 to specific cellular pathways. As a multifunctional hub connecting diverse cellular pathways and regulating protein homeostasis of cells, TER94 has been shown to be associated with numerous human neurodegenerative diseases and has aroused considerable interest as a target for controlling cancer cells (8–10).
Numerous studies have revealed that viruses can also hijack and exploit TER94 at different levels to establish productive infection in host cells. For example, TER94 is rearranged and colocalizes with viral proteins in viral replication organelles of enterovirus 71-infected cells (11). Conversely, knockdown of TER94 expression blocks the escape of the coronavirus infectious bronchitis virus from endosomes during the entry process (12). TER94 is also required for West Nile virus and poliovirus replication and possibly functions in the cellular secretion pathway for the latter (13, 14). Influenza virus, Rift Valley fever virus, and mouse mammary tumor virus depend on TER94 for the proper expression or transportation of viral proteins to virus assembly sites (15–18). In addition, a recent study on human cytomegalovirus showed that TER94 is required for viral protein expression and the onset of virus replication in human cells, further highlighting the potential antiviral activity of a TER94-specific inhibitor (16). However, the detailed mechanism of how TER94 functions in virus infection processes remains obscure.
Baculoviruses are insect-specific large DNA viruses that have been widely used as biological pesticides and protein expression vectors (19). Most baculoviruses have a unique biphasic life cycle characterized by the production of two distinct types of progeny virion phenotypes, budded virions (BVs) and occlusion-derived virions (ODVs). ODVs infect exclusively epithelium cells of the insect midgut to initiate primary infection, whereas BVs are subsequently produced and infect other cell types within larval bodies to spread systemic infection (20, 21). Following the entry of BV/ODV into cells, the incoming nucleocapsids will be transported into the nucleus, where the viral genome is released and DNA replication is initiated (22, 23). In the infected nuclei, a virus-induced subnuclear structure termed the virogenic stroma (VS) serves as a viral factory for successive viral DNA replication, gene transcription, and nucleocapsid assembly (19). A small fraction of progeny nucleocapsids will egress from the nuclear envelope and bud from the plasma membrane to form BVs, whereas others retained within the nucleus are enveloped to form mature ODVs at the ring zone region (19).
During these processes, many host factors are utilized by the baculovirus. For example, at the early stage of infection, host RNA polymerase II and transcription machinery are involved in baculovirus early gene transcription (19, 24, 25). Host cytoskeleton components, including actin and tubulin, are responsible for transporting baculovirus virions during virus entry and egress (26–