Large Polypeptide Research Articles

Molecular characterization of a novel fusarivirus infecting the plant-pathogenic fungus Nigrospora sphaerica.

Here, we describe a novel mycovirus, tentatively designated as "Nigrospora sphaerica fusarivirus 2" (NsFV2), which was isolated from the phytopathogenic fungus Nigrospora sphaerica strain HNXX-Ns20. NsFV2 has a single-stranded positive-sense RNA (+ ssRNA) genome of 6,156 nucleotides, excluding the poly(A) tail, and contains two putative open reading frames (ORFs). ORF1 encodes a large polypeptide of 1,509 amino acids (aa) containing a conserved RNA-dependent RNA polymerase (RdRp) domain and a viral helicase domain. The ORF1-encoded polypeptide shares 29.40-68.48% sequence identity with other fusariviruses and shares the highest sequence identity (68.48%) with Nigrospora sphaerica fusarivirus 1 (NsFV1). The small ORF2 encodes a polypeptide of 483 aa that contains a conserved chromosome segregation ATPase (Smc) domain. Multiple sequence alignments and phylogenetic analysis based on the ORF1-encoded polypeptide indicated that NsFV2 should be considered a new member of the genus Alphafusarivirus of the family Fusariviridae.

Towards a universal method for middle-down analysis of antibodies via proton transfer charge reduction-Orbitrap mass spectrometry.

Modern mass spectrometry technology allows for extensive sequencing of the ~ 25kDa subunits of monoclonal antibodies (mAbs) produced by IdeS proteolysis followed by disulfide bond reduction, an approach known as middle-down mass spectrometry (MD MS). However, the spectral congestion of tandem mass spectra of large polypeptides dramatically complicates fragment ion assignment. Here, we report the development and benchmark of an MD MS strategy based on the combination of different ion fragmentation techniques with proton transfer charge reduction (PTCR) to simplify the gas-phase sequencing of mAb subunits. Applied on the liquid chromatography time scale using an Orbitrap Tribrid mass spectrometer, PTCR produces easy-to-interpret mass spectra with limited ion signal overlap. We demonstrate that the accurate estimation of the number of charges submitted to the Orbitrap mass analyzer after PTCR allows for the detection of charge-reduced product ions over a wide mass-over-charge (m/z) window with low parts per million m/z accuracy. Therefore, PTCR-based MD MS analysis increases not only sequence coverage, number of uniquely identified fragments, and number of assigned complementary ion pairs, but also the general confidence in the assignment of subunit fragments. This data acquisition method can be readily applied to any class of mAbs without an apparent need for optimization, and benefits from the high resolving power of the Orbitrap mass analyzer to return sequence coverage of individual subunits exceeding 80% in a single run, and > 90% when just two experiments are combined.

Neurotrophic Hypothesis of Development of Depression

Currently, depression is a widespread mental disorder in modern society and is associated with significant impairments in the quality of life of patients. The review examines the main representatives of neurotrophic factors belonging to various families, in particular nerve growth factor, transforming growth factor beta, neurokines and non-neuronal factors. Neurotrophins, being large polypeptides, play an integrative role, fulfilling the signaling mission of intermediaries in a wide range of physiological processes. At the moment, a large number of studies have been carried out in order to understand the interaction between factors of various directions, including biological, psychological and environmental factors that determine the etiopathogenesis of this pathology. Based on the data obtained, one of the main hypotheses for the development of depression is considered - neurotrophic, which most fully explains the emerging pathogenetic changes. According to this hypothesis, the leading role in the etiology of depression is played by neurotrophic factors that ensure the maintenance of normal neuron-glial interaction, the processes of neurogenesis, angiogenesis, and synaptic plasticity. Neurotrophins have a high physiological activity due to the presence of several binding groups for different cell receptors and the regulatory ability to express other signaling molecules, the ability to penetrate the blood-brain barrier, showing trophic, anti-inflammatory, growth, mediator and effector properties. To date, the mechanisms of the onset of depression, despite the widespread prevalence of this pathology, remain largely unclear, hindering a directed search for targets for the development of effective therapy. The revealed disorders of neurotrophic factors in depression make it reasonable to consider neurotrophins as therapeutic agents in a multitarget approach to the treatment of depressive disorders.

Natural history of eukaryotic DNA viruses with double jelly-roll major capsid proteins

The phylum Preplasmiviricota (kingdom Bamfordvirae, realm Varidnaviria) is a broad assemblage of diverse viruses with comparatively short double-stranded DNA genomes (<50 kbp) that produce icosahedral capsids built from double jelly-roll major capsid proteins. Preplasmiviricots infect hosts from all cellular domains, testifying to their ancient origin, and, in particular, are associated with six of the seven supergroups of eukaryotes. Preplasmiviricots comprise four major groups of viruses, namely, polintons, polinton-like viruses (PLVs), virophages, and adenovirids. We used protein structure modeling and analysis to show that protein-primed DNA polymerases (pPolBs) of polintons, virophages, and cytoplasmic linear plasmids encompass an N-terminal domain homologous to the terminal proteins (TPs) of prokaryotic PRD1-like tectivirids and eukaryotic adenovirids that are involved in protein-primed replication initiation, followed by a viral ovarian tumor-like cysteine deubiquitinylase (vOTU) domain. The vOTU domain is likely responsible for the cleavage of the TP from the large pPolB polypeptide and is inactivated in adenovirids, in which TP is a separate protein. Many PLVs and transpovirons encode a distinct derivative of polinton-like pPolB that retains the TP, vOTU, and pPolB polymerization palm domains but lacks the exonuclease domain and instead contains a superfamily 1 helicase domain. Analysis of the presence/absence and inactivation of the vOTU domains and replacement of pPolB with other DNA polymerases in eukaryotic preplasmiviricots enabled us to outline a complete scenario for their origin and evolution.

Suppression of Innate Immunity by the Hepatitis C Virus (HCV): Revisiting the Specificity of Host-Virus Interactive Pathways.

The hepatitis C virus (HCV) is a major causative agent of hepatitis that may also lead to liver cancer and lymphomas. Chronic hepatitis C affects an estimated 2.4 million people in the USA alone. As the sole member of the genus Hepacivirus within the Flaviviridae family, HCV encodes a single-stranded positive-sense RNA genome that is translated into a single large polypeptide, which is then proteolytically processed to yield the individual viral proteins, all of which are necessary for optimal viral infection. However, cellular innate immunity, such as type-I interferon (IFN), promptly thwarts the replication of viruses and other pathogens, which forms the basis of the use of conjugated IFN-alpha in chronic hepatitis C management. As a countermeasure, HCV suppresses this form of immunity by enlisting diverse gene products, such as HCV protease(s), whose primary role is to process the large viral polyprotein into individual proteins of specific function. The exact number of HCV immune suppressors and the specificity and molecular mechanism of their action have remained unclear. Nonetheless, the evasion of host immunity promotes HCV pathogenesis, chronic infection, and carcinogenesis. Here, the known and putative HCV-encoded suppressors of innate immunity have been reviewed and analyzed, with a predominant emphasis on the molecular mechanisms. Clinically, the knowledge should aid in rational interventions and the management of HCV infection, particularly in chronic hepatitis.

Structure and Function of a Class III Metal-Independent Lanthipeptide Synthetase.

In bacteria, Ser/Thr protein kinase-like sequences are found as part of large multidomain polypeptides that biosynthesize lanthipeptides, a class of natural products distinguished by the presence of thioether cross-links. The kinase domain phosphorylates Ser or Thr residues in the peptide substrates. Subsequent β-elimination by a lyase domain yields electrophilic dehydroamino acids, which can undergo cyclase domain-catalyzed cyclization to yield conformationally restricted, bioactive compounds. Here, we reconstitute the biosynthetic pathway for a class III lanthipeptide from Bacillus thuringiensis NRRL B-23139, including characterization of a two-component protease for leader peptide excision. We also describe the first crystal structures of a class III lanthipeptide synthetase, consisting of the lyase, kinase, and cyclase domains, in various states including complexes with its leader peptide and nucleotide. The structure shows interactions between all three domains that result in an active conformation of the kinase domain. Biochemical analysis demonstrates that the three domains undergo movement upon binding of the leader peptide to establish interdomain allosteric interactions that stabilize this active form. These studies inform on the regulatory mechanism of substrate recognition and provide a framework for engineering of variants of biotechnological interest.

Complete genome sequence of a novel fusarivirus from the phytopathogenic fungus Fusarium sp.

Fusarium diseases include wilts, blights, rots, and cankers of many horticultural, field, ornamental, and forest crops in both agricultural and natural ecosystems, and they significantly hinder food plant production. Here, we describe a novel mycovirus, tentatively designated as "Fusarium fusarivirus 1" (FuFV1), which was discovered in an isolate of the phytopathogenic fungus Fusarium sp. FuFV1 has a positive-sense single-stranded RNA (+ssRNA) genome of 6,391 nucleotides (nt) containing three open reading frames (ORFs). ORF1 encodes a large polypeptide of 1,501 amino acids (aa) with conserved RNA-dependent RNA polymerase (RdRp) and helicase (Hel) domains. ORF2, overlapping ORF1 by 122 nucleotides, encodes a polypeptide with a conserved Smc domain. The third and smaller ORF (ORF3) encodes a polypeptide with an unknown function. BLASTp analysis of the ORF1-encoded polypeptide revealed that FuFV1 shares the highest aa sequence similarity (68.5% identity, E-value 0.0) with Fusarium poae fusarivirus 1 (FpFV1, genus Alphafusarivirus). Phylogenetic analysis of the RdRp and helicase (Hel) sequences indicated that FuFV1 clustered closely with FpFV1 in a separate branch within the clade containing members of the genus Alphafusarivirus. Based on these results, we propose that FuFV1 should be considered a novel mycovirus belonging to the genus Alphafusarivirus of the family Fusariviridae.

Development of an LC-MS/MS assay for quantification of intact INSL3 in rat plasma.

Background: Relatively large disulfide-linked polypeptides can serve as signaling molecules for a diverse array of biological processes and may be studied in animal models to investigate their function in vivo. The aim of this work was to develop an LC-MS/MS assay to measure a model peptide, INSL3, in rat plasma. Results: A dual enrichment strategy incorporating both protein precipitation and solid phase extraction was utilized to isolate INSL3 from rat plasma, followed by targeted LC-MS/MS detection. The method was able to measure full-length INSL3 (6.1 kDa) down to 0.2ng/ml with acceptable accuracy and precision. Conclusion: The final assay was applied to support an exploratory pharmacokinetic study to evaluate steady-state concentrations of dosed INSL3 in rat plasma.

Complete genome sequence of a novel fusarivirus from Rhizoctonia solani AG-3 PT strain 3P-2-2.

Here, we describe a novel mycovirus, tentatively designated as "Rhizoctonia solani fusarivirus 6" (RsFV6), which was discovered in Rhizoctonia solani AG-3 PT strain 3P-2-2. The virus has a single-stranded positive-sense RNA (+ssRNA) genome of 6141 nucleotides containing two open reading frames (ORFs) and a poly(A) tail. ORF1 encodes a large polypeptide of 1,862 amino acids (aa) with conserved RNA-dependent RNA polymerase (RdRp) and helicase (Hel) domains. ORF2 encodes a putative 167-aa protein of unknown function. BLASTp searches revealed that the ORF1-encoded polypeptide showed the highest sequence similarity (70.67% identity) to that of Rhizoctonia solani fusarivirus 3 (RsFV3), which was isolated from Rhizoctonia solani AG-2-2LP. Multiple sequence alignments and phylogenetic analysis based on RdRp and Hel sequences indicated that RsFV6 could be a novel member of the genus Alphafusarivirus family Fusariviridae.

Bulevirtide in the Treatment of Hepatitis Delta: Drug Discovery, Clinical Development and Place in Therapy.

It has been ten years since the identification of NTCP as the cell surface receptor for HBV and HDV entry into hepatocytes. The search for molecules interfering with the binding of NTCP and HBV/HDV led to design bulevirtide (BLV). This large polypeptide mimics a region of the pre-S1 HBsAg and blocks viral entry by inhibitory competition. BLV was initially tested in cell cultures, animal models and more recently in Phase I-III human trials (called 'MYRS'). As monotherapy or in combination with peginterferon, BLV is well tolerated and exhibits potent antiviral activity. Plasma viremia significantly declines and/or becomes undetectable in more than 75% of patients treated for >24 weeks. However, serum HBsAg concentrations remain unchanged. No selection of BLV resistance in HBV/HDV has been reported in vivo to date. BLV is administered subcutaneously once daily at doses between 2 and 10 mg. BLV received conditional approval in Europe in 2020 to treat chronic hepatitis delta. The advent of peginterferon lambda or new specific anti-HDV antivirals (lonafarnib, etc.) will open the door for combination therapies with BLV. Since there is no stable reservoir for HDV-RNA within infected hepatocytes, viral clearance might be achieved using antivirals for a minimum timeframe. This is what happens in hepatitis C combining several antivirals, curing nearly all patients treated for 3 months. Clearance of HDV-RNA genomes may occur despite HBV persistence as cccDNA or chromosome integrated HBV-DNA within hepatocytes. This is supported by cases of HDV elimination using BLV despite persistence of serum HBsAg. Another path for HDV cure will derive from achieving HBsAg clearance, the goal of new promising anti-HBV gene therapies (bepirovirsen, etc.). In summary, the advent of BLV has triggered a renovated interest for antiviral therapy in hepatitis delta. We envision combination therapies that will lead to HDV cure in the near future.

Molecular characterization of a novel victorivirus isolated from the phytopathogenic fungus Phaeobotryon rhois.

Phaeobotryon rhois is an important pathogenic fungus that causes dieback and canker disease of woody hosts. A novel mycovirus, tentatively named "Phaeobotryon rhois victorivirus 1" (PrVV1), was identified in P. rhois strain SX8-4. The PrVV1 has a double-stranded RNA (dsRNA) genome that is 5,224 base pairs long and contains two open reading frames (ORF1 and ORF2), which overlap at a AUGA sequence. ORF1 encodes a polypeptide of 786 amino acids (aa) that contains the conserved coat protein (CP) domain of victoriviruses, while ORF2, encodes a large polypeptide of 826 aa that contains the conserved RNA-dependent RNA polymerase (RdRp) domain of victoriviruses. Our analysis of genomic structure, homology, and phylogeny indicated that PrVV1 is a novel member of the genus Victorivirus in the family Totiviridae. This is the first report of the complete genome sequence of a victorivirus that infects P. rhois.

AlphaFold and Structural Mass Spectrometry Enable Interrogations on the Intrinsically Disordered Regions in Cyanobacterial Light-harvesting Complex Phycobilisome

Intrinsically disordered proteins/regions (IDPRs) are a very large and functionally important class of proteins that participate in weak multivalent interactions in protein complexes. They are recalcitrant for interrogations using X-ray crystallography and cryo-EM. The IDPRs observed at the interface of the photosynthetic pigment protein complexes (PPCs) remain much less clear, e.g., the major cyanobacterial light-harvesting complex (PBS) contains an unstructured PB-loop insertion in the phycocyanobilin domain (PB domain) of ApcE (the largest polypeptide in PBS). Here, a joint platform is built to probe such structural domains. This platform is characterized by two-round progressive justifications of in silico models by using the structural mass spectrometry data. First, the AlphaFold-generated 3D structure of the PB domain (containing PB-loop) was justified in the context of PBS. Second, docking the AlphaFold-generated ApcG (a ligand) into the first-step justified structure (a receptor). The final ligand-receptor complex was then subjected to a second-round justification, again, by using unequivocal isotopically-encoded cross-links identified in LC-MS/MS. This work reveals a full-length PB-loop structure modelled in the PBS basal cylinder, free from any spatial conflicts against the other subunits in PBS. The structure of PB domain highlights the close associations of the intrinsically disordered PB-loop with its binding partners in PBS, including ApcG, another IDPR. The PB-loop region involved in the binding of photosystem II (PSII) is also discussed in the context of excitation energy transfer regulation. This work calls attention to the highly disordered, yet interrogatable interface between the light-harvesting antenna complexes and the reaction centers.

The MCF Toxin of the Extracellular Pathogen Vibrio vulnificus is Activated by and Targets Host GTPases

Vibrio vulnificus (Vv) is a Gram‐negative bacterial pathogen typically found in warm coastal waters. This pathogen causes severe life‐threatening gastrointestinal and wound infections, most commonly from the consumption of contaminated seafood or an exposed wound. Infections progress quickly to sepsis, and death; approximately 90% of patients are hospitalized and &gt;50% die, often as fast as 48 hours after symptom onset. Of concern, the incidence of Vv is increasing, in part due to its expanding geographical distribution caused by climate change.Bacterial pathogens cause debilitating and lethal infections that are often difficult to treat. Understanding how to manage and control the infections they cause is immensely important for improving quality of life and preventing death. The toxins secreted by extracellular bacteria are frequently the primary virulence factors required to either establish colonization, promote maintenance, growth, and/or eventually dissemination. As such bacterial toxins are extremely important in pathogenesis, and they affect and manipulate many host cellular processes.The primary Vv virulence factor associated with sepsis and subsequent death is the composite MARTX toxin. The MARTX toxin is secreted as a single large polypeptide composed of glycine‐rich repeats at both the N‐ and C‐termini between which there are up to five variable effector domains and a cysteine protease domain (CPD). The repeat regions form a pore in the plasma membrane and the effectors and CPD are translocated across. Within the cell, the CPD auto‐cleaves at multiple sites to release each of the arrayed effectors individually into the host cytoplasm. The MARTX toxin thus serves as an effector delivery platform for translocation of multiple toxic virulence factors inside cells as a single bolus.The Makes Caterpillars Floppy‐like (MCF) effector domain is among the most commonly present Vv MARTX effector domains. MCF is unique from most other MARTX domains in that it utilizes its own autoproteolytic activity to activate itself once inside the host cell. MCF autoprocessing requires stimulation by host ARFs, small GTPases that cycle between GDP‐ or GTP‐bound forms and localize to specific subcellular locations. Once auto‐processed, MCF binds phosphatidylinositol‐5‐phosphate (PtdIns5P), a lipid enriched on Golgi membranes, and its exposed N‐terminal glycine is acetylated. Combined, this results in the generation of “active” MCF (aMCF). aMCF induces the intrinsic apoptotic pathway, causes cell rounding, and nuclear fragmentation. Furthermore, aMCF causes vesiculation of the Golgi apparatus, and destroys the mitochondria.Remarkably, while ARF stimulated autoprocessing results in aMCF, ARFs are not the direct target leading to the cytotoxic effects. The biochemical link between the activation of MCF and its cytotoxicity remains unknown. We explore how MCF activation leads to its disruption of host endocytic trafficking proteins. We examine how these proteins may be targeted and modified by MCF. Furthermore, we assess how the disruption of these host proteins ultimately leads to the dissolution of the endomembrane signaling network and the cytotoxicity we observe. This study begins to elucidate the biochemical activity of this important domain and gives insight into how it may promote disease progression. This work will be critical for advancing the understanding how these MARTX toxins function during Vv pathogenesis.

Molecular and Biological Characterization of the First Mymonavirus Identified in Fusarium oxysporum.

We characterized a negative sense single-stranded RNA mycovirus, Fusarium oxysporum mymonavirus 1 (FoMyV1), isolated from the phytopathogenic fungus Fusarium oxysporum. The genome of FoMyV1 is 10,114 nt, including five open reading frames (ORFs1–5) that are non-overlapping and linearly arranged. The largest, ORF5, encodes a large polypeptide L containing a conserved regions corresponding to Mononegavirales RNA-dependent RNA polymerase and mRNA-capping enzyme region V; the putative functions of the remaining four ORFs are unknown. The L protein encoded by ORF5 shared a high amino acid identity of 65% with that of Hubei rhabdo-like virus 4, a mymonavirus that associated with arthropods. However, the L protein of FoMyV1 also showed amino acid similarity (27–36%) with proteins of mynonaviruses that infect the phytopathogenic fungi Sclerotinia sclerotiorum and Botrytis cineaea. Phylogenetic analysis based on L protein showed that FoMyV1 is clustered with the members of the genus Hubramonavirus in the family Mymonaviridae. Moreover, we found that FoMyV1 could successfully transfer by hyphal anastomosis to a virus-free strain. FoMyV1 reduced the vegetative growth and conidium production of its fungal host but did not alter its virulence. To the best of our knowledge, this is not only the first mymonavirus described in the species F. oxysporum, but also the first Hubramonavirus species found to infect a fungus. However, the incidence of FoMyV1 infections in the tested F. oxysporum strains was only 1%.

Biphen[n]arenes: Modular Synthesis, Customizable Cavity Sizes, and Diverse Skeletons.

Macrocyclic compounds are fundamental tools in supramolecular chemistry and have been widely used in molecular recognition, biomedicine, and materials science. The construction of new macrocycles with distinctive structures and properties would unleash new opportunities for supramolecular chemistry. Traditionally popular macrocycles, e.g., cyclodextrins, calixarenes, cucurbiturils, and pillararenes, possess specific cavities that are usually less than 10 Å in diameter; they are normally suitable for accommodating small- or medium-sized guests but cannot engulf giant molecules or structures. Furthermore, the skeletons of traditional macrocycles are impoverished and incapable of being changed; functional substituents can be introduced only on their portals.Thus, it is very challenging to construct macrocycles with customizable cavity sizes and/or diverse backbones. We have developed a versatile and modular strategy for synthesizing macrocycles, namely, biphen[n]arenes (n = 3-8), based on the structure- or function-oriented replacement of reaction modules, functional modules, and linking modules. First, two reaction modules and one functional module are connected by Suzuki-Miyaura coupling to obtain a monomer having two reaction sites. Then Friedel-Crafts alkylation between the monomer and an aldehyde (linking module) serves to afford diversely functionalized macrocycles. Moreover, large macrocycles can be achieved by using long and rigid oligo(para-phenylene) monomers. Because of the modular synthesis and plentiful molecular supplies, the biphen[n]arenes showed interesting recognition properties for both small molecules and large polypeptides. Customizable functional backbones and binding sites endowed this new family of macrocycles with peculiar self-assembly properties and potential applications in gas chromatography, pollutant capture, and physisorptive separation. Biphen[n]arenes would be a promising family of workhorses in supramolecular chemistry.In this Account, we summarize our recent work on the chemistry of biphen[n]arenes. We introduce their design and modular synthesis, including systematic exploration for reaction modules, customizable cavity sizes, skeleton functionalization, pre- and postmodification, and molecular cages. Thereafter, we discuss their host-guest properties, involving the binding for small guests by cationic/anionic/neutral biphen[n]arenes, as well as the complexation of polypeptides by large quaterphen[n]arenes. In addition, we outline the self-assembly and potential applications of this new family of macrocycles. Finally, we forecast their further development. The chemistry of biphen[n]arenes is still in its infancy. Continued exploration will not only further expand the supramolecular toolbox but also open new avenues for the use of biphen[n]arenes in the fields of biology, pharmaceutical science, and materials science.

Structure-based engineering of minimal proline dehydrogenase domains for inhibitor discovery.

Proline dehydrogenase (PRODH) catalyzes the FAD-dependent oxidation of l-proline to Δ1-pyrroline-5-carboxylate and is a target for inhibitor discovery because of its importance in cancer cell metabolism. Because human PRODH is challenging to purify, the PRODH domains of the bacterial bifunctional enzyme proline utilization A (PutA) have been used for inhibitor development. These systems have limitations due to large polypeptide chain length, conformational flexibility and the presence of domains unrelated to PRODH activity. Herein, we report the engineering of minimal PRODH domains for inhibitor discovery. The best designs contain one-third of the 1233-residue PutA from Sinorhizobium meliloti and include a linker that replaces the PutA α-domain. The minimal PRODHs exhibit near wild-type enzymatic activity and are susceptible to known inhibitors and inactivators. Crystal structures of minimal PRODHs inhibited by S-(-)-tetrahydro-2-furoic acid and 2-(furan-2-yl)acetic acid were determined at 1.23 and 1.72Å resolution. Minimal PRODHs should be useful in chemical probe discovery.

Purification of DNA-Dependent Protein Kinase Catalytic Subunit (DNA-PKcs) from HeLa Cells.

With a predicted molecular mass of 469kDa, expression of recombinant DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is challenging. However, DNA-PKcs is relatively abundant in human cells, making it possible to purify the endogenous protein. Here we describe a method to purify DNA-PKcs and its binding partner Ku70/80 from HeLa cells and describe conditions for transfer of DNA-PKcs and other large polypeptides for immunoblotting.

Drug Repurposing for the SARS-CoV-2 Papain-Like Protease.

As the pathogen of COVID‐19, SARS‐CoV‐2 encodes two essential cysteine proteases that process the pathogen's two large polypeptide products pp1a and pp1ab in the human cell host to form 15 functionally important, mature nonstructural proteins. One of the two enzymes is papain‐like protease or PLPro. It possesses deubiquitination and deISGylation activities that suppress host innate immune responses toward SARS‐CoV‐2 infection. To repurpose drugs for PLPro, we experimentally screened libraries of 33 deubiquitinase and 37 cysteine protease inhibitors on their inhibition of PLPro. Our results showed that 15 deubiquitinase and 1 cysteine protease inhibitors exhibit strong inhibition of PLPro at 200 μM. More comprehensive characterizations revealed seven inhibitors GRL0617, SJB2‐043, TCID, DUB‐IN‐1, DUB‐IN‐3, PR‐619, and S130 with an IC50 value below 40 μM and four inhibitors GRL0617, SJB2‐043, TCID, and PR‐619 with an IC50 value below 10 μM. Among four inhibitors with an IC50 value below 10 μM, SJB2‐043 is the most unique in that it does not fully inhibit PLPro but has a noteworthy IC50 value of 0.56 μM. SJB2‐043 likely binds to an allosteric site of PLPro to convene its inhibition effect, which needs to be further investigated. As a pilot study, the current work indicates that COVID‐19 drug repurposing by targeting PLPro holds promise, but in‐depth analysis of repurposed drugs is necessary to avoid omitting critical allosteric inhibitors.

Molecular cloning, characterization and expression patterns of HSP70 and HSP60 in the pufferfish (Takifugu rubripes)

Takifugu rubripes is a commercially important fish in the Chinese aquaculture industry due to its excellent meat quality. In eukaryotic cells, heat shock proteins (HSPs) play a crucial role in maintaining intracellular protein homeostasis. They play significant roles in response to adverse environmental stresses in aquatic animals. In this study, we used bioinformatics to comprehensively compare two different full-length cDNA sequences at the molecular level in the HSP family of T. rubripes. We performed various physicochemical studies such as race cloning and RT-qPCR, and found that TrHSP60 (575 amino acids) has a larger polypeptide, followed by TrHSP70–13 (441 amino acids). Homology and phylogenetic studies showed that these sequences have the greatest homology to Takifugu bimaculatus. The mRNA of the two genes were constitutively expressed in all tested tissues of untreated T. rubripes, including brain, muscle, kidney, liver, spleen, gill, and intestine, with highest expression levels in the intestine. Three experimental groups were established in this study, the H1 group (20 °C), H2 group (24 °C) and H3 group (28 °C), in order to analyze the expressed levels of TrHSP70 in T. rubripes. To understand the role of HSPs in temperature stress in T. rubripes, we analyzed the expression levels of HSPs in T. rubripes at different temperatures. The results showed that the mRNA expression of the HSP gene were influenced by temperature stress. Gene expression of TrHSP70 and TrHSP60 were mainly up-regulated at 12 h and 48 h in the H1 group, respectively. Meanwhile, gene expression of TrHSP70 was highest at 48 h in the H3 group and TrHSP60 reached was lowest at 24 h in the H3 group. The results showed that the two genes were constitutively expressed genes, which play an important role in maintaining normal physiological function and stress responses. The results show that TrHSP70 and TrHSP60 are constitutively expressed genes, which plays important role in maintaining normal physiological function and coping with stress.

Molecular characterization of a novel fusarivirus infecting the plant-pathogenic fungus Alternaria solani.

A novel mycovirus belonging to the proposed family "Fusariviridae" was discovered in Alternaria solani by sequencing a cDNA corresponding to double-stranded RNA extracted from this phytopathogenic fungus. The virus was tentatively named "Alternaria solani fusarivirus 1" (AsFV1). AsFV1 has a single-stranded positive-sense (+ssRNA) genome of 6845 nucleotides containing three open reading frames (ORFs) and a poly(A) tail. The largest ORF, ORF1, encodes a large polypeptide of 1,556 amino acids (aa) with conserved RNA-dependent RNA polymerase and helicase domains. The ORF2 and ORF3 have overlapping regions, encoding a putative protein of 522 amino acids (aa) and a putative protein of 105 amino acids (aa), respectively, both of unknown function. A multiple sequence alignment and phylogenetic analysis revealed that AsFV1 could be a new member of the "Fusariviridae". This is the first report of the full-length nucleotide sequence of a fusarivirus that infects Alternaria solani.