Molecular Cloning and Characterization of a New Cold-active Extradiol Dioxygenase from a Metagenomic Library Derived from Polychlorinated Biphenyl-contaminated Soil

To find new extradiol dioxygenases（EDOs,EC 1.13.11.2）,a metagenomics library was constructed from polychlorinated biphenyl-contaminated soil and was screened for some dioxygenase with aromatic ring cleavage activity.A novel EDO,designated as BphC_A,was identified and heterologously expressed in Escherichia coli.The deduced amino acid sequence of BphC_A exhibited a homology of less than 60% with other known EDOs.Phylogenetic analysis of BphC_A suggests that the protein is a novel member of the EDO family.The enzyme exhibits higher substrate affinity and catalytic efficiency toward 3-methylcatechol than toward 2,3-dihydroxybiphenyl or catechol,the preferred substrate of other known EDOs.The optimum activity of purified BphC_A occurred at pH=8.5 and 35 °C,and BphC_A showed more than 40% of its initial activity at 5 °C.The activity of purified BphC_A was significantly induced by Mn^2＋ and slightly reduced by Al^3＋,Cu^2＋ and Zn^2＋.

AZD1775 and anti-PD-1 antibody synergistically sensitize hepatoma to radiotherapy

Background:Radiation(IR)-induced DNA damage triggers cell cycle arrest and has a suppressive effect on the tumor microenvironment(TME).Wee1,a cell cycle regulator,can eliminate G2/M arrest by phosphorylating cyclin-dependent kinase 1(CDK1).Meanwhile,programed death-1/programed death ligand-1(PD-1/PDL-1)blockade is closely related to TME.This study aims to investigate the effects and mechanisms of Wee1 inhibitor AZD1775 and anti-PD-1 antibody(anti-PD-1 Ab)on radiosensitization of hepatoma.Methods:The anti-tumor activity of AZD1775 and IR was determined by 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide(MTT)assay on human and mouse hepatoma cells HepG2,Hepa1-6,and H22.The anti-hepatoma mechanism of AZD1775 and IR revealed by flow cytometry and Western blot in vitro.A hepatoma subcutaneous xenograft mice model was constructed on Balb/c mice,which were divided into control group,IR group,AZD1775 group,IR+AZD1775 group,IR+anti-PD-1 Ab group,and the IR+AZD1775+anti-PD-1 Ab group.Cytotoxic CD8^(+)T cells in TME were analyzed by flow cytometry.Results:Combining IR with AZD1775 synergistically reduced the viability of hepatoma cells in vitro.AZD1775 exhibited antitumor effects by decreasing CDK1 phosphorylation to reverse the IR-induced G2/M arrest and increasing IR-induced DNA damage.AZD1775 treatment also reduced the proportion of PD-1^(+)/CD8^(+)T cells in the spleen of hepatoma subcutaneous xenograft mice.Further studies revealed that AZD1775 and anti-PD-1 Ab could enhance the radiosensitivity of hepatoma by enhancing the levels of interferonγ(IFNγ)^(+)or Ki67^(+)CD8 T cells and decreasing the levels of CD8^(+)Tregs cells in the tumor and spleen of the hepatoma mice model,indicating that the improvement of TME was manifested by increasing the cytotoxic factor IFNγexpression,enhancing CD8^(+)T cells proliferation,and weakening CD8^(+)T cells depletion.Conclusions:This work suggests that AZD1775 and anti-PD-1 Ab synergistically sensitize hepatoma to radiotherapy by enhancing IR-induced DNA damage and improving cytotoxic CD8^(+)T cells in TME.

Ectopic Expression of TRIM25 Restores RIG-I Expression and IFN Production Reduced by Multiple Enteroviruses 3Cpro

Enteroviruses(EVs) 3C proteins suppress type I interferon(IFN) responses mediated by retinoid acid-inducible gene I(RIG-I), while an E3 ubiquitin ligase, tripartite motif protein 25(TRIM25)-mediated RIG-I ubiquitination is essential for RIG-I antiviral activity. Therefore, whether the effect of EVs 3C on RIG-I is associated with TRIM25 expression is worth to be further investigated. Here, we demonstrate that 3C proteins of EV71 and coxsackievirus B3(CVB3) reduced not only RIG-I expression but also TRIM25 expression through protease cleavage activity, while overexpression of TRIM25 restored RIG-I expression and IFN-b production reduced by 3C proteins. Further investigation confirmed that the two amino acids and functional domains in TRIM25 required for RIG-I ubiquitination and TRIM25 structural conformation were essential for the recovery of RIG-I expression. Moreover, we also observed that TRIM25 could rescue RIG-I expression reduced by 3C proteins of CVA6 and EV-D68 but not CVA16. Our findings provide an insightful interpretation of 3C-mediated host innate immune suppression and support TRIM25 as an attractive target against multiple EVs infection.

Regulation of Virus Replication and T Cell Homeostasis by N^6-Methyladenosine

RNA modifications are abundant in eukaryotes, bacteria, and archaea. N^6-methyladenosine(m^6A), a type of RNA modification mainly found in messenger RNA(mRNA), has significant effects on the metabolism and function of m RNAs. This modification is governed by three types of proteins, namely methyltransferases as ‘‘writers' ', demethylases as ‘‘erasers' ',and specific m^6A-binding proteins(YTHDF1-3) as ‘‘readers' '. Further, it is important for the regulation of cell fate and has a critical function in many biological processes including virus replication, stem cell differentiation, and cancer development, and exerts its effect by controlling gene expression. Herein, we summarize recent advances in research on m^6A in virus replication and T cell regulation, which is a rapidly emerging field that will facilitate the development of antiviral therapies and the study of innate immunity.

A case report of autochthonous Q fever with pneumonia and hepatitis in northeastern China

There are only a few case reports of Q fever caused by Coxiella burnetii in China,despite the nature as a ubiquitous zoonotic disease worldwide.In the northeast part of China,a 52-year-old male presented with fever,cough,shortness of breath,and sputum production,accompanied by headache,dizziness,chill,myalgia,and arthralgia.Chest computed tomography images showed pneumonia accompanied by bilateral scattered infiltrates and localized upper-lobe emphysema.The abnormal liver function was indicated by the increased levels of alanine aminotransferase and aspartate aminotransferase.Through high-throughput sequencing and molecular detection,Coxiella burnetii was positive in the patient's blood specimens.After treatment with moxifloxacin hydrochloride and vidarabine monophosphate for 12 days,the patient completely recovered.To our knowledge,this was the first reported case of Q fever with pneumonia and hepatitis in this country.

The SARS-CoV-2 main protease induces neurotoxic TDP-43 cleavage and aggregates

Dear Editor,Neurologic manifestations associated with many COVID-19 patients,including acute infection with severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)and long COVID,have been proven by increasing evidence.1 To date,most studies have focused on how SARS-CoV-2 invades the nervous system and the consequent neuropathological changes.2,3 In contrast,the specific mechanism by which SARS-CoV-2 infection leads to neurological disease remains unclear.

LncRNA NKILA inhibits HBV replication by repressing NF-κB signalling activation

Hepatitis B virus(HBV)infection results in liver cirrhosis and hepatocellular carcinoma(HCC).HBx/nuclear factor(NF)-κB pathway plays a role in HBV replication.However,whether NF-κB-interacting long noncoding RNA(NKILA),a suppressor of NF-κB activation,regulates HBV replication remains largely unknown.In this study,gain-and-loss experiments showed that NKILA inhibited HBV replication by inhibiting NF-κB activity.In turn,HBV infection down-regulated NKILA expression.In addition,expression levels of NKILA were lower in the peripheral blood-derived monocytes(PBMCs)of HBV-positive patients than in healthy individuals,which were correlated with HBV viral loads.And a negative correlation between NKILA expression level and HBV viral loads was observed in blood serum from HBV-positive patients.Lower levels of endogenous NKILA were also observed in HepG2 cells expressing a 1.3-fold HBV genome,HBV-infected HepG2-NTCP cells,stable HBV-producing HepG2.2.15 and HepAD38 cells,compared to those HBV-negative cells.Furthermore,HBx was required for NKILA-mediated inhibition on HBV replication.NKILA decreased HBx-induced NF-κB activation by interrupting the interaction between HBx and p65,whereas NKILA mutants lack of essential domains for NF-κB inhibition,lost the ability to inhibit HBV replication.Together,our data demonstrate that NKILA may serve as a suppressor of HBV replication via NF-κB signalling.

EV713C protease cleaves host anti-viral factor OAS3 and enhances virus replication

The global spread of enteroviruses(EVs)has become more frequent,severe and life-threatening.Intereron(IFN)I has been proved to control EVs by regulating IFN-stimulated genes(ISG)expression.20-50-oligoadenylate synthetases 3(OAS3)is an important ISG in the OAS/RNase L antiviral system.The relationship between OAS3 and EVs is still unclear.Here,we reveal that OAS3,superior to OAS1 and OAS2,significantly inhibited EV71 replication in vitro.However,EV71 utilized autologous 3C protease(3C^(pro))to cleave intracellular OAS3 and enhance viral replication.Rupintrivir,a human rhinovirus 3C protease inhibitor,completely abolished the cleavage of EV713C^(pro)on OAS3.And the proteolytically deficient mutants H40G,E71A,and C147G of EV713C^(pro)also lost the ability of OAS3 cleavage.Mechanistically,the Q982-G983 motif in C-terminal of OAS3 was identified as a crucial 3C^(pro)cutting site.Further investigation indicated that OAS3 inhibited not only EV71 but also Coxsackievirus B3(CVB3),Coxsackievirus A16(CA16),Enterovirus D68(EVD68),and Coxsackievirus A6(CA6)subtypes.Notably,unlike other four subtypes,CA163C^(pro)could not cleave OAS3.Two key amino acids variation Ile36 and Val86 in CA163C^(pro)might result in weak and delayed virus replication of CA16 because of failure of OAS and 3AB cleavage.Our works elucidate the broad anti-EVs function of OAS3,and illuminate a novel mechanism by which EV71 use 3C^(pro)to escape the antiviral effect of OAS3.These findings can be an important entry point for developing novel therapeutic strategies for multiple EVs infection.

ATP1B3 Restricts Hepatitis B Virus Replication Via Reducing the Expression of the Envelope Proteins

Our recent study reported that ATP1B3 inhibits hepatitis B virus(HBV)replication via inducing NF-κB activation.However,ATP1B3 mutants which were defective in NF-κB activation still maintained the moderate degree of suppression on HBV replication,suggesting that another uncharacterized mechanism is also responsible for ATP1B3-mediated HBV suppression.Here,we demonstrated that ATP1B3 reduced the expression of HBV envelope proteins LHBs,MHBs and SHBs,but had no effect on intracellular HBV DNA,RNA levels as well as HBV promoter activities.Further investigation showed that proteasome inhibitor MG132 rescued ATP1B3-mediated envelope proteins degradation,demonstrating that proteasome-dependent pathway is involved in ATP1B3-induced degradation of envelope proteins.Co-IP showed that ATP1B3 interacts with LHBs and MHBs and induces LHBs and MHBs polyubiquitination.Immunofluorescence colocalization analysis confirmed LHBs and MHBs colocalized with ATP1B3 together.Our work provides important information for targeting ATP1B3 as a potential therapeutic molecule for HBV infection.

Inhibition of the Neddylation Pathway Suppresses Enterovirus Replication

Dear Editor,Human enteroviruses(HEVs)comprise polioviruses,coxsackieviruses,echoviruses,rhinoviruses,and the enterovirus subgroups and belong to the genus Enterovirus in the family Picornaviridae(Baggen et al.2018).The HEV genome was thought to contain a single open reading frame(ORF)encoding one polyprotein,which was post-translationally processed into structural capsid proteins(VP1-4)and non-structural accessory proteins(2A,2B,2C,3A,3B,3C,3D)(Baggen et al.2018).Recently,we and others,have identified an additional upstream ORF(Guo et al.2019a;Lulla et al.2019)(ORF2/uORF).

Deubiquitinase ubiquitin-specific protease 3 (USP3) inhibits HIV-1 replication via promoting APOBEC3G (A3G) expression in both enzyme activity-dependent and -independent manners

Background: Ubiquitination plays an essential role in many biological processes, including viral infection, and can be reversed by deubiquitinating enzymes (DUBs). Although some studies discovered that DUBs inhibit or enhance viral infection by various mechanisms, there is lack of information on the role of DUBs in virus regulation, which needs to be further investigated.Methods: Immunoblotting, real-time polymerase chain reaction,in vivo/in vitro deubiquitination, protein immunoprecipitation, immunofluorescence, and co-localization biological techniques were employed to examine the effect of ubiquitin-specific protease 3 (USP3) on APOBEC3G (A3G) stability and human immunodeficiency virus (HIV) replication. To analyse the relationship between USP3 and HIV disease progression, we recruited 20 HIV-infected patients to detect the levels of USP3 and A3G in peripheral blood and analysed their correlation with CD4^(+) T-cell counts. Correlation was estimated by Pearson correlation coefficients (for parametric data).Results: The results demonstrated that USP3 specifically inhibits HIV-1 replication in an A3G-dependent manner. Further investigation found that USP3 stabilized 90% to 95% of A3G expression by deubiquitinating Vif-mediated polyubiquitination and blocking its degradation in an enzyme-dependent manner. It also enhances the A3G messenger RNA (mRNA) level by binding to A3G mRNA and stabilizing it in an enzyme-independent manner. Moreover, USP3 expression was positively correlated with A3G expression (r= 0.5110) and CD4^(+) T-cell counts (r= 0.5083) in HIV-1-infected patients.Conclusions: USP3 restricts HIV-1 viral infections by increasing the expression of the antiviral factor A3G. Therefore, USP3 may be an important target for drug development and serve as a novel therapeutic strategy against viral infections.

Characterization of the amino-terminal domain of Mx2/MxB-dependent interaction with the HIV-1 capsid

Dear Editor More than 50 years have passed since the myxovirus resis- tance （MX） genes were first discovered and found to suppress infection with influenza viruses in mice （Lindenmann, 1962）. Like most mammals, mice carry two MXgenes, MX1 and MX2, which have arisen by gene duplication; both of these genes exhibit antiviral activity against a wide range of viruses （Liu et al., 2013）. Humans also have two MX genes, encoding the MxA and MxB proteins, which are interferon-induced, dynamin- like large molecular weight guanosine triphosphatases （GTP- ases）. The antiviral functions of MxA have been deeply explored： MxA can protect cells from infection by multiple groups of pathogenic DNA and RNA viruses, such as influenza A virus and hepatitis B virus （Liu et al., 2013）.

The E3 ligase RNF5 restricts SARS-CoV-2 replication by targeting its envelope protein for degradation

The coronavirus disease 2019(COVID-19)pandemic caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has caused a severe global health crisis;its structural protein envelope(E)is critical for viral entry,budding,production,and induction of pathology which makes it a potential target for therapeutics against COVID-19.Here,we find that the E3 ligase RNF5 interacts with and catalyzes ubiquitination of E on the 63rd lysine,leading to its degradation by the ubiquitin-proteasome system(UPS).Importantly,RNF5-induced degradation of E inhibits SARS-CoV-2 replication and the RNF5 pharmacological activator Analog-1 alleviates disease development in a mouse infection model.We also found that RNF5 is distinctively expressed in different age groups and in patients displaying different disease severity,which may be exploited as a prognostic marker for COVID-19.Furthermore,RNF5 recognized the E protein from various SARS-CoV-2 strains and SARS-CoV,suggesting that targeting RNF5 is a broad-spectrum antiviral strategy.Our findings provide novel insights into the role of UPS in antagonizing SARS-CoV-2 replication,which opens new avenues for therapeutic intervention to combat the COVID-19 pandemic.

Correction To:The E3 ligase RNF5 restricts SARS-CoV-2 replication by targeting its envelope protein for degradation

Correction to:Signal Transduction and Targeted Therapy https://doi.org/10.1038/s41392-023-01335-5,published online 03 February 2023 In the process of collating the raw data,the authors noticed 7 inadvertent mistakes occurred in Figs.3a,3b,3e,5d,6b,6d and 6e that need to be corrected after online publication of the article.1 The correct data are provided as follows.The key findings of the article are not affected by these corrections.The original article has been corrected.

IFN-β1b induces OAS3 to inhibit EV71 via IFN-β1b/JAK/STAT1 pathway

Enterovirus 71(EV71) caused hand, foot and mouth disease(HFMD) is a serious threat to the health of young children. Although type I interferon(IFN-I) has been proven to control EV71 replication, the key downstream IFNstimulated gene(ISG) remains to be clarified and investigated. Recently, we found that 2’-5’-oligoadenylate synthetases 3(OAS3), as one of ISG of IFN-β1b, was antagonized by EV71 3C protein. Here, we confirm that OAS3is the major determinant of IFN-β1b-mediated EV71 inhibition, which depends on the downstream constitutive RNase L activation. 2’-5’-oligoadenylate(2-5A) synthesis activity deficient mutations of OAS3 D816A, D818A,D888A, and K950A lost resistance to EV71 because they could not activate downstream RNase L. Further investigation proved that EV71 infection induced OAS3 but not RNase L expression by IFN pathway. Mechanically, EV71 or IFN-β1b-induced phosphorylation of STAT1, but not STAT3, initiated the transcription of OAS3 by directly binding to the OAS3 promoter. Our works elucidate the immune regulatory mechanism of the host OAS3/RNase L system against EV71 replication.

HIV-1 Vif suppresses antiviral immunity by targeting STING

HIV-1 infection-induced cGAS–STING–TBK1–IRF3 signaling activates innate immunity to produce type I interferon(IFN).The HIV-1 nonstructural protein viral infectivity factor(Vif)is essential in HIV-1 replication,as it degrades the host restriction factor APOBEC3G.However,whether and how it regulates the host immune response remains to be determined.In this study,we found that Vif inhibited the production of type I IFN to promote immune evasion.HIV-1 infection induced the activation of the host tyrosine kinase FRK,which subsequently phosphorylated the immunoreceptor tyrosine-based inhibitory motif(ITIM)of Vif and enhanced the interaction between Vif and the cellular tyrosine phosphatase SHP-1 to inhibit type I IFN.Mechanistically,the association of Vif with SHP-1 facilitated SHP-1 recruitment to STING and inhibited the K63-linked ubiquitination of STING at Lys337 by dephosphorylating STING at Tyr162.However,the FRK inhibitor D-65495 counteracted the phosphorylation of Vif to block the immune evasion of HIV-1 and antagonize infection.These findings reveal a previously unknown mechanism through which HIV-1 evades antiviral immunity via the ITIM-containing protein to inhibit the posttranslational modification of STING.These results provide a molecular basis for the development of new therapeutic strategies to treat HIV-1 infection.