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1.
Comput Biol Med ; 145: 105395, 2022 06.
Article in English | MEDLINE | ID: covidwho-1894895

ABSTRACT

The identification of DNA-binding proteins (DBPs) has always been a hot issue in the field of sequence classification. However, considering that the experimental identification method is very resource-intensive, the construction of a computational prediction model is worthwhile. This study developed and evaluated a hybrid kernel alignment maximization-based multiple kernel model (HKAM-MKM) for predicting DBPs. First, we collected two datasets and performed feature extraction on the sequences to obtain six feature groups, and then constructed the corresponding kernels. To ensure the effective utilisation of the base kernel and avoid ignoring the difference between the sample and its neighbours, we proposed local kernel alignment to calculate the kernel between the sample and its neighbours, with each sample as the centre. We combined the global and local kernel alignments to develop a hybrid kernel alignment model, and balance the relationship between the two through parameters. By maximising the hybrid kernel alignment value, we obtained the weight of each kernel and then linearly combined the kernels in the form of weights. Finally, the fused kernel was input into a support vector machine for training and prediction. Finally, in the independent test sets PDB186 and PDB2272, we obtained the highest Matthew's correlation coefficient (MCC) (0.768 and 0.5962, respectively) and the highest accuracy (87.1% and 78.43%, respectively), which were superior to the other predictors. Therefore, HKAM-MKM is an efficient prediction tool for DBPs.


Subject(s)
Algorithms , DNA-Binding Proteins , DNA-Binding Proteins/metabolism , Support Vector Machine
2.
J Virol ; 96(10): e0007022, 2022 05 25.
Article in English | MEDLINE | ID: covidwho-1832352

ABSTRACT

In global infection and serious morbidity and mortality, porcine epidemic diarrhea virus (PEDV) has been regarded as a dreadful porcine pathogen, but the existing commercial vaccines are not enough to fully protect against the epidemic strains. Therefore, it is of great necessity to feature the PEDV-host interaction and develop efficient countermeasures against viral infection. As an RNA/DNA protein, the trans-active response DNA binding protein (TARDBP) plays a variety of functions in generating and processing RNA, including transcription, splicing, transport, and mRNA stability, which have been reported to regulate viral replication. The current work aimed to detect whether and how TARDBP influences PEDV replication. Our data demonstrated that PEDV replication was significantly suppressed by TARDBP, regulated by KLF16, which targeted its promoter. We observed that through the proteasomal and autophagic degradation pathway, TARDBP inhibited PEDV replication via the binding as well as degradation of PEDV-encoded nucleocapsid (N) protein. Moreover, we found that TARDBP promoted autophagic degradation of N protein via interacting with MARCHF8, an E3 ubiquitin ligase, as well as NDP52, a cargo receptor. We also showed that TARDBP promoted host antiviral innate immune response by inducing interferon (IFN) expression through the MyD88-TRAF3-IRF3 pathway during PEDV infection. In conclusion, these data revealed a new antiviral role of TARDBP, effectively suppressing PEDV replication through degrading virus N protein via the proteasomal and autophagic degradation pathway and activating type I IFN signaling via upregulating the expression of MyD88. IMPORTANCE PEDV refers to the highly contagious enteric coronavirus that has quickly spread globally and generated substantial financial damage to the global swine industry. During virus infection, the host regulates the innate immunity and autophagy process to inhibit virus infection. However, the virus has evolved plenty of strategies with the purpose of limiting IFN-I production and autophagy processes. Here, we identified that TARDBP expression was downregulated via the transcription factor KLF16 during PEDV infection. TARDBP could inhibit PEDV replication through the combination as well as degradation of PEDV-encoded nucleocapsid (N) protein via proteasomal and autophagic degradation pathways and promoted host antiviral innate immune response by inducing IFN expression through the MyD88-TRAF3-IRF3 pathway. In sum, our data identify a novel antiviral function of TARDBP and provide a better grasp of the innate immune response and protein degradation pathway against PEDV infection.


Subject(s)
Coronavirus Infections , DNA-Binding Proteins , Interferon Type I , Porcine epidemic diarrhea virus , Virus Replication , Animals , Coronavirus Infections/veterinary , DNA-Binding Proteins/metabolism , Immunity, Innate , Interferon Regulatory Factor-3/metabolism , Interferon Type I/metabolism , Myeloid Differentiation Factor 88/metabolism , Nucleocapsid Proteins/metabolism , Porcine epidemic diarrhea virus/genetics , Porcine epidemic diarrhea virus/physiology , RNA/metabolism , Signal Transduction , Swine , TNF Receptor-Associated Factor 3/metabolism
3.
Sci Rep ; 12(1): 4082, 2022 03 08.
Article in English | MEDLINE | ID: covidwho-1735288

ABSTRACT

The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), also known as 2019 novel coronavirus (2019-nCoV), is a highly infectious RNA virus. A percentage of patients develop coronavirus disease 2019 (COVID-19) after infection, whose symptoms include fever, cough, shortness of breath and fatigue. Acute and life-threatening respiratory symptoms are experienced by 10-20% of symptomatic patients, particularly those with underlying medical conditions. One of the main challenges in the containment of COVID-19 is the identification and isolation of asymptomatic/pre-symptomatic individuals. A number of molecular assays are currently used to detect SARS-CoV-2. Many of them can accurately test hundreds or even thousands of patients every day. However, there are presently no testing platforms that enable more than 10,000 tests per day. Here, we describe the foundation for the REcombinase Mediated BaRcoding and AmplificatioN Diagnostic Tool (REMBRANDT), a high-throughput Next Generation Sequencing-based approach for the simultaneous screening of over 100,000 samples per day. The REMBRANDT protocol includes direct two-barcoded amplification of SARS-CoV-2 and control amplicons using an isothermal reaction, and the downstream library preparation for Illumina sequencing and bioinformatics analysis. This protocol represents a potentially powerful approach for community screening of COVID-19 that may be modified for application to any infectious or non-infectious genome.


Subject(s)
COVID-19/diagnosis , DNA-Binding Proteins/metabolism , Membrane Proteins/metabolism , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , Viral Proteins/metabolism , COVID-19/virology , High-Throughput Nucleotide Sequencing , Humans , Mass Screening , RNA, Viral/analysis , RNA, Viral/metabolism , SARS-CoV-2/isolation & purification
4.
Elife ; 112022 01 25.
Article in English | MEDLINE | ID: covidwho-1662829

ABSTRACT

The human proteome is replete with short linear motifs (SLiMs) of four to six residues that are critical for protein-protein interactions, yet the importance of the sequence surrounding such motifs is underexplored. We devised a proteomic screen to examine the influence of SLiM sequence context on protein-protein interactions. Focusing on the EVH1 domain of human ENAH, an actin regulator that is highly expressed in invasive cancers, we screened 36-residue proteome-derived peptides and discovered new interaction partners of ENAH and diverse mechanisms by which context influences binding. A pocket on the ENAH EVH1 domain that has diverged from other Ena/VASP paralogs recognizes extended SLiMs and favors motif-flanking proline residues. Many high-affinity ENAH binders that contain two proline-rich SLiMs use a noncanonical site on the EVH1 domain for binding and display a thermodynamic signature consistent with the two-motif chain engaging a single domain. We also found that photoreceptor cilium actin regulator (PCARE) uses an extended 23-residue region to obtain a higher affinity than any known ENAH EVH1-binding motif. Our screen provides a way to uncover the effects of proteomic context on motif-mediated binding, revealing diverse mechanisms of control over EVH1 interactions and establishing that SLiMs can't be fully understood outside of their native context.


Subject(s)
Actins/metabolism , Binding Sites , DNA-Binding Proteins/metabolism , Microfilament Proteins/metabolism , Proline/metabolism , Cell Adhesion Molecules/metabolism , HEK293 Cells , Humans , Proteomics
5.
Nat Commun ; 12(1): 2735, 2021 05 12.
Article in English | MEDLINE | ID: covidwho-1241460

ABSTRACT

Inflammasomes are filamentous signaling platforms integral to innate immunity. Currently, little is known about how these structurally similar filaments recognize and distinguish one another. A cryo-EM structure of the AIM2PYD filament reveals that the architecture of the upstream filament is essentially identical to that of the adaptor ASCPYD filament. In silico simulations using Rosetta and molecular dynamics followed by biochemical and cellular experiments consistently demonstrate that individual filaments assemble bidirectionally. By contrast, the recognition between AIM2 and ASC requires at least one to be oligomeric and occurs in a head-to-tail manner. Using in silico mutagenesis as a guide, we also identify specific axial and lateral interfaces that dictate the recognition and distinction between AIM2 and ASC filaments. Together, the results here provide a robust framework for delineating the signaling specificity and order of inflammasomes.


Subject(s)
CARD Signaling Adaptor Proteins/metabolism , DNA-Binding Proteins/metabolism , Immunity, Innate/physiology , Inflammasomes/metabolism , CARD Signaling Adaptor Proteins/genetics , Cryoelectron Microscopy , DNA-Binding Proteins/genetics , HEK293 Cells , Humans , Molecular Dynamics Simulation , Mutation/genetics , Protein Structure, Secondary , Signal Transduction/physiology
6.
Biochem Biophys Res Commun ; 567: 195-200, 2021 08 27.
Article in English | MEDLINE | ID: covidwho-1263226

ABSTRACT

Recombinase polymerase amplification (RPA) is an isothermal reaction that amplifies a target DNA sequence with a recombinase, a single-stranded DNA-binding protein (SSB), and a strand-displacing DNA polymerase. In this study, we optimized the reaction conditions of RPA to detect SARS-CoV-2 DNA and RNA using a statistical method to enhance the sensitivity. In vitro synthesized SARS-CoV-2 DNA and RNA were used as targets. After evaluating the concentration of each component, the uvsY, gp32, and ATP concentrations appeared to be rate-determining factors. In particular, the balance between the binding and dissociation of uvsX and DNA primer was precisely adjusted. Under the optimized condition, 60 copies of the target DNA were specifically detected. Detection of 60 copies of RNA was also achieved. Our results prove the fabrication flexibility of RPA reagents, leading to an expansion of the use of RPA in various fields.


Subject(s)
DNA, Viral/analysis , DNA-Directed DNA Polymerase/metabolism , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Amplification Techniques/standards , RNA, Viral/analysis , Recombinases/metabolism , SARS-CoV-2/genetics , Statistics as Topic , DNA Primers/metabolism , DNA-Binding Proteins/metabolism , Membrane Proteins/metabolism , SARS-CoV-2/isolation & purification , Viral Proteins/metabolism
7.
Biochem Biophys Res Commun ; 554: 94-98, 2021 05 21.
Article in English | MEDLINE | ID: covidwho-1157142

ABSTRACT

The post-infection of COVID-19 includes a myriad of neurologic symptoms including neurodegeneration. Protein aggregation in brain can be considered as one of the important reasons behind the neurodegeneration. SARS-CoV-2 Spike S1 protein receptor binding domain (SARS-CoV-2 S1 RBD) binds to heparin and heparin binding proteins. Moreover, heparin binding accelerates the aggregation of the pathological amyloid proteins present in the brain. In this paper, we have shown that the SARS-CoV-2 S1 RBD binds to a number of aggregation-prone, heparin binding proteins including Aß, α-synuclein, tau, prion, and TDP-43 RRM. These interactions suggests that the heparin-binding site on the S1 protein might assist the binding of amyloid proteins to the viral surface and thus could initiate aggregation of these proteins and finally leads to neurodegeneration in brain. The results will help us to prevent future outcomes of neurodegeneration by targeting this binding and aggregation process.


Subject(s)
Amyloid/metabolism , COVID-19/metabolism , Heparin/metabolism , Neurodegenerative Diseases/metabolism , Protein Aggregation, Pathological , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism , Amyloid beta-Peptides/metabolism , Brain/metabolism , Brain/pathology , Brain/virology , COVID-19/virology , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Humans , Molecular Docking Simulation , Neurodegenerative Diseases/virology , Prions/metabolism , Protein Binding , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , alpha-Synuclein/metabolism , tau Proteins/metabolism
8.
Drug Discov Today ; 26(5): 1302-1310, 2021 05.
Article in English | MEDLINE | ID: covidwho-1077854

ABSTRACT

The synthetic antibacterial drug clofoctol (CFT) has long been used to treat respiratory tract infections in Europe. In recent years, the drug was found to target two biologically important proteins, the Cdc7/Dbf4 protein kinase complex and the mRNA-binding protein cold shock domain containing E1 (CSDE1), also known as upstream-of-N-Ras protein (UNR). These interactions are at the origin of the antitumor activity of CFT, recently evidenced in prostate cancer and neuroglioma. Drug-protein binding models provide a structural basis to guide the design of more potent anticancer compounds. A renewed interest in CFT can be anticipated for the treatment of cancers, and possibly Coronavirus 2019 (COVID-19).


Subject(s)
Anti-Bacterial Agents/therapeutic use , Antineoplastic Agents/therapeutic use , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Drug Repositioning , Neoplasms/drug therapy , Animals , Anti-Bacterial Agents/adverse effects , Antineoplastic Agents/adverse effects , Antiviral Agents/adverse effects , COVID-19/metabolism , COVID-19/virology , Cell Cycle Proteins/metabolism , Chlorobenzenes , Cresols/adverse effects , Cresols/therapeutic use , DNA-Binding Proteins/metabolism , Humans , Neoplasms/metabolism , Neoplasms/pathology , RNA-Binding Proteins/metabolism
9.
Sci Adv ; 7(1)2021 01.
Article in English | MEDLINE | ID: covidwho-1066781

ABSTRACT

Despite past extensive studies, the mechanisms underlying pulmonary fibrosis (PF) still remain poorly understood. Here, we demonstrated that lungs originating from different types of patients with PF, including coronavirus disease 2019, systemic sclerosis-associated interstitial lung disease, and idiopathic PF, and from mice following bleomycin (BLM)-induced PF are characterized by the altered methyl-CpG-binding domain 2 (MBD2) expression in macrophages. Depletion of Mbd2 in macrophages protected mice against BLM-induced PF. Mbd2 deficiency significantly attenuated transforming growth factor-ß1 (TGF-ß1) production and reduced M2 macrophage accumulation in the lung following BLM induction. Mechanistically, Mbd2 selectively bound to the Ship promoter in macrophages, by which it repressed Ship expression and enhanced PI3K/Akt signaling to promote the macrophage M2 program. Therefore, intratracheal administration of liposomes loaded with Mbd2 siRNA protected mice from BLM-induced lung injuries and fibrosis. Together, our data support the possibility that MBD2 could be a viable target against PF in clinical settings.


Subject(s)
COVID-19/metabolism , DNA-Binding Proteins/metabolism , Macrophages/metabolism , Pulmonary Fibrosis/metabolism , Animals , Bleomycin/pharmacology , Carcinoma, Non-Small-Cell Lung/metabolism , Fibrosis , Gene Expression Profiling , Gene Expression Regulation , Humans , Liposomes/chemistry , Lung Diseases, Interstitial/metabolism , Lung Neoplasms/metabolism , Macrophages/virology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Pulmonary Fibrosis/virology , RNA, Small Interfering/metabolism , Scleroderma, Systemic/metabolism , Signal Transduction , Transforming Growth Factor beta1/metabolism
10.
Int J Mol Sci ; 22(2)2021 Jan 16.
Article in English | MEDLINE | ID: covidwho-1040994

ABSTRACT

The inflammasome is a three-component (sensor, adaptor, and effector) filamentous signaling platform that shields from multiple pathogenic infections by stimulating the proteolytical maturation of proinflammatory cytokines and pyroptotic cell death. The signaling process initiates with the detection of endogenous and/or external danger signals by specific sensors, followed by the nucleation and polymerization from sensor to downstream adaptor and then to the effector, caspase-1. Aberrant activation of inflammasomes promotes autoinflammatory diseases, cancer, neurodegeneration, and cardiometabolic disorders. Therefore, an equitable level of regulation is required to maintain the equilibrium between inflammasome activation and inhibition. Recent advancement in the structural and mechanistic understanding of inflammasome assembly potentiates the emergence of novel therapeutics against inflammasome-regulated diseases. In this review, we have comprehensively discussed the recent and updated insights into the structure of inflammasome components, their activation, interaction, mechanism of regulation, and finally, the formation of densely packed filamentous inflammasome complex that exists as micron-sized punctum in the cells and mediates the immune responses.


Subject(s)
DNA-Binding Proteins/metabolism , Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Animals , CARD Signaling Adaptor Proteins/chemistry , CARD Signaling Adaptor Proteins/metabolism , Caspase 1/chemistry , Caspase 1/metabolism , DNA-Binding Proteins/chemistry , Humans , Inflammasomes/chemistry , NLR Family, Pyrin Domain-Containing 3 Protein/chemistry , Protein Domains , Protein Multimerization
11.
Int J Mol Sci ; 21(24)2020 Dec 15.
Article in English | MEDLINE | ID: covidwho-1024586

ABSTRACT

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease-19 (COVID-19) being associated with severe pneumonia. Like with other viruses, the interaction of SARS-CoV-2 with host cell proteins is necessary for successful replication, and cleavage of cellular targets by the viral protease also may contribute to the pathogenesis, but knowledge about the human proteins that are processed by the main protease (3CLpro) of SARS-CoV-2 is still limited. We tested the prediction potentials of two different in silico methods for the identification of SARS-CoV-2 3CLpro cleavage sites in human proteins. Short stretches of homologous host-pathogen protein sequences (SSHHPS) that are present in SARS-CoV-2 polyprotein and human proteins were identified using BLAST analysis, and the NetCorona 1.0 webserver was used to successfully predict cleavage sites, although this method was primarily developed for SARS-CoV. Human C-terminal-binding protein 1 (CTBP1) was found to be cleaved in vitro by SARS-CoV-2 3CLpro, the existence of the cleavage site was proved experimentally by using a His6-MBP-mEYFP recombinant substrate containing the predicted target sequence. Our results highlight both potentials and limitations of the tested algorithms. The identification of candidate host substrates of 3CLpro may help better develop an understanding of the molecular mechanisms behind the replication and pathogenesis of SARS-CoV-2.


Subject(s)
COVID-19/virology , Coronavirus 3C Proteases/metabolism , SARS-CoV-2/enzymology , Alcohol Oxidoreductases/metabolism , Amino Acid Sequence , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/genetics , DNA-Binding Proteins/metabolism , Host-Pathogen Interactions , Humans , SARS-CoV-2/genetics , Substrate Specificity
12.
EMBO J ; 39(24): e106478, 2020 12 15.
Article in English | MEDLINE | ID: covidwho-927779

ABSTRACT

Tightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and assemble within viral factories, dynamic compartments formed within the host cells associated with human stress granules. Here, we test the possibility that the multivalent RNA-binding nucleocapsid protein (N) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) condenses with RNA via liquid-liquid phase separation (LLPS) and that N protein can be recruited in phase-separated forms of human RNA-binding proteins associated with SG formation. Robust LLPS with RNA requires two intrinsically disordered regions (IDRs), the N-terminal IDR and central-linker IDR, as well as the folded C-terminal oligomerization domain, while the folded N-terminal domain and the C-terminal IDR are not required. N protein phase separation is induced by addition of non-specific RNA. In addition, N partitions in vitro into phase-separated forms of full-length human hnRNPs (TDP-43, FUS, hnRNPA2) and their low-complexity domains (LCs). These results provide a potential mechanism for the role of N in SARS-CoV-2 viral genome packing and in host-protein co-opting necessary for viral replication and infectivity.


Subject(s)
COVID-19/virology , Coronavirus Nucleocapsid Proteins/chemistry , SARS-CoV-2/chemistry , COVID-19/pathology , Coronavirus Nucleocapsid Proteins/metabolism , Cytoplasmic Granules/virology , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/chemistry , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism , Host-Pathogen Interactions , Humans , Phosphoproteins/chemistry , Phosphoproteins/metabolism , RNA-Binding Protein FUS/chemistry , RNA-Binding Protein FUS/metabolism , SARS-CoV-2/metabolism , Virus Assembly
13.
J Comput Aided Mol Des ; 34(12): 1237-1259, 2020 12.
Article in English | MEDLINE | ID: covidwho-841071

ABSTRACT

Computational protein-ligand docking is well-known to be prone to inaccuracies in input receptor structures, and it is challenging to obtain good docking results with computationally predicted receptor structures (e.g. through homology modeling). Here we introduce a fragment-based docking method and test if it reduces requirements on the accuracy of an input receptor structures relative to non-fragment docking approaches. In this method, small rigid fragments are docked first using AutoDock Vina to generate a large number of favorably docked poses spanning the receptor binding pocket. Then a graph theory maximum clique algorithm is applied to find combined sets of docked poses of different fragment types onto which the complete ligand can be properly aligned. On the basis of these alignments, possible binding poses of complete ligand are determined. This docking method is first tested for bound docking on a series of Cytochrome P450 (CYP450) enzyme-substrate complexes, in which experimentally determined receptor structures are used. For all complexes tested, ligand poses of less than 1 Å root mean square deviations (RMSD) from the actual binding positions can be recovered. Then the method is tested for unbound docking with modeled receptor structures for a number of protein-ligand complexes from different families including the very recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protease. For all complexes, poses with RMSD less than 3 Å from actual binding positions can be recovered. Our results suggest that for docking with approximately modeled receptor structures, fragment-based methods can be more effective than common complete ligand docking approaches.


Subject(s)
Betacoronavirus/enzymology , Coronavirus Infections/drug therapy , Cysteine Endopeptidases/drug effects , Molecular Docking Simulation , Pandemics , Pneumonia, Viral/drug therapy , Viral Nonstructural Proteins/drug effects , ATPases Associated with Diverse Cellular Activities/chemistry , ATPases Associated with Diverse Cellular Activities/metabolism , COVID-19 , Coronavirus 3C Proteases , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/metabolism , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Humans , Ligands , Models, Chemical , Models, Molecular , Molecular Chaperones/chemistry , Molecular Chaperones/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Binding , Protein Conformation , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/metabolism , SARS-CoV-2 , Transcription Factors/chemistry , Transcription Factors/metabolism , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism
14.
J Virol ; 94(6)2020 02 28.
Article in English | MEDLINE | ID: covidwho-827743

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.


Subject(s)
Adenosine Triphosphatases/metabolism , Nucleocapsid/metabolism , Nucleopolyhedroviruses/physiology , Virus Replication , Animals , Cell Nucleus/virology , Cytoplasm/virology , DNA Helicases/metabolism , DNA, Viral/biosynthesis , DNA-Binding Proteins/metabolism , Host-Pathogen Interactions , Sf9 Cells/virology , Vacuoles/virology , Viral Proteins/metabolism , Virion
15.
Sci Rep ; 10(1): 4481, 2020 03 11.
Article in English | MEDLINE | ID: covidwho-7753

ABSTRACT

Severe acute respiratory syndrome coronavirus nonstructural protein 13 (SCV nsP13), a superfamily 1 helicase, plays a central role in viral RNA replication through the unwinding of duplex RNA and DNA with a 5' single-stranded tail in a 5' to 3' direction. Despite its putative role in viral RNA replication, nsP13 readily unwinds duplex DNA by cooperative translocation. Herein, nsP13 exhibited different characteristics in duplex RNA unwinding than that in duplex DNA. nsP13 showed very poor processivity on duplex RNA compared with that on duplex DNA. More importantly, nsP13 inefficiently unwinds duplex RNA by increasing the 5'-ss tail length. As the concentration of nsP13 increased, the amount of unwound duplex DNA increased and that of unwound duplex RNA decreased. The accumulation of duplex RNA/nsP13 complexes increased as the concentration of nsP13 increased. An increased ATP concentration in the unwinding of duplex RNA relieved the decrease in duplex RNA unwinding. Thus, nsP13 has a strong affinity for duplex RNA as a substrate for the unwinding reaction, which requires increased ATPs to processively unwind duplex RNA. Our results suggest that duplex RNA is a preferred substrate for the helicase activity of nsP13 than duplex DNA at high ATP concentrations.


Subject(s)
Adenosine Triphosphate/metabolism , Methyltransferases/metabolism , RNA Helicases/metabolism , RNA, Double-Stranded/metabolism , RNA, Viral/metabolism , SARS Virus/enzymology , Viral Nonstructural Proteins/metabolism , DNA/metabolism , DNA Helicases/metabolism , DNA, Single-Stranded/metabolism , DNA, Viral/metabolism , DNA-Binding Proteins/metabolism , Hydrolysis , Kinetics , Protein Binding , RNA-Binding Proteins/metabolism , Substrate Specificity , Virus Replication/physiology
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