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 application of RNA interference (RNAi) technology for pest control is environmentally friendly and accurate. However, the efficiency of RNAi is often inconsistent and unreliable, and finding a suitable carrier element is considered critical to success in overcoming biotic and abiotic barriers to reach the target site. The fall armyworm, Spodoptera frugiperda (FAW), which is one of most important global agricultural pests, has recently spread rapidly to other parts of the world. In this study, a method to improve the stability and RNAi efficiency of the dsRNA carrier complex was reported. Methoprene-tolerant gene (Met) was selected as a target, a gene which is critical to the growth and development of FAW. Biomaterials nanoliposomes (LNPs) were modified with polyethylenimine (PEI) to deliver the dsRNA of Met. The synthesized Met3@PEI@LNPs reached a size of 385 nm and were found to load dsRNA effectively. Through stability and protection assays, it was found that LNPs provided reliable protection. In addition, the release curve also demonstrated that LNPs were able to prevent premature release under alkaline condition of the insect midgut but accelerate the release after entering the acidic environment of the target cells. The cell transfection efficiency of the prepared LNPs reached 96.4%. Toxicity tests showed that the use of LNPs could significantly improve the interference efficiency, with 91.7% interference efficiency achieved when the concentration of dsRNA in LNPs was only 25% of that of the control. Successful interference of Met demonstrated it could significantly shorten the larval period and make the larvae pupate earlier, thus achieving the purpose of control. In this study, we have demonstrated the use of nanotechnology to provide a novel RNAi delivery method for pest control.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) GP64 is a class III viral fusion protein that mediates low-pH triggered membrane fusion during virus entry. Although the structure of GP64 in a postfusion conformation has been solved, its prefusion structure and the mechanism of how the protein refolds to execute fusion are unknown. In postfusion structure, GP64 is composed of five domains (domain I-V). Domain IV (374-407 aa) contains two loops (loop 1 and loop 2) that form a hydrophobic pocket at the membrane-distal end of the molecule. To determine the roles of domain IV, we used alanine-scanning mutagenesis to substitute each of the residues and the contacts within domain IV and evaluate their contributions to GP64-mediated membrane fusion and virus infection. In many cases, substitution of a single amino acid has no significant impact on GP64. However, substitution of R392 or disrupting the contact N381-N385, N384-Y388, N385-W393, or K389-W393 resulted in poor cell surface expression and fusion loss of the modified GP64, whereas substitution of E390 or G391, or disrupting the contact N381-K389, N381-Q401, or N381-I403 reduced the cell surface level of the constructs and the ability of GP64 to mediate fusion pore expansion. In contrast, substitution of N407 or disrupting the contact D404-S406 appears to restrict fusion pore expansion without affecting expression. Combined with the identification of these constructs remaining stable prefusion conformation or dramatically less efficient transition from a prefusion to postfusion state under acidic conditions, we proposed that domain IV is necessary for refolding of GP64 during membrane fusion. Importance Baculoviruses GP64 is grouped with rhabdoviruses G, herpesviruses gB, and thogotoviruses glycoproteins as class III viral fusion proteins. In their postfusion structures, these proteins contain five domains (domain I-V). Distinguished from domain IV of rhabdoviruses G and herpesviruses gB that composed of β-sheets, domain IV of GP64 is a loop region and the same domain in thogotovirues glycorproteins has not been solved. In addition, domain IV is proximal to domain I (fusion domain) in prefusion structures of vesicular stomatitis virus (VSV) G and human cytomegalovirus (HCMV) gB but resides at the domain I-distal end of the molecule in a postfusion conformation. In this study, we identified that the highly conserved residues and the contacts within domain IV of AcMNPV GP64 are necessary for low-pH triggered conformational change and fusion pore expansion. Our results highlight the roles of domain IV of class III viral fusion proteins in refolding during membrane fusion.

Peptidoglycan recognition proteins (PGRPs) are a class of molecules that play a critical role in insect immunity. Understanding the function of PGRPs is important to improve the efficiency of microbial insecticides. In this study, we investigated the role of PGRP-LB (a long type PGRP) in insect immunity against viruses using Spodoptera exigua and Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) as an insectvirus model. We cloned and identified a PGRP-LB gene from S. exigua; the gene consisted of 7 exons that encoded a polypeptide of 234 amino acids with a signal peptide and a typical amidase domain. Expression analysis revealed that the abundance of SePGRP-LB transcripts in the fat body was greater than in other tissues. Overexpression of SePGRP-LB resulted in a significant decrease of 49% in the rate of SeMNPV-infected cells. In addition, the multiplication of SeMNPV was significantly decreased: a decrease of 79% in the production of occlusion-derived virion (ODV), and a maximum decrease of 50% in the production of budded virion (BV). In contrast, silencing of SePGRP-LB expression by RNA interference resulted in a significant 1.65-fold increase in the rate of SeMNPVinfected cells, a significant 0.54-fold increase in ODV production, a maximum 1.57-fold increase in BV production, and the larval survival dropped to 21%. Our findings show that SePGRP-LB has an antiviral function against SeMNPV, and therefore this gene may provide a target for lepidopteran pest control using virus insecticides.

The Autographa californica multiple nucleopolyhedrovirus GP64 is a class III viral fusion protein. Although the post-fusion structure of GP64 has been solved, its pre-fusion structure and the detailed mechanism of conformational change are unknown. In GP64, domain V is predicted to interact with two domain I segments that flank fusion loop 2. To evaluate the significance of the amino acids involved in these interactions, we examined 24 amino acid positions that represent interacting and conserved residues within domains I and V. In several cases, substitution of a single amino acid involved in a predicted interaction disrupted membrane fusion activity, but no single amino acid pair appears to be absolutely required. We identified 4 critical residues in domain V (G438, W439, T452, and T456) that are important for membrane fusion, and two residues (G438 and W439) that appear to be important for formation or stability of the pre-fusion conformation of GP64.

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