K64 acetylation of heat shock protein 90 suppresses nucleopolyhedrovirus replication in Bombyx mori.

HSP90 is a highly conserved chaperone that facilitates the proliferation of many viruses, including silkworm (bombyx mori) nucleopolyhedrovirus (BmNPV), but the underlying regulatory mechanism was unclear. We found that suppression of HSP90 by 17-AAG, a HSP90-specific inhibitor, significantly reduced the expression of BmNPV capsid protein gp64 and viral genome replication, whereas overexpression of B. mori HSP90(BmHSP90) promoted BmNPV replication. Furthermore, in a recent study of the lysine acetylome of B. mori infected with BmNPV, we focused on the reduced viral proliferation due to changes of BmHSP90 lysine acetylation. Site-directed introduction of acetylated (K/Q) or deacetylated (K/R) mimic mutations into BmHSP90 revealed that lysine 64 (K64) acetylation activated the JAK/STAT pathway and reduced BmHSP90 ATPase activity, leading to diminished chaperone activity and ultimately inhibiting BmNPV proliferation. In this study, a single lysine 64 acetylation change of BmHSP90 was elucidated as a model of posttranslational modifications occurring in the wake of host-virus interactions, providing novel insights into potential antiviral strategies.

Deacetylation of ACO2 Is Essential for Inhibiting Bombyx mori Nucleopolyhedrovirus Propagation.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a specific pathogen of Bombyx mori that can significantly impede agricultural development. Accumulating evidence indicates that the viral proliferation in the host requires an ample supply of energy. However, the correlative reports of baculovirus are deficient, especially on the acetylation modification of tricarboxylic acid cycle (TCA cycle) metabolic enzymes. Our recent quantitative analysis of protein acetylome revealed that mitochondrial aconitase (ACO2) could be modified by (de)acetylation at lysine 56 (K56) during the BmNPV infection; however, the underlying mechanism is yet unknown. In order to understand this regulatory mechanism, the modification site K56 was mutated to arginine (Lys56Arg; K56R) to mimic deacetylated lysine. The results showed that mimic deacetylated mitochondrial ACO2 restricted enzymatic activity. Although the ATP production was enhanced after viral infection, K56 deacetylation of ACO2 suppressed BmN cellular ATP levels and mitochondrial membrane potential by affecting citrate synthase and isocitrate dehydrogenase activities compared with wild-type ACO2. Furthermore, the deacetylation of exogenous ACO2 lowered BmNPV replication and generation of progeny viruses. In summary, our study on ACO2 revealed the potential mechanism underlying WT ACO2 promotes the proliferation of BmNPV and K56 deacetylation of ACO2 eliminates this promotional effect, which might provide novel insights for developing antiviral strategies.

Effects of LEF-11 acetylation modification on the regulation of baculovirus infection.

Late expression factor 11 (LEF-11) is an essential protein in the regulation of Bombyx mori nucleopolyhedrovirus (BmNPV) DNA replication and late gene expression. Our recent quantitative analysis of protein acetylome revealed for the first time that LEF-11 can be acetylated at one lysine residue (K83) during viral infection, but the underlying mechanism is unclear. The acetylation level for K83 was down-regulated after 36 h post-infection by approximately 30%. To clarify the regulatory function of this modification, overlap PCR was used for site-specific mutagenesis for acetylated (K83Q) or deacetylated (K83R) mimic mutants of LEF-11. The results of viral titration and quantitative polymerase chain reaction showed that after K83 acetylation, budding virion production and the viral genome replication level were significantly upregulated. Meanwhile, the results of yeast two-hybrid (Y2H) system confirmed that K83 deacetylation modification inhibited the interaction between LEF-11 and immediate early gene 1 (IE-1). In conclusion, the acetylation of LEF-11 at K83 might enhance the interaction with IE-1 in the host cell nucleus to promote viral DNA replication, and might be one of the antiviral strategies of the silkworm host. The host inhibits virus proliferation by deacetylating LEF-11. Keywords: BmNPV; LEF-11; acetylation; virus replication; protein interaction.

Meticulous Graded and Early Warning System of Coal Spontaneous Combustion Based on Index Gases and Characteristic Temperature.

The accurate prediction of coal spontaneous combustion (CSC) in the goaf areas of coal mines is a vital aspect of the migration from passive to active fire prevention and control. However, CSC is highly complicated and existing technologies cannot accurately monitor coal temperatures over wide expanses. Thus, it may be beneficial to assess CSC based on various index gases produced by the reactions of coal. In the present study, the CSC process was simulated by temperature-programmed experiments, and the relationships between index gas concentrations with the coal temperature were determined using logistic fitting functions. CSC was divided into seven stages, and a coal seam spontaneous ignition early warning system involving six criteria was established. Field trials demonstrated that this system is a viable approach to predicting coal seam fires and meets the requirements for the active prevention and control of coal combustion. This work establishes an early warning system based on specific theoretical guidelines that permits the identification of CSC and the implementation of active fire prevention and extinguishing measures.

Acetylation of fructose-bisphosphate aldolase-mediated glycolysis is essential for Bombyx mori nucleopolyhedrovirus infection.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a baculovirus that infects silkworms, and its interaction with silkworm has been considered an important model in the field of insect virology. Accumulating evidence indicates that most viruses promote glycolytic metabolism in host cells to favor infection. However, similar reports are lacking in insects, especially in the area of post-translational modifications of proteins. In this study, we found that BmNPV infection induced the acetylation of fructose-bisphosphate aldolase (ALDO) on lysine 42 (K42) to promote its enzyme activity. To explore the underlying mechanisms, site-directed mutagenesis of deacetylated mimic (K/R) was performed. The results demonstrated that K42 acetylation promoted viral proliferation by exacerbating the glycolytic flux induced by BmNPV infection, which resulted in increased ATP, glucose uptake and lactate accumulation. Inhibiting glycolysis with 2-deoxygucose (2DG) revealed that glycolysis was essential for optimal BmNPV infection. Finally, we showed that BmNPV-infected cells enhanced the transcription of glycolysis-related genes, including Glut1, Hk2 and Ldh. In parallel, K42 acetylation of ALDO also promoted the expression of these genes. Therefore, acetylation of ALDO could be considered a regulator of BmNPV-induced glycolysis. These finding provide insights into the interaction between silkworm and BmNPV.

Enhancing methanogenesis of anaerobic granular sludge by incorporating Fe/Fe oxides nanoparticles aided with biofilm disassembly agents and mediating redox activity of extracellular polymer substances.

Anaerobic granular sludge (AGS) is a promising technology for organic wastewater treatment and energy recovery. In this study, three different kinds of Fe and Fe oxides nanoparticles (Fe3O4, Fe2O3 and ZVI) were tried to be incorporated into AGS through direct loading or aided with biofilm disassembly agents of norspermidine and D-tyrosine, which was aimed to enhance methane production capacity of AGS via increasing redox activity of extracellular polymer substance (EPS) and interspecies electron transfer. Despite the loading methods, incorporation of Fe and Fe oxides nanoparticles into AGS increased methane production capacity remarkably, with an enhancement of 36.49-85.17%, 20.37-204.95% and 189.71-243.32%, respectively, for the Fe3O4, Fe2O3 and ZVI loaded AGS. Pretreatment of AGS using biofilm disassembly agents helped to incorporate more Fe and Fe oxides into the inner structure of AGS, which further enhanced methane production capacity by 48.68% and 184.58%, respectively, for the Fe3O4 and Fe2O3 loaded AGS. Loading Fe and Fe oxides into AGS not only introduced exogenous conductive substances and Fe(III)/Fe(II) redox couples into EPS matrix of AGS, but also stimulated the production of redox active components of flavins and c-Cyts. All these factors may contribute to the reduced resistance of EPS, enhanced interspecies electron transfer and methane production capacity of AGS. This study provides a novel strategy and facile method to accelerate interspecies electron transfer and enhance methane production for matured AGS.

Role of the Bombyx mori nucleopolyhedrovirus LEF3 acetylation on viral replication.

Late expression factor 3 (LEF3) is a single-stranded DNA binding protein of Bombyx mori nucleopolyhedrovirus (BmNPV) with multiple functions. It is an essential factor for viral DNA replication and plays an important regulatory role during BmNPV infection. Our recent quantitative analysis of protein acetylome revealed for the first time that LEF3 can be acetylated at four lysine residues during the viral infection, but the underlying mechanism is unknown. Among the modification sites, two of them (K18 and K27) are located in the conserved nuclear localization sequence region. The acetylation level for K18 especially was up-regulated approximately 7.4 times after 36 h of post-infection. To understand the regulatory function of this modification, site-direct mutagenesis for acetylated mimic (K18Q) or deacetylated mimic (K18R) mutants was performed on LEF3. The fluorescence analysis results showed that the replication capacity of the virus was significantly reduced after K18 acetylation. Meanwhile, co-localization analysis revealed that acetylation at K18 caused LEF3 to lose its nuclear targeting ability and affected the interaction between LEF3 and P143, retaining P143 in the cytoplasm. And further Yeast two-hybrid analysis results also confirmed that the acetylation at K18 did affect the interaction between LEF3 and P143. In conclusion, the acetylation of LEF3 at K18 might act as one of the antiviral strategies for silkworm host by affecting nuclear localization of LEF3, interaction with P143, and then blocking viral replication.

In-situ formation and self-immobilization of biogenic Fe oxides in anaerobic granular sludge for enhanced performance of acidogenesis and methanogenesis.

Addition of ferric oxides into flocculent anaerobic sludge was reported to enhance methanogenesis due to accelerated direct interspecies electron transfer (DIET) between syntrophic microbial communities. However, it is generally hard to incorporate Fe oxides into already matured anaerobic granular sludge (AGS) due to its special aggregated structure. In this study, a novel method was attempted to fast incorporate Fe oxides into AGS through in-situ microbial formation and immobilization of biogenic Fe oxides. Factors influencing the formation of Fe oxides were investigated and effects of Fe oxides on the acidogenic and methanogenic performance of AGS were assessed. Results showed that AGS could form Fe oxides mainly in the form of magnetite and hematite through biological reduction of Fe(III) oxyhydroxide. A maximum loading amount of 83.9 mg Fe/g MLVSS was obtained at pH 7 after contacting with 60 mM Fe(III) oxyhydroxide. The efficiency of electron donors which supported Fe(III) reduction followed the order of pyruvate > propionate > glucose > acetate > lactate > formate. Addition of electron transfer mediators (ETMs) promoted the formation of Fe oxides and their performance followed the order of AQDS > AQC > humics > FMN > riboflavin. Presence of Fe oxides in AGS (134.6 Fe/g VSS) increased the production of volatile fatty acids (VFAs) and methane by 16.28% and 41.94% respectively, comparing to the control. The enhancement may be attributed to increased conductivity and stimulated growth of exoelectrogens (Clostridium and Anaerolinea) and methanogenic endoelectrogens Methanosaeta in granular sludge which may strengthen direct interspecies electron transfer between syntrophic microbial communities. Overall, this study provides an alternative strategy to improve the digestion performance of AGS through in-situ formation and immobilization of biogenic Fe oxides.

Deacetylation of HSC70-4 Promotes Bombyx mori Nucleopolyhedrovirus Proliferation via Proteasome-Mediated Nuclear Import.

Silkworm (Bombyx mori) is a model organism with great agricultural economic value that plays a crucial role in biological studies. B. mori nucleopolyhedrovirus (BmNPV) is a major viral pathogen found in silkworms, which leads to huge silk loss annually. In a recent lysine acetylome of silkworm infected with BmNPV, we focused on the heat shock cognate protein 70-4 (HSC70-4) lysine acetylation change due to the consequent nuclear accumulation and viral structure assembly. In this study, the genome replication, proliferation, and production of budded viruses (BVs) were arrested by HSP/HSC70 inhibitor treatment. However, HSC70-4 overexpression enhanced BmNPV reproduction. Furthermore, site-direct mutagenesis for acetylated mimic (K/Q) or deacetylated mimic (K/R) mutants of HSC70-4 demonstrated that lysine 77 (K77) deacetylation promotes HSC70-4 stability, viral DNA duplication, and HSC70-4 nuclear entry upon BmNPV challenge, and the nuclear propulsion of HSC70-4 after viral stimulus might be dependent on the interaction with the carboxyl terminus of HSC70-interacting protein (CHIP, an E3 ubiquitin ligase), followed by ubiquitin-proteasome system assistance. In this study, single lysine 77 deacetylation of HSC70-4 was deemed a part of the locomotive pathway for facilitating BmNPV proliferation and provided novel insights into the antiviral strategic development.

HSP/HSC70 activity is required for Bombyx mori nucleopolyhedrovirus replication at the early infectious phase.

Bombyx mori nucleopolyhedrovirus caused large amounts of silk loss annually, although it also could be used as silkworm bioreactor expression vector effectively and efficiently. Many heat shock (cognate) proteins 70 (HSP/HSC70) were induced by baculovirus and found existence in viral structure assembly. However, the concrete mechanism still need further elucidation for understanding host and virus interaction. In this study, the application of HSP/HSC70 inhibitor VER155008 is virus infectious phase-dependent for figuring out the role of intact molecular chaperone HSP/HSC70 activity in different stages of BmNPV proliferation progress. All the data had shown that HSP/HSC70 played a vital role in viral genome replication, virus protein abundance, BmNPV proliferation and budded virus production at the early infectious phase. This finding may provide new insights to unravel the interaction between baculovirus and silkworm in the initial infectious stage.

Coordinated targeting of CK2 and KIT in gastrointestinal stromal tumours.

BACKGROUND: Most gastrointestinal stromal tumours (GIST) are driven by activating oncogenic mutations of KIT/PDGFRA, which provide a compelling therapeutic target. Our previous studies showed that CDC37, regulated by casein kinase 2 (CK2), is a crucial HSP90 cofactor for KIT oncogenic function and a promising and more selective therapeutic target in GIST. METHODS: Biologic mechanisms of CK2-mediated CDC37 regulation were assessed in GISTs by immunoblotting, immunoprecipitations, knockdown and inactivation assays. The effects of a combination of KIT and CK2 inhibition were assessed by immunoblotting, cell viability, colony growth, cell cycle analysis, apoptosis, migration and invasiveness. RESULTS: CK2 overexpression was demonstrated by immunoblotting in GIST cell lines and patient biopsies. Treatment with a specific CK2 inhibitor, CX4945, leads to CDC37 dephosphorylation and inhibits KIT signalling in imatinib-sensitive and in imatinib-resistant GIST cell lines. Immunoprecipitation demonstrated that CK2 inhibition blocks KIT:HSP90:CDC37 interaction in GIST cells. Coordinated inhibition of CK2 and KIT by CX4945 (or CK2 shRNA) and imatinib, respectively, leads to increased apoptosis, anti-proliferative effects and cell cycle arrest and decreased p-AKT and p-S6 expression, migration and invasiveness in all GIST cell lines compared with either intervention alone, indicating additive effects of inhibiting these two important regulators of GIST biology. CONCLUSION: Our findings suggest that combinatorial inhibition of CK2 and KIT warrants evaluation as a novel therapeutic strategy in GIST, especially in imatinib-resistant GIST.

HN1L is essential for cell growth and survival during nucleopolyhedrovirus infection in silkworm, Bombyx mori.

Hematological and neurological expressed 1-like (HN1L) protein is an evolutionarily conserved protein that plays an important role in embryonic development. It has been reported that HN1L is involved in the process of cell growth and cancer formation and that cell cycle arrest occurs during suppression of HN1L expression. Previous studies have demonstrated that the expression levels of the Bombyx mori HN1L protein were significantly downregulated in Bombyx mori Nucleopolyhedrovirus (BmNPV) infected silkworm cells. Transient transfections were performed with plasmids for pIEX-1-HN1L expression in Bombyx mori ovarian cells (BmN) in order to explore the effect of the HN1L protein on the growth of silkworm cells and its regulatory role in the process of viral infection. Cellular localization analysis revealed that HN1L was localized in the cytoplasm and that its upregulation could significantly enhance cellular activity. Furthermore, HN1L could promote G1/S phase conversion, thereby contributing to cell proliferation. Upon infection of BmN cells with BmNPV, the induction of apoptosis increased, although HN1L overexpression could inhibit DNA fragmentation, suggesting that the HN1L protein could inhibit cell apoptosis induced by viral invasion. In addition, Western blotting indicated that the HN1L protein inhibited the activation of caspase-9 zymogen and the expression of Bax protein, although it promoted Bcl-2 expression. Flow cytometry analysis further confirmed that overexpression of HN1L significantly inhibited apoptosis induced by BmNPV infection. Consequently, we demonstrated that BmN HN1L is a protein with multiple functions, which enhanced cell activity, regulated the cell cycle and induced an anti-apoptotic response by BmNPV infection.

Acetylation of BmAtg8 inhibits starvation-induced autophagy initiation.

Silkworm (Bombyx mori) is not only a model organism for scientific studies, but also a commercial insect for agricultural production. BmAtg8 (a B. mori homolog of yeast Atg8) plays crucial roles in macroautophagy (hereafter referred to autophagy), which is helpful for silkworm metamorphosis. Relevant mechanism about BmAtg8 currently remains ambiguous. Based on our previous acetylome of B. mori after BmNPV infection, we focused on that acetylation of BmAtg8 K13 was changed upon virus challenge. Subsequently, anti-BmAtg8 antibody was generated, and EBSS-induced BmN cellular autophagy model was established. Next, by constructing acetylation-mimic K13Q or deacetylation-mimic K13R mutant BmAtg8, we further examined that K13 of BmAtg8 was acetylated after BmNPV infection and chose 3 h as an appropriate point after EBSS treatment for autophagy initiation. Furthermore, acetylation of BmAtg8 K13 significantly reduced BmAtg8-PE formation in the presence of EBSS, thereby interfering autophagy initiation. Interestingly, acetylated K13 of BmAtg8 contributed to weaken interaction with Atg7, which may influence BmAtg8-PE conjugation. Eventually, acetylation of BmAtg8 K13 is critical for attenuating cell rescue through impaired autophagy initiation. Taken together, our data support an acetylated molecular function for BmAtg8 during starvation-induced autophagy, and provide insights into the modulating mechanisms that potentially reveal the LC3 (a mammalian homolog of Atg8) function in mammal.

Quantitative proteomics of Bombyx mori after BmNPV challenge.

The domesticated silkworm is an ideal and economic insect model that plays crucial roles in sericulture and bioreactor. Bombyx mori nucleopolyhedrovirus (BmNPV) is not only an infectious pathogen to B. mori, but also an efficient vector expressing recombinant proteins. Although, the proteomics of silkworm and BmN cell membrane lipid raft towards BmNPV infection had been investigated, proteome results of BmN cells upon BmNPV challenge currently remain ambiguous. In order to explore the interaction between silkworm and BmNPV, we analyzed several pivotal processes of BmNPV infected BmN cell by quantitative mass spectrometry. Our study indicated that a total of 4205 identified proteins, among which 4194 were with quantitative level. Concretely, during BmNPV infection, several transcription factors and epigenetically modified proteins showed substantially different abundance levels. Especially, proteins with binding activity, displayed significant changes in their molecular functions. Disabled non-homologous end joining by BmNPV reflects irreversible breakage of DNA. Nevertheless, highly abundant superoxide dismutase suggests that the cellular defense system is persistently functional in maintaining biochemical homeostasis. Our comparative and quantitative proteomics will be helpful to unravel the dynamics of B.mori after BmNPV infection and could provide new insights to decipher the mechanism of interaction between BmN cell and BmNPV.

Comprehensive Profiling of Lysine Acetylome in Baculovirus Infected Silkworm (Bombyx mori) Cells.

Bombyx mori is one of the key lepidopteran model species, and is economically important for silk production and proteinaceous drug expression. Baculovirus and insect host are important natural biological models for studying host-pathogen interactions. The impact of Bombyx mori nucleopolyhedrovirus (BmNPV) infection on the proteome and acetylome of Bombyx mori ovarian (BmN) cells are explored to facilitate a better understanding of infection-driven interactions between BmNPV and host in vitro. The proteome and acetylome are profiled through six-plex Tandem mass tag (TMT) labeling-based quantitative proteomics. A total of 4194 host proteins are quantified, of which 33 are upregulated and 47 are downregulated in BmN cells at 36 h post-infection. Based on the proteome, quantifiable differential Kac proteins are identified and functionally annotated to gene expression regulation, energy metabolism, substance synthesis, and metabolism after BmNPV infection. Altogether, 644 Kac sites in 431 host proteins and 39 Kac sites in 22 viral proteins are identified and quantified in infected BmN cells. Our study demonstrates that BmNPV infection globally impacts the proteome and acetylome of BmN cells. The viral proteins are also acetylated by the host acetyltransferase. Protein acetylation is essential for cellular self-regulation and response to virus infection. This study provides new insights for understanding the host-virus interaction mechanisms, and the role of acetylation in BmN cellular response to viral infection.

Quantitative phosphoproteome on the silkworm (Bombyx mori) cells infected with baculovirus.

BACKGROUND: Bombyx mori has become an important model organism for many fundamental studies. Bombyx mori nucleopolyhedrovirus (BmNPV) is a significant pathogen to Bombyx mori, yet also an efficient vector for recombinant protein production. A previous study indicated that acetylation plays many vital roles in several cellular processes of Bombyx mori while global phosphorylation pattern upon BmNPV infection remains elusive. METHOD: Employing tandem mass tag (TMT) labeling and phosphorylation affinity enrichment followed by high-resolution LC-MS/MS analysis and intensive bioinformatics analysis, the quantitative phosphoproteome in Bombyx mori cells infected by BmNPV at 24 hpi with an MOI of 10 was extensively examined. RESULTS: Totally, 6480 phosphorylation sites in 2112 protein groups were identified, among which 4764 sites in 1717 proteins were quantified. Among the quantified proteins, 81 up-regulated and 25 down-regulated sites were identified with significant criteria (the quantitative ratio above 1.3 was considered as up-regulation and below 0.77 was considered as down-regulation) and with significant p-value (p < 0.05). Some proteins of BmNPV were also hyperphosphorylated during infection, such as P6.9, 39 K, LEF-6, Ac58-like protein, Ac82-like protein and BRO-D. CONCLUSION: The phosphorylated proteins were primary involved in several specific functions, out of which, we focused on the binding activity, protein synthesis, viral replication and apoptosis through kinase activity.

[Enhanced Removal of Herbicide 2,4-dichlorophenoxyacetic Acid and Simultaneous Power Generation in Microbial Fuel Cells].

This study investigated the effects of a widely used herbicide 2,4-dichlorophenoxyacetic acid on power generation, pollutants removal from microbial fuel cells (MFCs) and microbial community changes, and also explored anode pre-aeration for enhanced 2,4-D removal and power generation. The results showed that when 2,4-D was inputted to the anode chamber of MFCs which was previously enriched with acetate sodium as the fuel, the voltage output and power density declined and the internal resistance increased apparently. The maximum power density declined to 0.057 W·m-2 in the presence of 300 mg·L-1 2,4-D comparing to 0.151 W·m-2 obtained with acetate alone (850 mg·L-1), and the internal resistance increased from 524 Ω to 1230 Ω correspondingly. To accelerate 2,4-D removal rate and reduce its inhibition to anode exoelectrogens, 6h pre-aeration was applied to the anode chamber. Fast removal of 2,4-D was achieved during aeration period and simultaneous high maximum voltage output (0.42-0.47 V) was obtained. Anode microbial community changed after 2,4-D addition and several 2,4-D degrading bacteria and 2,4-D tolerant exoelectrogen were enriched. MFCs could be used for 2,4-D removal and simultaneous power generation through anode pre-aeration.

A DNA Binding Protein Is Required for Viral Replication and Transcription in Bombyx mori Nucleopolyhedrovirus.

A DNA-binding protein (DBP) [GenBank accession number: M63416] of Bombyx mori nuclear polyhedrosis virus (BmNPV) has been reported to be a regulatory factor in BmNPV, but its detailed functions remain unknown. In order to study the regulatory mechanism of DBP on viral proliferation, genome replication, and gene transcription, a BmNPV dbp gene knockout virus dbp-ko-Bacmid was generated by the means of Red recombination system. In addition, dbp-repaired virus dbp-re-Bacmid was constructed by the means of the Bac to Bac system. Then, the Bacmids were transfected into BmN cells. The results of this viral titer experiment revealed that the TCID50 of the dbp-ko-Bacmid was 0; however, the dbp-re-Bacmid was similar to the wtBacmid (p>0.05), indicating that the dbp-deficient would lead to failure in the assembly of virus particles. In the next step, Real-Time PCR was used to analyze the transcriptional phases of dbp gene in BmN cells, which had been infected with BmNPV. The results of the latter experiment revealed that the transcript of dbp gene was first detected at 3 h post-infection. Furthermore, the replication level of virus genome and the transcriptional level of virus early, late, and very late genes in BmN cells, which had been transfected with 3 kinds of Bacmids, were analyzed by Real-Time PCR. The demonstrating that the replication level of genome was lower than that of wtBacmid and dbp-re-Bacmid (p<0.01). The transcriptional level of dbp-ko-Bacmid early gene lef-3, ie-1, dnapol, late gene vp39 and very late gene p10 were statistically significantly lower than dbp-re-Bacmid and wtBacmid (p<0.01). The results presented are based on Western blot analysis, which indicated that the lack of dbp gene would lead to low expressions of lef3, vp39, and p10. In conclusion, dbp was not only essential for early viral replication, but also a viral gene that has a significant impact on transcription and expression during all periods of baculovirus life cycle.

CARMA3 Is a Host Factor Regulating the Balance of Inflammatory and Antiviral Responses against Viral Infection.

Host response to RNA virus infection is sensed by RNA sensors such as RIG-I, which induces MAVS-mediated NF-κB and IRF3 activation to promote inflammatory and antiviral responses, respectively. Here, we have found that CARMA3, a scaffold protein previously shown to mediate NF-κB activation induced by GPCR and EGFR, positively regulates MAVS-induced NF-κB activation. However, our data suggest that CARMA3 sequesters MAVS from forming high-molecular-weight aggregates, thereby suppressing TBK1/IRF3 activation. Interestingly, following NF-κB activation upon virus infection, CARMA3 is targeted for proteasome-dependent degradation, which releases MAVS to activate IRF3. When challenged with vesicular stomatitis virus or influenza A virus, CARMA3-deficient mice showed reduced disease symptoms compared to those of wild-type mice as a result of less inflammation and a stronger ability to clear infected virus. Altogether, our results reveal the role of CARMA3 in regulating the balance of host antiviral and pro-inflammatory responses against RNA virus infection.