Prediction of coronavirus 3C-like protease cleavage sites using machine-learning algorithms

The coronavirus 3C-like(3CL)protease,a cysteine protease,plays an important role in viral infection and immune escape.However,there is still a lack of effective tools for determining the cleavage sites of the 3CL protease.This study systematically investigated the diversity of the cleavage sites of the coronavirus 3CL protease on the viral polyprotein,and found that the cleavage motif were highly conserved for viruses in the genera of Alphacoronavirus,Betacoronavirus and Gammacoronavirus.Strong residue preferences were observed at the neighboring positions of the cleavage sites.A random forest(RF)model was built to predict the cleavage sites of the coronavirus 3CL protease based on the representation of residues in cleavage motifs by amino acid indexes,and the model achieved an AUC of 0.96 in cross-validations.The RF model was further tested on an independent test dataset which were composed of cleavage sites on 99 proteins from multiple coronavirus hosts.It achieved an AUC of 0.95 and predicted correctly 80%of the cleavage sites.Then,1,352 human proteins were predicted to be cleaved by the 3CL protease by the RF model.These proteins were enriched in several GO terms related to the cytoskeleton,such as the microtubule,actin and tubulin.Finally,a webserver named 3CLP was built to predict the cleavage sites of the coronavirus 3CL protease based on the RF model.Overall,the study provides an effective tool for identifying cleavage sites of the 3CL protease and provides insights into the molecular mechanism underlying the pathogenicity of coronaviruses.

Procleave: Predicting Protease-specific Substrate Cleavage Sites by Combining Sequence and Structural Information

Proteases are enzymes that cleave and hydrolyse the peptide bonds between two specific amino acid residues of target substrate proteins.Protease-controlled proteolysis plays a key role in the degradation and recycling of proteins,which is essential for various physiological processes.Thus,solving the substrate identification problem will have important implications for the precise understanding of functions and physiological roles of proteases,as well as for therapeutic target identification and pharmaceutical applicability.Consequently,there is a great demand for bioinformatics methods that can predict novel substrate cleavage events with high accuracy by utilizing both sequence and structural information.In this study,we present Procleave,a novel bioinformatics approach for predicting protease-specific substrates and specific cleavage sites by taking into account both their sequence and 3D structural information.Structural features of known cleavage sites were represented by discrete values using a LOWESS data-smoothing optimization method,which turned out to be critical for the performance of Procleave.The optimal approximations of all structural parameter values were encoded in a conditional random field(CRF)computational framework,alongside sequence and chemical group-based features.Here,we demonstrate the outstanding performance of Procleave through extensive benchmarking and independent tests.Procleave is capable of correctly identifying most cleavage sites in the case study.Importantly,when applied to the human structural proteome encompassing 17,628 protein structures,Procleave suggests a number of potential novel target substrates and their corresponding cleavage sites of different proteases.Procleave is implemented as a webserver and is freely accessible at http://procleave.erc.monash.edu/.

Tau truncation in the pathogenesis of Alzheimer's disease:a narrative review

Alzheimer's disease is characterized by two major neuropathological hallmarks—the extracellularβ-amyloid plaques and intracellular neurofibrillary tangles consisting of aggregated and hyperphosphorylated Tau protein.Recent studies suggest that dysregulation of the microtubuleassociated protein Tau,especially specific proteolysis,could be a driving force for Alzheimer's disease neurodegeneration.Tau physiologically promotes the assembly and stabilization of microtubules,whereas specific truncated fragments are sufficient to induce abnormal hyperphosphorylation and aggregate into toxic oligomers,resulting in them gaining prion-like characteristics.In addition,Tau truncations cause extensive impairments to neural and glial cell functions and animal cognition and behavior in a fragment-dependent manner.This review summarizes over 60 proteolytic cleavage sites and their corresponding truncated fragments,investigates the role of specific truncations in physiological and pathological states of Alzheimer's disease,and summarizes the latest applications of strategies targeting Tau fragments in the diagnosis and treatment of Alzheimer's disease.

Roles of host proteases in the entry of SARS-CoV-2

The spike protein(S)of SARS-CoV-2 is responsible for viral attachment and entry,thus a major factor for host suscep-tibility,tissue tropism,virulence and pathogenicity.The S is divided with S1 and S2 region,and the S1 contains the receptor-binding domain(RBD),while the S2 contains the hydrophobic fusion domain for the entry into the host cell.Numerous host proteases have been implicated in the activation of SARS-CoV-2 S through various c leavage sites.In this article,we review host proteases including furin,trypsin,transmembrane protease serine 2(TMPRSS2)and cathepsins in the activation of SARS-CoV-2 S.Many betacoronaviruses including SARS-CoV-2 have polybasic residues at the S1/S2 site which is subjected to the cleavage by furin.The S1/S2 cleavage facilitates more assessable RBD to the receptor ACE2,and the binding triggers further conformational changes and exposure of the S2'site to proteases such as type Il transmembrane serine proteases(TTPRs)including TMPRSS2.In the presence of TMPRSS2 on the target cells,SARS-CoV-2 can utilize a direct entry route by fusion of the viral envelope to the cellular membrane.In the absence of TMPRSS2,SARS-CoV-2 enter target cells via endosomes where multiple cathepsins cleave the S for the successful entry.Additional host proteases involved in the cleavage of the S were discussed.This article also includes roles of 3C-like protease inhibitors which have inhibitory activity against cathepsin L in the entry of SARS-CoV-2,and discussed the dual roles of such inhibitors in virus replication.

Enhanced Protective Efficacy of H5 Subtype Influenza Vaccine with Modification of the Multibasic Cleavage Site of Hemagglutinin in Retroviral Pseudotypes

Traditionally, the multibasic cleavage site （MBCS） of surface protein H5-hemagglutinin （HA） is converted to a monobasic one so as to weaken the virulence of recombinant H5N1 influenza viruses and to produce inactivated and live attenuated vaccines. Whether such modification benefits new candidate vaccines has not been adequately investigated. We previously used retroviral vectors to generate wtH5N1 pseudotypes containing the wild-type HA （wtH5） from A/swine/Anhui/ca/2004 （H5N1） virus. Here, we generated mtH5N1 pseudotypes, which contained a mutant-type HA （mtH5） with a modified monobasic cleavage site. Groups of mice were subcutaneously injected with the two types of influenza pseudotypes. Compared to the group immunized with wtH5N1 pseudotypes, the inoculation of mtH5N1 pseudotypes induced significantly higher levels of HA specific IgG and IFN-y in immunized mice, and enhanced protection against the challenge of mouse-adapted avian influenza virus A/Chicken/Henardl2/2004 （H5N1）. This study suggests modification of the H5-hemagglutinin MBCS in retroviral pseudotypes enhances protection efficacy in mice and this information may be helpful for development of vaccines from mammalian cells to fight against H5N 1 influenza viruses.

Sequence-specific Hydrolysis of Single-stranded DNA by PNA-Cerium （Ⅳ） Adduct

A novel artificial site specific cleavage reagent, with peptide nucleic acid （PNA） as sequence-recognizing moiety and cerium （IV） ions as “scissors” for cleaving target DNA, was synthesized. Subsequently, it was employed in the cleavage of target 26-mer single-stranded DNA （ssDNA）, which has 10-mer sequence complementary with PNA recognizer in the hybrids, under physiological conditions. Reversed-phase high-performance liquid chromatogram （RPHPLC） experiments indicated that the artificial site specific cleavage reagent could cleave the target DNA specifically.

A novel bat coronavirus with a polybasic furin-like cleavage site

The current pandemic of COVID-19 caused by a novel coronavirus,severe acute respiratory syndrome coronavirus-2(SARS-CoV-2),threatens human health around the world.Of particular concern is that bats are recognized as one of the most potential natural hosts of SARS-CoV-2;however,coronavirus ecology in bats is still nascent.Here,we performed a degenerate primer screening and next-generation sequencing analysis of 112 bats,collected from Hainan Province,China.Three coronaviruses,namely bat betacoronavirus(Bat CoV)CD35,Bat CoV CD36 and bat alpha-coronavirus CD30 were identified.Bat CoV CD35 genome had 99.5%identity with Bat CoV CD36,both sharing the highest nucleotide identity with Bat Hp-betacoronavirus Zhejiang2013(71.4%),followed by SARS-CoV-2(54.0%).Phylogenetic analysis indicated that Bat CoV CD35 formed a distinct clade,and together with Bat Hp-betacoronavirus Zhejiang2013,was basal to the lineage of SARS-CoV-1 and SARS-CoV-2.Notably,Bat CoV CD35 harbored a canonical furin-like S1/S2 cleavage site that resembles the corresponding sites of SARS-CoV-2.The furin cleavage sites between CD35 and CD36 are identical.In addition,the receptor-binding domain of Bat CoV CD35 showed a highly similar structure to that of SARS-CoV-1 and SARS-CoV-2,especially in one binding loop.In conclusion,this study deepens our understanding of the diversity of coronaviruses and provides clues about the natural origin of the furin cleavage site of SARS-CoV-2.

Cleavage of the Carboxyl-Terminus of LEACS2, a Tomato 1-Aminocyclopropane-l-Carboxylic Acid Synthase Isomer, by a 64-kDa Tomato Metalloprotease Produces a Truncated but Active Enzyme

1-Aminocyclopropane-1-carboxylic acid （ACC） synthase （ACS） is the principal enzyme in phytohormone ethylene biosynthesis. Previous studies have shown that the hypervariable C-terminus of ACS is proteolytically processed in vivo. However, the protease responsible for this has not yet been identified. In the present study, we investigated the processing of the 55-kDa full-length tomato ACS （LeACS2） into 52-, 50- and 49-kDa truncated isoforms in ripening tomato （Lycopersicon esculentum Mill. cv. Cooperation 903） fruit using the sodium dodecyl sulfate-boiling method. Meanwhile, an LeACS2-processing protease was purified via multi-step column chromatography from tomato fruit. Subsequent biochemical analysis of the 64-kDa purified protease revealed that it is a metalloprotease active at multiple cleavage sites within the hypervariable C-terminus of LeACS2. N-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight analysis indicated that the LeACS2-processing metalloprotease cleaves at the C-terminal sites Lys^438, Glu^447, Lys^448, Asn^456, Ser^460, Ser^462, Lys^463, and Leu^474, but does not cleave the N- terminus of LeACS2. Four C-terminus-deleted （26-50 amino acids） LeACS2 fusion proteins were overproduced and subjected to proteolysis by this metalloprotease to identify the multiple cleavage sites located on the N-terminal side of the phosphorylation site Ser^460. The results indisputably confirmed the presence of cleavage sites within the region between the α-helix domain （H14） and Ser^460 for this metalloprotease. Furthermore, the resulting C-terminally truncated LeACS2 isoforms were active enzymatically. Because this protease could produce LeACS2 isoforms in vitro similar to those detected in vivo, it is proposed that this metalloprotease may be involved in the proteolysis of LeACS2 in vivo.

Combined insertion of basic and non-basic amino acids at hemagglutinin cleavage site of highly pathogenic H7N9 virus promotes replication and pathogenicity in chickens and mice

Since mid-2016,the low pathogenic H7N9 influenza virus has evolved into a highly pathogenic(HP)phenotype in China,raising many concerns about public health and poultry industry.The insertion of a“KRTA”motif at hemagglutinin cleavage site(HACS)occurred in the early stage of HP H7N9 variants.During the co-circulation,the HACS of HP-H7N9 variants were more polymorphic in birds and humans.Although HP-H7N9 variants,unlike the H5 subtype virus,exhibited the insertions of basic and non-basic amino acids,the underlying function of those insertions and substitutions remains unclear.The results of bioinformatics analysis indicated that the PEVPKRKRTAR/G motif of HACS had become the dominant motif in China.Then,we generated six H7N9 viruses bearing the PEIPKGR/G,PEVPKGR/G,PEVPKRKRTAR/G,PEVPKGKRTAR/G,PEVPKGKRIAR/G,and PEVPKRKRR/G motifs.Interestingly,after the deletion of threonine and alanine(TA)at HACS,the H7N9 viruses manifested decreased thermostability and virulence in mice,and the PEVPKRKRTAR/G-motif virus is prevalent in birds and humans probably due to its increased transmissibility and moderate virulence.By contrast,the insertion of non-basic amino acid isoleucine and alanine(IA)decreased the transmissibility in chickens and virulence in mice.Remarkably,the I335V substitution of H7N9 virus enhanced infectivity and transmission in chickens,suggesting that the combination of mutations and insertions of amino acids at the HACS promoted replication and pathogenicity in chickens and mice.The ongoing evolution of H7N9 increasingly threatens public health and poultry industry,so,its comprehensive surveillance and prevention of H7N9 viruses should be pursued.