Multifaceted interactions between host ESCRT-III and budded virus-related proteins involved in entry and egress of the baculovirus Autographa californica multiple nucleopolyhedrovirus.

The endosomal sorting complex required for transport (ESCRT) is a conserved protein machine mediating membrane remodeling and scission. In the context of viral infection, different components of the ESCRT-III complex, which serve as the core machinery to catalyze membrane fission, are involved in diverse viruses' entry, replication, and/or budding. However, the interplay between ESCRT-III and viral factors in the virus life cycle, especially for that of large enveloped DNA viruses, is largely unknown. Recently, the ESCRT-III components Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60 were determined for entry and/or egress of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Here, we identified the final three ESCRT-III components Chm7, Ist1, and Vps2A of Spodoptera frugiperda. Overexpression of the dominant-negative forms of these proteins or RNAi downregulation of their transcripts significantly reduced infectious budded viruses (BVs) production of AcMNPV. Quantitative PCR together with confocal and transmission electron microscopy analysis revealed that these proteins were required for internalization and trafficking of BV during entry and egress of nucleocapsids. In infected Sf9 cells, nine ESCRT-III components were distributed on the nuclear envelope and plasma membrane, and except for Chm7, the other components were also localized to the intranuclear ring zone. Y2H and BiFC analysis revealed that 42 out of 64 BV-related proteins including 35 BV structural proteins and 7 non-BV structural proteins interacted with single or multiple ESCRT-III components. By further mapping the interactome of 64 BV-related proteins, we established the interaction networks of ESCRT-III and the viral protein complexes involved in BV entry and egress.IMPORTANCEFrom archaea to eukaryotes, the endosomal sorting complex required for transport (ESCRT)-III complex is hijacked by many enveloped and nonenveloped DNA or RNA viruses for efficient replication. However, the mechanism of ESCRT-III recruitment, especially for that of large enveloped DNA viruses, remains elusive. Recently, we found the ESCRT-III components Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60 are necessary for the entry and/or egress of budded viruses (BVs) of Autographa californica multiple nucleopolyhedrovirus. Here, we demonstrated that the other three ESCRT-III components Chm7, Ist1, and Vps2A play similar roles in BV infection. By determining the subcellular localization of ESCRT-III components in infected cells and mapping the interaction of nine ESCRT-III components and 64 BV-related proteins, we built the interaction networks of ESCRT-III and the viral protein complexes involved in BV entry and egress. These studies provide a fundamental basis for understanding the mechanism of the ESCRT-mediated membrane remodeling for replication of baculoviruses.

Aphid Viruses: A Brief View of a Long History.

Aphids are common agricultural pests with a wide range of hosts from agriculture to forestry plants. As known, aphids also serve as the major vectors to transmit plant viruses. Although numerous studies have focused on interactions between aphids and plant viruses, little is known about the aphid viruses, i.e., the insect viruses that are infectious to aphids. In the past four decades, several aphid viruses have been identified in diverse aphid species. In this review, we present a brief view of the aphid pathogenic viruses from several aspects, including classification of aphid viruses and characters of the viral genome, integration of viral sequences in host genomes, infection symptoms and influence on aphids, as well as host range and transmission modes. Taken together, these studies have increased our understanding of the rarely known aphid viruses, and will potentially contribute to the development of new strategies for controlling aphid populations.

Rapid analysis and identification of dianthrone glycosides in Polygoni Multiflori Caulis based on enrichment of macroporous absorbent resin and UPLC-Q-TOF-MS/MS.

INTRODUCTION: Polygoni Multiflori Caulis (PMC) has been used as a traditional Chinese medicine for a long time in China. However, hepatotoxic events of PMC have been reported in recent years, but the potential toxic compounds have remained unclear. Dianthrones as the secondary plant metabolites were revealed to potential hepatotoxicity in a previous study. However, no reports focused on dianthrones in PMC. OBJECTIVE: In the quest for exploring potential hepatotoxic compounds in PMC, the aim of this work was to undertake a comprehensive characterisation of dianthrones in PMC. METHODS: A simple and effective macroporous absorbent resin column chromatography method was established in this study to enrich the minor dianthrones from PMC extracts. Exploration and characterisation of dianthrones in PMC was conducted by an ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS/MS) method and information dependent acquisition (IDA) mode. The aglycones of dianthrone glycosides were further verified by acid hydrolysis method. RESULTS: Seventy-two dianthrone glycosides and their five aglycones were discovered and tentatively characterised in PMC for the first time, of which 29 dianthrones were identified as potential new compounds. Dianthrone glycosides could be classified into three types according to their aglycone structures, and their fragmentation pathway rules and diagnosed ions were also summarised comprehensively. CONCLUSION: This was the first comprehensive investigation on dianthrones in PMC. The result would help to fully understand the phytochemical constituents and toxic components in PMC, and highlight the need for further toxicological investigations of the dianthrones in PMC due to their potential hepatotoxicity correlation.

Structural differences of polysaccharides from Astragalus before and after honey processing and their effects on colitis mice.

Honey-processed Astragalus is a dosage form of Radix Astragali processed with honey, which exhibits better efficacy of tonifying Qi than the raw product. Polysaccharides are its main water-soluble active components. This work was designed to study the structural differences of homogeneous honey-processed Astragalus polysaccharides (HAPS3a) and Astragalus polysaccharides (APS3a) and their effects on colitis mice. The results showed that HAPS3a (Mw = 2463.5 kDa) and APS3a (Mw = 3373.2 kDa) differed in molecular weight, monosaccharide compositions, glycosidic bonds and degree of branching (DB). Notably, the molar ratios of galactose and galacturonic acid in HAPS3a were 22.66% and 33.24%, while those in APS3a were 11.87% and 49.55%, respectively. The uronic acid residues 1,4-ß-GalpA and 1,6-α-GlcpA of the backbone in APS3a were converted into the corresponding neutral residues in HAPS3a after honey processing. The different DB of HAPS3a (15.35%) and APS3a (25.13%) suggested that the chain conformation became smoother. The anti-inflammatory effects on colitis mice revealed that HAPS3a exhibited better effects than APS3a by protecting intestinal mucosa, regulating the expression of cytokines and influencing microbiota diversity. Taken together, the differences in anti-inflammatory activity might be related to structural differences caused by honey processing. Our findings have laid a foundation for the processing mechanism of Astragalus.

Enhanced hydrolysis of ß-cypermethrin caused by deletions in the glycin-rich region of carboxylesterase 001G from Helicoverpa armigera.

BACKGROUND: Carboxylesterase (CarE) is a major class of enzyme involved in the detoxification of toxic xenobiotics in various insect species. Previous work has shown that the carboxylesterase gene CarE001G found in Helicoverpa armigera is more active and can metabolize synthesized pyrethroids, such as ß-cypermethrin, one of the commonly used commercial insecticides for lepidopteran pest control. In addition, CarE001G is very special as it has a very specific glycine-rich region located adjacent to its C-terminal. But whether mutations in this unique sequence can change the biochemistry and function of CarE001G are unknown. RESULTS: In this study, four variants of CarE001G with different deletions in the glycine-rich region were obtained and functionally expressed in Escherichia coli. The recombinant proteins were purified and confirmed by Western blot and mass spectrometry analyses. These mutant enzymes showed high catalytic efficiency toward the model substrate α-naphthyl acetate. Inhibition study showed that ß-cypermethrin had relatively strong inhibition on CarE activities. In vitro metabolism assay showed that the mutant enzymes significantly enhanced their metabolic activities toward ß-cypermethrin with specific activities between 4.0 and 5.6 nmol L-1 min-1 mg-1 protein. Molecular docking analyses consistently demonstrated that deletion mutations in the glycine-rich region may facilitate the anchoring of the ß-cypermethrin molecule in the active binding pocket of the mutant enzymes. CONCLUSION: The data show that deletion mutations can cause qualitative change in the capacity of CarEs in the detoxification of ß-cypermethrin. This indicates that deletion mutations in the glycine-rich region may have the potential to cause synthesized pyrethroid (SP) resistance in H. armigera in the future. © 2020 Society of Chemical Industry.

Critical Residues and Contacts within Domain IV of Autographa californica Multiple Nucleopolyhedrovirus GP64 Contribute to Its Refolding during Membrane Fusion.

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 its postfusion structure, GP64 is composed of five domains (domains I to V). Domain IV (amino acids [aa] 374 to 407) 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 replace each of the individual residues and the contact-forming residues within domain IV and evaluate their contributions to GP64-mediated membrane fusion and virus infection. In many cases, replacement of a single amino acid had no significant impact on GP64. However, replacement of R392 or disruption of the N381-N385, N384-Y388, N385-W393, or K389-W393 contact resulted in poor cell surface expression and fusion loss of the modified GP64, whereas replacement of E390 or G391 or disruption of the N381-K389, N381-Q401, or N381-I403 contact reduced the cell surface expression level of the constructs and the ability of GP64 to mediate fusion pore expansion. In contrast, replacement of N407 or disruption of contact D404-S406 appeared to restrict fusion pore expansion without affecting expression. Combined with the finding that these constructs remain in the prefusion conformation or have a dramatically less efficient transition from the prefusion to the postfusion state under acidic conditions, we proposed that domain IV is necessary for refolding of GP64 during membrane fusion.IMPORTANCE Baculovirus GP64 is grouped with rhabdovirus G, herpesvirus gB, and thogotovirus glycoproteins as a class III viral fusion protein. In their postfusion structures, these proteins contain five domains (domains I to V). Distinct from domain IV of rhabdovirus G and herpesvirus gB proteins, which is composed of ß-sheets, domain IV of GP64 is a loop region; the same domain in thogotovirus glycoproteins 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 highly conserved residues and 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.

Functional Characterization of Two Carboxylesterase Genes Involved in Pyrethroid Detoxification in Helicoverpa armigera.

Insect carboxylesterases are major enzymes involved in metabolism of xenobiotics including insecticides. Two carboxylesterase genes, CarE001A and CarE001H, were cloned from the destructive agricultural pest Helicoverpa armigera. Quantitative real-time polymerase chain reaction showed that CarE001A and CarE001H were predominantly expressed in fat body and midgut, respectively; developmental expression analyses found that the expression levels of both CarEs were significantly higher in fifth-instar larvae than in other life stages. Recombinant CarE001A and CarE001H expressed in the Escherichia coli exhibited high enzymatic activity toward α-naphthyl acetate. Inhibition assays showed that organophosphates had strong inhibition on CarEs activity compared to pyrethroids. Metabolism assays indicated that CarE001A and CarE001H were able to metabolize ß-cypermethrin and λ-cyhalothrin. Homology modeling and molecular docking analyses demonstrated that ß-cypermethrin could fit nicely into the active pocket of both carboxylesterases. These results suggested that CarE001A and CarE001H could play important roles in the detoxification of pyrehtroids in H. armigera.

Identification and biochemical characterization of carboxylesterase 001G associated with insecticide detoxification in Helicoverpa armigera.

Carboxylesterases (CarEs) are a major class of detoxification enzymes involved in insecticide resistance in various insect species. In this study, a novel CarE 001G was isolated from the cotton bollworm Helicoverpa armigera, one of the most destructive agricultural insect pests. The open reading frame of 001G has 2244 nucleotides and putatively encodes 747 amino acid residues. The deduced CarE possessed the highly conserved catalytic triads(Ser-Glu-His) and pentapeptide motifs (Gly-X-Ser-X-Gly), suggesting 001G is biologically active. The truncated 001G was successfully expressed in Escherichia coli, and the recombinant proteins were purified and tested. The enzyme kinetic assay showed the purified proteins could catalyze two model substrates, α-naphthyl acetate and ß-naphthyl acetate, with a kcat of 8.8 and 2.3 s-1, a Km of 9.6 and 16.2 µM, respectively. The inhibition study with pyrethroid, organophosphate and neonicotinoid insecticides showed different inhibition profile against the purified CarE. The HPLC assay demonstrated that the purified proteins were able to metabolize ß-cypermethrin, λ-cyhalothrin and fenvalerate insecticides, exhibiting respective specific activities of 1.7, 1.4 and 0.5 nM/min/mg protein. However, the purified proteins were not able to metabolize the chlorpyrifos, parathion-methyl, paraoxon-ethyl and imidacloprid. The modeling and docking analyses consistently demonstrated that the pyrethroid molecule fits snugly into the catalytic pocket of the CarE 001G. Collectively, our results suggest that 001G may play a role in pyrethroids detoxification in H. armigera.

Broadband light trapping based on periodically textured ZnO thin films.

Transparent conductive front electrodes (TCFEs) deployed in photovoltaic devices have been extensively studied for their significance in transporting carriers, coupling and trapping the incident photons in high-performing solar cells. The trade-off between the light-transmission, electrical, and scattering properties for TCFEs to achieve a broadband improvement in light absorption in solar cells while maintaining a high electrical performance has become the key issue to be tackled. In this paper, we employ self-assembled polystyrene (PS) spheres based on a sauna-like method as a template, followed by a double-layer deposition and then successfully fabricate highly-transparent, well-conductive, and large-scale periodically-textured ZnO TCFEs with broadband light trapping properties. A sheet resistance below 15 Ω sq(-1) was achieved for the periodically-textured ZnO TCFEs, with a concomitant average transmission of 81% (including the glass substrate) in the 400-1100 nm spectral range, a haze improvement in a broadband spectral range, and a wider scattering angular domain. The proposed approach affords a promising alternative method to prepare periodically-textured TCFEs, which are essential for many optoelectronic device semiconductors, such as photovoltaic and display applications.