Zika Virus Non-structural Protein 4B Interacts With DHCR7 Kom Yooj Yim Rau Kev Tiv Thaiv Kab Mob

TSAB NTAWV

Tus kab mob Zika (ZIKV) hloov pauv cov proteins uas tsis yog cov txheej txheem los tiv thaiv lub cev tiv thaiv kab mob thiab ua kom muaj txiaj ntsig zoo hauv cov tswv tsev. Cov roj (cholesterol) metabolic enzyme 7-dehydrocholesterol reductase (DHCR7) tsis ntev los no tau tshaj tawm tias cuam tshuam rau lub cev tiv thaiv kab mob hauv ZIKV kab mob. Txawm li cas los xij, qhov tseem ceeb ntawm cov protein uas tsis yog qauv thiab cov txheej txheem koom nrog hauv DHCR7-kev tawm tsam tus kab mob sib kis tsis tau piav qhia zoo. Hauv txoj kev tshawb no, peb tau pom tias tus kab mob ZIKV tau yooj yim DHCR7 qhia. Qhov tseem ceeb, kev tswj hwm DHCR7 nyob rau hauv lem tau yooj yim ZIKV kab mob, thiab thaiv DHCR7 suppressed ZIKV kab mob. Mechanically, ZIKV non-structural protein 4B (NS4B) cuam tshuam nrog DHCR7 kom ntxias DHCR7 qhia. Ntxiv mus, DHCR7 inhibited TANK-binding kinase 1 (TBK1) thiab interferon regulatory factor 3 (IRF3) phosphorylation, uas ua rau txo qis ntawm interferon-beta (IFN-) thiab interferon-stimulated noob (ISGs). Yog li ntawd, peb npaj siab tias ZIKV NS4B khi rau DHCR7 los tawm tsam TBK1 thiab IRF3 ua kom, uas tig mus inhibits IFN- thiab ISGs, yog li pab txhawb ZIKV evasion. Txoj kev tshawb no nthuav dav cov kev nkag siab txog yuav ua li cas cov kab mob uas tsis yog cov qauv ntawm cov proteins antagonize innate tiv thaiv kab mob los pab txhawb kev kis kab mob los ntawm cov roj cholesterol metabolic enzymes thiab nruab nrab.

cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob

1. Taw qhia

Raws li yoov tshaj cum-yug flavivirus, Zika tus kab mob (ZIKV) tau pib txheeb xyuas los ntawm rhesus macaque hauv Uganda xyoo 1947. Muaj ntau qhov kev sib kis thoob ntiaj teb, ZIKV kis tau dhau los ua kev hem thawj rau kev noj qab haus huv thoob ntiaj teb vim kev nthuav dav los ntawm yoov tshaj cum, kev mus ncig thoob ntiaj teb. asymptomatic transporters, thiab kev sib deev kis. Feem ntau cov kab mob ZIKV hauv cov kab mob sib kis dhau los yog asymptomatic lossis mob me (Wang li al., 2016), txawm li cas los xij, nyob rau hauv ob peb qhov kev sib kis dhau los, tus kab mob ZIKV tau ua rau muaj kev puas tsuaj loj, nrog rau cov kab mob neurological sequelae thaum cev xeeb tub (microcephaly thiab fetal demise) thiab paj hlwb. (Guillain-Barre syndrome) hauv cov neeg laus (Cao-Lormeau li al., 2016; Pierson thiab Pob Zeb Diamond, 2020; Rasmussen li al., 2016). Lub cev tiv thaiv kab mob hauv lub cev yog thawj thiab tseem ceeb kab ntawm tus tswv tsev tiv thaiv kab mob tiv thaiv kab mob. Tom qab tus kab mob ZIKV, tus kab mob RNA tau lees paub los ntawm intracellular retinoic acid-inducible gene I (RIG-I) (Chazal et al., 2018; Hertzog et al., 2018; Kato et al., 2006), uas tau pib lub cev tsis muaj zog. cov lus teb, suav nrog TANK binding kinase 1 (TBK1) ua kom muaj zog, interferon regulatory factor 3 (IRF3) phosphorylation thiab tom qab ntawd ua rau hom I interferons (IFN-I) ntau lawm (Fitzgerald li al., 2003; Grant et al., 2016; Xia li al., 2018) thiab kev qhia ntawm kaum ob ntawm interferon-stimulated noob (ISGs) nrog ntau yam tshuaj tiv thaiv kab mob (Savidis li al., 2016; Schneider li al., 2014). Tus tswv tsev tiv thaiv kab mob ua rau IFNs thiab ISGs txwv tsis pub kis tus kab mob, txawm li cas los xij, ZIKV nws tus kheej tau hloov kho ntau yam kev khiav tawm kom ntseeg tau tias muaj kev vam meej thiab rov ua dua tshiab hauv lub xov tooj ntawm tes, xws li encoding tshwj xeeb uas tsis yog qauv (NS) cov proteins. ZIKV genomic RNA tsim xya cov proteins uas tsis yog qauv, suav nrog NS1, NS2A, NS2B, NS3, NS4A, NS4B, thiab NS5 (Berthoux, 2020). ZIKV NS1 recruits host deubiquitinase USP8 kom ruaj khov caspase-1 thiab attenuates hom I IFN signaling kom muaj txiaj ntsig rau tus kab mob ZIKV (Zheng li al., 2018). Ib qho mutant NS1 tau raug tshaj tawm los khi rau TBK1 thiab txo cov phosphorylation ntawm TBK1, ua rau txo IFN- ntau lawm (Xia li al., 2018). ZIKV NS3 khi rau thiab sequesters human scaffold proteins (14-3-3ε thiab 14-3-3η) (Riedl li al., 2019), thaum NS4A cuam tshuam nrog MAVS kom txo qis RIG-I- thiab MDA{{50} }Mediated innate immune teb (Ma et al., 2018). Qhov tseem ceeb, ZIKV NS5 khi thiab degrades teeb liab transducer thiab activator ntawm transcription 2 (STAT2) kom attenuate hom I IFN signaling (Grant li al., 2016; Kumar et al., 2016). Ntxiv mus, NS5 kuj tau raug pov thawj los cuam tshuam nrog RIG-I thiab txwv cov K63-txuas polyubiquitination ntawm RIG-I, yog li txwv IFN- ntau lawm (Li et al., 2020). Tsis tas li ntawd, ZIKV NS2A, NS2B, NS4A, thiab NS4B kuj tau pom tias muaj zog tshaj IFN- ntau lawm los ntawm kev tsom cov ntsiab lus tsis sib xws hauv RIG-I txoj hauv kev (Xia li al., 2018). Txhawm rau ua kom tau txais txiaj ntsig zoo hauv cov qe ntshav, ZIKV kuj tso siab rau cov tswv tsev cellular lipid metabolic Cheebtsam los tsim ib lub hnab ntawv thiab ua tiav cov kab mob sib kis (Chen li al., 2020; Leier et al., 2020). Cov roj (cholesterol) yog ib qho ntawm cov lipid Cheebtsam ntawm cov cell membranes thiab koom nrog ntau yam kev ua haujlwm lom neeg (Ikonen, 2008). Cellular cholesterol homeostasis yog nyob ze hloov kho los ntawm cov roj cholesterol synthesis, nrog rau kev nqus los ntawm lipoprotein hais thiab tso tawm rau cov neeg txais kev pabcuam ntxiv. Yuav luag txhua lub hlwb ntawm cov tsiaj nyeg yog nyob ntawm cov roj cholesterol metabolism (Luo li al., 2020). Cov ntaub ntawv pov thawj tau qhia tias cov roj cholesterol metabolism koom nrog hauv lub cev tiv thaiv kab mob tiv thaiv kab mob (Blanc li al., 2013; Petersen li al., 2014; York et al., 2015). Thaum kis tus kab mob, cov roj cholesterol synthesis tau hloov pauv thiab nrog rau kev nthuav qhia ntawm IFN-I thiab ISGs hauv macrophages (Li et al., 2017; Liu et al., 2008; York et al., 2015). Raws li cov enzyme tseem ceeb hauv cov roj cholesterol metabolism, cov roj (cholesterol) -25-hydroxylase (CH25H) hloov cov roj cholesterol rau hauv 25-hydroxycholesterol (25HC), uas tau txheeb xyuas tias yog cov khoom siv roj ntsha uas ua rau muaj kev tiv thaiv kab mob hauv lub cev tawm tsam feem coob. ntawm cov kab mob, suav nrog human immunodeficiency virus 1 (HIV-1), vesicular stomatitis virus (VSV), herpes simplex virus 1 (HSV-1), Ebola virus (EBOV), ZIKV, murine gamma herpes virus ( MHV68), Rift valley fever virus (RVFV), thiab Lavxias teb sab caij nplooj ntoos hlav-lub caij ntuj sov encephalitis virus (RSSEV) (Blanc li al., 2013; Li et al., 2017; Liu et al., 2013). Cov kev tshawb fawb tam sim no raug txwv hauv kev tshawb xyuas seb cov khoom lag luam roj cholesterol (xws li 25HC) tuaj yeem tswj cov teeb liab hloov pauv thiab koom nrog kev tiv thaiv hauv lub cev. Txog niaj hnub no, cov enzymes lossis cov nruab nrab nruab nrab ntawm cov roj cholesterol biosynthesis tseem tsis tau piav qhia zoo. Raws li qhov tseem ceeb ntawm cov roj cholesterol metabolic enzyme, 7-dehydrocholesterol reductase (DHCR7) tuaj yeem tshem tawm C (7-8) ob daim ntawv cog lus hauv B nplhaib ntawm sterols thiab catalyze cov cholesterol los ntawm 7-dehydrocholesterol ({100} }DHC) (Luu et al., 2015). 7-DHC kuj yog ib tug thawj coj ntawm cov vitamin D, thiab DHCR7 kev hloov pauv tau txuas nrog cov vitamin D ntau dua, qhia tias DHCR7 qhia cov teebmeem lom lom hauv kev hloov cov roj cholesterol thiab vitamin D (Kuan et al., 2013; Prabhu li al. , 2016 a). Ib txoj kev tshawb fawb qhia tau zoo tau pom tias DHCR7 silence tuaj yeem qhib txoj hauv kev PI3K-AKT3, ua rau IRF3 Ser385 phosphorylation thiab txhawb IFN- ntau lawm los tiv thaiv ntau yam kab mob hauv vitro thiab hauv vivo (Xiao li al., 2020). Txawm li cas los xij, kev nkag siab ntawm DHCR{116}}kev kho cov roj cholesterol metabolism hauv ZIKV NS protein-mediated immune evasion yog txhais tsis zoo. Ntawm no, peb piav qhia txog cov txheej txheem sib txawv uas ZIKV NS4B lub hom phiaj ntawm DHCR7, enzyme tseem ceeb hauv kev tsim cov roj cholesterol, kom txo qis cov lus teb IFN-I thiab pab txhawb ZIKV kab mob. Peb pom tias tus kab mob ZIKV nce DHCR7 qhia. Interestingly, txhim kho DHCR7 txhawb nqa tus kab mob ZIKV, thaum tsoo lossis tsom DHCR7 nrog cov tshuaj inhibitors tuaj yeem tiv thaiv tus kab mob ZIKV. Ntxiv mus, ZIKV NS4B tuaj yeem khi DHCR7 thiab yog li ua rau DHCR7 qhia, uas inhibit TBK1 thiab IRF3 activation thiab ua rau txo IFN- thiab ISG ntau lawm. Yog li ntawd, peb npaj siab tias ZIKV NS4B khi rau DHCR7 txo qis IRF3 kev ua haujlwm, uas ua rau inhibit IFN- thiab ISGs los pab txhawb ZIKV evasion. Txoj kev tshawb no nthuav dav cov kev nkag siab tshiab txog yuav ua li cas ZIKV tsis yog cov qauv protein tshem tawm kev tiv thaiv hauv lub cev los pab txhawb kev kis kab mob los ntawm kev cuam tshuam nrog cov roj cholesterol metabolic enzymes.

cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob

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2. Cov ntaub ntawv thiab cov txheej txheem

2.1. Cell kab

U251 hlwb tau yuav los ntawm Tuam Tshoj Chaw rau Hom Kab lis kev cai sau (CCTCC) (Wuhan, Suav). Cercopithecus aethiops raum (Vero) hlwb, tib neeg lub raum embryonic (HEK 293T) hlwb, thiab tib neeg lub ntsws adenocarcinoma (A549) hlwb tau yuav los ntawm American Tissue Culture Collection (ATCC). Cells tau khaws cia hauv Dulbecco's Modified Eagle Medium (DMEM) (Gibco) ntxiv nrog 10% fetal bovine serum (FBS) (Gibco), penicillin (100 U / mL), thiab streptomycin sulfate (100 ug / mL) ntawm 37 C thiab 5. % CO2. Aedes albopictus (C6/36) hlwb tau coj mus kuaj hauv Yam Tsawg Tseem Ceeb Nruab Nrab (MEM) ntxiv nrog 10% FBS, penicillin (100 U / mL), thiab streptomycin sulfate (100 ug / mL) ntawm 28 C nrog 5% CO2.

2.2. ZIKV amplification, titration, thiab kab mob

ZIKV cais z16006 (GenBank tus lej nkag, KU955589.1) tau cog rau hauv C6 / 36 hlwb, thiab ZIKV tau aliquoted hauv khov vials thiab khaws cia ntawm 80 C. ZIKV titers tau ntsuas los ntawm cov quav hniav. Cov hlwb tau kis tus kab mob ZIKV ntawm qhov qhia tau hais tias muaj ntau yam kab mob (MOI) rau 2 h, tom qab ntawd ntxuav nrog phosphate buffer saline (PBS), thiab tom qab ntawd coj mus kuaj, sau, thiab tshuaj xyuas.

2.3. Plasmids thiab reagents

Kev nthuav qhia plasmid pcDNA3.1(þ)-3 Chij tau siv los clone ib tus neeg ZIKV noob los ntawm cov khoom sib txuas ntawm ZIKV cDNA. Txhawm rau tsim pGEX6p-1-NS4B, ZIKV NS4B tau sub-clloned rau hauv pGEX6p-1 vector siv ClonExpress II Ib Kauj Ruam Cloning Kit (Vazyme Biotech, Nanjing, Suav). Cov cDNAs encoding tib neeg DHCR7, RIG-I, TBK1, thiab IRF3 cloned rau hauv pCAGGS-HA vector lossis pcDNA3.1 (þ)-3 Chij vector yog tsim los ntawm tus qauv molecular cloning method. Qhov kev hloov pauv tsis zoo G410S ntawm DHCR7 tau tsim los siv qhov chaw-qhia mutagenesis txoj kev. Monoclonal luav anti-ZIKV hnab ntawv (GTX133314) tau yuav los ntawm GeneTex (Irvine, CA, USA). Luav polyclonal anti-DHCR7 (A8049), luav monoclonal anti-ISG15 (A2416), luav monoclonal anti-TBK1 (A3458), luav monoclonal anti-P-TBK1 ntawm Ser172 (AP1026), luav polyclonal anti-IRF3 (A11118), -rabbit IgG (ac005), thiab anti-nas IgG (ac011) tau yuav los ntawm ABclonal Technology (Wuhan, Suav). Antimouse FITC thiab tshuaj tiv thaiv luav Cy3 cov tshuaj tiv thaiv kab mob thib ob tau yuav los ntawm Abbkine (Wuhan, Suav). Luav monoclonal anti-P-IRF3 ntawm Ser396 (29047S) thiab luav monoclonal anti-ISG56 (14769S) tau yuav los ntawm Cell Signaling Technology (CST, Boston, MA, USA). Mouse monoclonal anti-Flag (F3165), luav monoclonal anti-HA (H6908), thiab nas monoclonal anti-GAPDH (G9295) tau yuav los ntawm Sigma (St Louis, MO, USA). Mouse monoclonal antiFlavivirus pawg antigen-antibody (MAB10216) tau yuav los ntawm Millipore (Mass, USA). Lipofectamine 2000 (11668-027) thiab Trizol (15596018) tau yuav los ntawm Invitrogen Corporation (Carlsbad, CA, USA). Poly (I: C) tau yuav los ntawm InvivoGen (San Diego, CA, USA).

2.4. Plaque assay

ZIKV uas muaj cov tshuaj supernatant tau diluted nrog DMEM tsis muaj ntshav qab zib thiab muaj kab mob Vero hlwb hauv 12-zoo phaj rau 2 teev, tom qab ntawd ntxuav nrog 1 mL PBS ob zaug. DMEM muaj 2% FBS thiab 2% qis melting point agarose tau tov zoo raws li 1: 1 ntim piv, thiab 1 mL sib tov ntxiv rau txhua qhov dej. Cov kab mob kis tau raug coj los ntawm 37 C nrog 5% CO2 rau 5-7 hnub. Cov hlwb raug kho nrog 4% paraformaldehyde rau 1 h thiab dyed nrog 0.5% crystal violet rau 30 min. 12- lub phaj dej tau ntxuav nrog dej, thiab cov plaques raug xam.

2.5. Lentivirus ntau lawm thiab kab mob

Lub hom phiaj ntawm shRNAs rau tib neeg DHCR7 yog raws li nram no: sh-DHCR7-1: 50 -ATTGCCAGCACAGACGGATTT-30, sh-DHCR7-2: 50 -CGTGATTGACTTTCTGGAA{ {8}}. Lub pLKO.1 vector bearing a scrambled shRNA rau kev tswj tsis zoo (Sigma-Aldrich, St. Louis, MO, USA) lossis cov phiaj xwm tshwj xeeb tau kis mus rau HEK293T hlwb ua ke nrog psPAX2 thiab pMD2.G nrog Lipofectamine 2{{19} }00. Cov kab mob uas muaj cov supernatants tau sau thaum 36 teev tom qab hloov pauv thiab tom qab ntawd centrifuged ntawm 210 g rau 10 min. Lub cell-free supernatant theem tau pom los ntawm 0.45 μm lim kom tshem tawm cov khib nyiab ntawm tes. U251 hlwb tau kis cov kab mob lentiviral nyob rau hauv muaj 8 ug / mL polybene. Tom qab 48 teev ntawm kab lis kev cai, U251 hlwb raug xaiv nrog puromycin (2 ug / mL, Sigma). Lub khob-down efficiency ntawm sh-DHCR7 tau txiav txim los ntawm RT-PCR thiab Western blot tsom xam.

cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob

2.6. Western blot

Cells tau lysed los ntawm RIPA tsis ntxiv nrog cov protease inhibitors thiab phosphatase inhibitors ntawm dej khov rau 1 h, ces centrifuged ntawm 4 C, 13, 000 g rau 1{{10}} min los sau cov cell lysate. Protein concentration hauv cell lysate yog ntsuas los ntawm bicinchoninic acid (BCA) protein assay. Cov protein tau sib cais los ntawm SDS-PAGE thiab tom qab ntawd xa mus rau polyvinyl difluoride (PVDF) daim nyias nyias. PVDF daim nyias nyias tau thaiv nrog kev tiv thaiv kab mob tiv thaiv kab mob TBST (10 mmol/L Tris-HCl, pH 7.4, 0.15 mol/L NaCl, thiab 0.05% Tween-20) muaj 5% BSA rau 1 h. Tom qab ntxuav nrog TBST, daim nyias nyias tau incubated nrog thawj cov tshuaj tiv thaiv thiab cov tshuaj tiv thaiv thib ob thiab tom qab kuaj pom siv Chemiluminescence imaging system (Bio-Rad).

2.7. Kev ntsuam xyuas tag nrho cov cholesterol

Tag nrho cov roj (cholesterol) hauv hlwb tau kuaj pom los ntawm kev siv cov khoom siv tshuaj ntsuam xyuas cov roj (cholesterol) tag nrho los ntawm Applygen Technologies Inc. (Beijing, Suav). Tus qauv 5 mmol / L cov roj (cholesterol) tau raug suav nrog kev sib xyaw ua ke nrog anhydrous ethanol los kos tus qauv nkhaus, thiab cov lej ntawm cov roj cholesterol tag nrho tau suav nrog raws li tus qauv nkhaus. Qhov kev sim tau ua raws li cov txheej txheem muab los ntawm cov chaw tsim khoom.

2.8. Co-immunoprecipitation kev soj ntsuam

HEK293T hlwb tau hloov nrog cov plasmids qhia. Cells tau sau thiab lysed nrog RIPA lysis tsis ntxiv nrog protease inhibitors. Tom qab centrifugation ntawm 18, 000 g rau 15 min ntawm 4 C, cell lysates tau sau thiab immunoprecipitated los ntawm Flag-Trap ProteinG Sepharose (GE Healthcare, Milwaukee, WI, USA) rau 4 h ntawm 4 C. Cov proteins uas khi tau eluted nrog 2 loading tsis thiab soj ntsuam los ntawm immunoblotting.

2.9. Real-time PCR

Tag nrho RNAs tau muab rho tawm los ntawm cov hlwb los ntawm kev siv TRIzol reagent (Invitrogen Life Technologies), thiab cDNA tau tsim nrog cov khoom siv M-MLV thim rov qab transcriptase (Promega, Madison, WI, USA). RT-PCR tau ua tiav siv SYBR RT-PCR Cov Khoom Siv (DBI Bio-science) ntawm Roche LC480. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA tau teeb tsa raws li kev siv endogenous rau normalize mRNAs thiab cov qib qhia noob tau suav nrog siv 2 ΔΔCT txoj kev. Cov kab ke ntawm RT-PCR primers tau qhia hauv Cov Lus Ntxiv S1. 2.10. Confocal microscopy

U251 hlwb tau hloov nrog plasmid thiab kab lis kev cai rau 30 h, ces ntxuav peb zaug nrog PBS. Cells raug kho nrog 4% paraformaldehyde rau 15 feeb thiab permeated nrog PBS muaj 0.1% TritonX-100 rau 5 min. Tom qab ntxuav nrog PBS, cov hlwb raug kaw nrog PBS uas muaj 5% BSA rau 30 feeb. Cells raug ntxuav peb zaug nrog PBS thiab incubated nrog anti-HA, anti-FLAG, thiab anti-flavivirus, ces incubated nrog anti-nas FITC thiab anti-luv Cy3 thib ob antibodies. Cells tau stained nrog 40,6-Diamidino-2-phenylindole dihydrochloride (DAPI) thiab ua los ntawm Olympus confocal microscope.

2.11. GST rub-down kev soj ntsuam

Lub plasmid pGEX6p-1-NS4B tau kis mus rau Escherichia coli (E. coli) hom BL21. GST thiab GST-NS4B raug ntxias los ntawm IPTG nrog qhov kawg siab ntawm 0.5 mmol / L, thiab cov kab lis kev cai tau loj hlob ntxiv rau 6-8 teev ntawm 37 C. Thiab tom qab ntawd GST protein thiab GST- NS4B protein raug ntxuav los ntawm cov kab mob E. coli. GST los yog GST-NS4B protein tau incubated nrog glutathione Sepharose hlaws (Novagen) thiab ntxuav peb zaug nrog PBS. GST thiab GST-NS4B protein tau incubated nrog eukaryotic fusion protein HA-DHCR7, uas yog los ntawm plasmids encoding HA-DHCR7- qhia HEK293T cell lysates rau 4 h ntawm 4 C. Cov precipitates tau ntxuav tsib zaug, boiled nyob rau hauv 2 SDS loading tsis, thiab kuaj los ntawm Western blot nrog anti-GST thiab anti-HA cov tshuaj tiv thaiv.

2.12. HA-DHCR7 site-directed mutagenesis

Cov khub uas tsim nyog ntawm mutagenic primers (Table Ntxiv S1) tau tsim thiab siv los tsim cov kev hloov pauv los ntawm PCR. HA-DHCR7 tau siv los ua tus qauv rau PrimeSTAR® HS Premix (Takara). Tom qab PCR, cov tsiaj qus-hom plasmid tseem tshuav hauv cov khoom PCR tau xaiv zom los ntawm DpnI (New England Biolabs). Cov khoom digested PCR tau kis mus rau E. coli strain DH5 . Qhov xav tau mutant tau lees paub los ntawm kev soj ntsuam ntawm Sanger.

cistanche tsob nroj-nce kev tiv thaiv kab mob

2.13. Kev txheeb cais

Txhua qhov kev sim tau rov ua dua tsawg kawg peb zaug nrog cov txiaj ntsig zoo sib xws. Cov ntaub ntawv raug nthuav tawm raws li tus qauv sib txawv (SD). Qhov tseem ceeb ntawm qhov sib txawv tau txiav txim siab los ntawm Tus Tub Kawm Ntawv ob-tailed unpaired t-test rau ob pawg lossis ib-txoj kev ANOVA nrog Dunnett ntau qhov kev sib piv los yog ob txoj kev ANOVA nrog Sidak ntau qhov kev sib piv rau ntau pab pawg sib piv siv GraphPad Prism software (version 8.0.1). Tus nqi ntawm P < 0.{{10}}5 tau suav tias yog qhov tseem ceeb, thiab P > 0.05 tau raug txheeb xyuas tias tsis tseem ceeb. Rau tag nrho cov ntaub ntawv, qhov tseem ceeb tau nthuav tawm raws li * P < 0.05, ** P < 0.01 thiab *** P < 0.001.

3. Cov txiaj ntsig

3.1. Tus kab mob ZIKV induces DHCR7 qhia

Cov ntaub ntawv pov thawj tau pom tias cov roj cholesterol metabolism cuam tshuam rau lub cev tiv thaiv kab mob hauv lub cev tiv thaiv kab mob flavivirus, xws li ZIKV thiab Dengue virus (DENV) (Leier li al., 2020; Randall, 2018). DHCR7 encodes ib qho enzyme uas hloov 7-DHC rau hauv cov roj cholesterol, cov kauj ruam kawg hauv biosynthesis ntawm cov cholesterol (Moebius li al., 1998). Peb thawj zaug kuaj pom DHCR7 qhia thaum tus kab mob ZIKV. Tib neeg astrocyte U251 tau kis tus kab mob ZIKV, uas yog nyob ntawm koob tshuaj ntau ntxiv DHCR7 qhia ntawm ob qho tib si mRNA thiab protein (Fig. 1A–C). Txhawm rau kom paub meej qhov tshwm sim, peb tau tshuaj xyuas DHCR7 tom ntej hauv Vero hlwb thiab pom tias tus kab mob ZIKV tau ua rau DHCR7 qhia ntau heev (Fig. 1D-F), uas tau ua raws li cov txiaj ntsig hauv U251 hlwb.

cistanche cov txiaj ntsig-ua kom muaj zog tiv thaiv kab mob

3.2. Targeting DHCR7 elicits ib qho tseem ceeb heev anti-ZIKV kev ua ub no

Txhawm rau ntsuas qhov cuam tshuam ntawm DHCR7 tiv thaiv tus kab mob ZIKV, U251 thiab A549 hlwb tau kis nrog HA-DHCR7 encoding plasmid thiab tom qab ntawd kis tus kab mob ZIKV (MOI ¼ 1). Cov txiaj ntsig tau pom tias DHCR7 overexpression ho txhim kho ZIKV mRNA qhia (Fig. 2A thiab B). Qhov tseem ceeb, nrog rau kev sib sau ntawm DHCR7 protein, kev qhia ntawm ZIKV structural protein E kuj tau nce ntxiv (Daim duab 2C thiab D). Tsis tas li ntawd, peb tau soj ntsuam cov quav hniav ntxiv uas siv cov supernatant los ntawm daim duab 2C thiab pom tias DHCR7 overexpression ho nce plaque-forming units, piv rau pawg tswj (Fig. 2E thiab F). Kev hloov pauv tsis zoo G410S ntawm DHCR7 tau tshaj tawm kom tshem tawm cov haujlwm enzyme (Fitzky li al., 1998; Shim li al., 2004; Witsch-Baumgartner li al., 2000). Txhawm rau txiav txim siab ntxiv qhov cuam tshuam ntawm DHCR7 ntawm tus kab mob ZIKV, peb txuas ntxiv tsim qhov tsis muaj zog ntawm DHCR7 (G410S). Piv rau cov tsiaj qus-hom DHCR7, G410S mutant tuaj yeem cuam tshuam ZIKV kab mob (Daim duab 2G). Peb kuj tau ua qhov kev ntsuam xyuas qhov ntsuas qhov ntsuas qhov ntsuas kom paub meej qhov kev soj ntsuam. Raws li pom nyob rau hauv daim duab 2H, overexpression ntawm DHCR7 nce ZIKV kab mob. Txhawm rau txiav txim siab seb qhov kev ua haujlwm endogenous DHCR7 yog qhov tseem ceeb rau tus kab mob ZIKV, peb tau tsim ob lub hairpin RNAs (shDHCR7-1 thiab sh-DHCR7-2) tshwj xeeb rau DHCR7 thiab tsim DHCR7-silenced U251 cell. kab nrog shRNA system (Fig. 3A). Thaum kawg, peb tau xaiv ib qho sh-DHCR7-1 rau nws txoj kev ua kom ntsiag to ntau dua. Piv rau cov kab hluav taws xob hloov pauv kev tswj hwm shRNA, cov DHCR7 knockdown hlwb tau pom qis dua rau ZIKV kab mob (Fig. 3B thiab C). Peb tom ntej no siv cov kev xaiv DHCR7 inhibitor AY9944 uas tiv thaiv 7-DHC sib tham rau cov roj cholesterol (Xu li al., 2011), uas tau pom tias inhibit DHCR7 enzymic kev ua haujlwm (Horlick, 1966; Moebius et al., 1998). Piv rau kev tswj hwm, AY9944 kev tswj hwm koob tshuaj-raws li inhibited ZIKV kab mob hauv U251 hlwb (Fig. 3D-F). Plaque assay kuj tau ua nyob rau hauv Vero hlwb los soj ntsuam ntxiv cov teebmeem ntawm DHCR7, thiab qhia tias AY9944 tuaj yeem cuam tshuam tus kab mob ZIKV (Daim duab 3G thiab H). Qhov tseem ceeb, confocal microscopy tau pom tias ZIKV E protein tau txo qis hauv qhov muaj AY9944 (Fig. 3I). Cov kev tshawb pom no tau qhia tias lub hom phiaj DHCR7 tuaj yeem cuam tshuam tus kab mob ZIKV.

Fig. 1. Tus kab mob ZIKV induces DHCR7 qhia. A–F U251 thiab Vero hlwb tau kis ZIKV (MOI ¼ 0, 1 thiab 2) rau 24 h, thiab cov qib RNA intracellular ZIKV (A, D) thiab DHCR7 (B, E) tau txiav txim los ntawm RT-PCR nrog GAPDH raws li kev tswj hwm sab hauv. Cov qib protein txheeb ze ntawm DHCR7 thiab ZIKV hnab ntawv tau kuaj pom los ntawm Western blot (C, F). Cov ntaub ntawv yog los ntawm tsawg kawg peb qhov kev sim ywj pheej (txhais tau tias SD) lossis cov ntaub ntawv sawv cev (C, F). Kev txheeb xyuas qhov tseem ceeb tau suav nrog siv ib-txoj kev ANOVA nrog Dunnett ntau qhov kev sib piv (A, B, D, E). **P < 0 01, ***P < 0.001. SD, tus qauv sib txawv.

3.3. DHCR7 inhibits TBK1 thiab IRF3 ua kom txo tau IFN- thiab ISGs ntau lawm

Txij li thaum DHCR7 yog qhov tseem ceeb enzyme hloov pauv 7-DHC rau cov roj cholesterol (Moebius li al., 1998) thiab tus kab mob ZIKV tuaj yeem ua rau DHCR7 qhia, peb ntsuas qib roj cholesterol raws li tus kab mob ZIKV. Peb tau tshaj tawm tias tus kab mob ZIKV muaj kev cuam tshuam me ntsis ntawm cov roj cholesterol synthesis hauv U251 thiab Vero hlwb (Fig. 4A thiab B), txawm hais tias DHCR7 tau nce (Fig. 1A–D). Muab qhov txhim kho DHCR7 raws li tus kab mob ZIKV thiab lub luag haujlwm tseem ceeb ntawm IFNs hauv kev tawm tsam ZIKV tus kab mob, peb tom ntej tau txiav txim siab qhov cuam tshuam ntawm DHCR7 ntawm IFN-I txoj kev taw qhia. Kev tswj hwm thiab DHCR7-silenceing U251 hlwb tau kis tus kab mob ZIKV lossis kis nrog poly(I: C), thiab IFN- qhia tau tshawb nrhiav. Raws li piv nrog rau pawg tswj hwm, ZIKV kab mob los yog kev kho mob poly(I: C) tau txhim kho IFN- qhia hauv DHCR7-silences U251 hlwb (Fig. 4C thiab D). ISGs yog cov txiaj ntsig ntawm kev ua haujlwm interferon thiab ua lub luag haujlwm tseem ceeb hauv kev tiv thaiv kab mob hauv lub cev tiv thaiv kab mob ZIKV. Peb kuj tau pom tias thaiv DHCR7 tuaj yeem txhawb nqa ISG15 thiab ISG56 kev qhia ntawm ob qho tib si mRNA thiab protein ntau (Fig. 4E–J). Raws li kev kho, DHCR7 inhibitor AY9944 tuaj yeem ua rau IFN-, ISG15, thiab ISG56 qhia (Fig. 4K, L). Qhov tseem ceeb, qhov tsis muaj zog mutant G410S tuaj yeem thim rov qab cov teebmeem inhibitory ntawm DHCR7 ntawm IFN- thiab ISG qhia (Fig. 4M, N). Kev ua kom TBK1 thiab IRF3 yog qhov tseem ceeb rau IFNs-ISGs induction, yog li, tom ntej no peb tau soj ntsuam cov txiaj ntsig ntawm DHCR7 ntawm TBK1 thiab IRF3 qhia thiab ua kom. Peb pom tau hais tias DHCR7 tsis yog tsuas yog siv tshuaj tua kab mob inhibited qib protein, tab sis kuj inhibited qib ntawm TBK1 thiab IRF3 phosphorylation (Fig. 5A–C). Ntxiv mus, qhov tsis muaj zog mutant G410S ua tsis tiav qhov cuam tshuam inhibitory ntawm DHCR7 ntawm IRF3 thiab TBK1 phosphorylation (Fig. 5D). Tsis sib haum nrog qhov kev tshawb pom ntawm DHCR7 overexpression, AY9944 kev kho mob tuaj yeem ua rau IRF3 thiab TBK1 ua rau hauv U251 cell (Fig. 5E).

3.4. ZIKV NS4B cuam tshuam nrog DHCR7 rau inhibit TBK1 thiab IRF3 phosphorylation

Mounting pov thawj tau qhia tias ZIKV hloov kho cov NS cov proteins tshwj xeeb los pab txhawb kev rov ua haujlwm zoo hauv cov hlwb los ntawm kev tawm tsam tus tswv tsev hauv lub cev thiab kev tiv thaiv kab mob los ntawm ntau yam ntawm cov txheej txheem sib cais. Peb tau ua qhov kev ntsuam xyuas co-IP los tshuaj xyuas tus neeg ZIKV NS protein uas tuaj yeem cuam tshuam nrog DHCR7. HEK293T hlwb tau koom ua ke nrog pCMV-DHCR7-HA thiab plasmid qhia tus neeg NS protein fused nrog Flag-tag. Raws li qhia hauv daim duab 6A, DHCR7 tsuas khi rau NS4B tab sis tsis cuam tshuam nrog lwm cov NS proteins. Reciprocal co-IP kev soj ntsuam tau ua ntxiv thiab pom tias NS4B protein cuam tshuam nrog DHCR7 hauv HEK293T hlwb (Fig. 6B thiab C). Txhawm rau kom paub meej tias qhov kev sib cuam tshuam ntawm NS4B thiab DHCR7, peb tom ntej no tau ua lub tshuab tsom iav thiab pom tias NS4B protein thiab DHCR7 protein tau colocalized hauv cytoplasm (Fig. 6D). GST rub-down assay ntxiv tau qhia txog kev sib cuam tshuam ntawm NS4B thiab DHCR7 (Fig. 6E). Ntxiv mus, transient transfection ntawm NS4B hauv U251 hlwb tuaj yeem ua rau DHCR7 qhia (Fig. 6F). Qhov tseem ceeb, NS4B tuaj yeem txhaj tshuaj tiv thaiv phosphorylation ntawm ob qho tib si TBK1 thiab IRF3, uas muaj kev cuam tshuam zoo nrog kev ua kom zoo dua ntawm TBK1 thiab IRF3 hauv cov lus teb rau DHCR7 (Fig. 6G thiab H). Txawm li cas los xij, NS4B overexpression hauv HEK293T hlwb txo qis cov protein ntau ntawm TBK1, tab sis tsis tshua muaj txiaj ntsig rau IRF3 protein (Fig. 6G thiab H). Txhawm rau kom paub meej ntxiv txog cov kev soj ntsuam no, NS4B tau kis mus rau hauv DHCR7 knockdown U251 hlwb thiab pom tias muaj kev cuam tshuam inhibitory ntawm endogenous protein thiab phosphorylation theem ntawm TBK1 thiab IRF3 (Fig. 6I). Ua ke, cov txiaj ntsig no tau pom tias ZIKV NS4B cuam tshuam nrog DHCR7 thiab txhim kho DHCR7 qhia los thaiv TBK1 thiab IRF3 ua kom muaj zog thiab pab txhawb ZIKV kab mob.

Daim duab 2. Overexpression ntawm DHCR7 txhawb ZIKV replication. A-D U251 thiab A549 hlwb tau kis nrog HA-vector lossis HA-DHCR7 ntawm qhov qhia tau tias muaj ntau dua 12 teev thiab tom qab ntawd kis nrog ZIKV rau 36 teev ntxiv. Cov qib ZIKV RNA tau kuaj pom los ntawm RT-PCR nrog GAPDH raws li kev tswj hwm sab hauv (A, B), thiab qib protein ntawm ZIKV lub hnab ntawv, HA-DHCR7, thiab GAPDH tau kuaj pom los ntawm Western blot (C, D). E, F Vero hlwb tau kis tus kab mob ZIKV-muaj supernatant los ntawm daim duab 2C rau 48 teev los txiav txim ZIKV cov ntawv luam los ntawm cov tshuaj plaque. G U251 hlwb tau kis nrog HA-vector, HA-DHCR7, los yog HA-DHCR7 (G410S) rau 12 h thiab kis ZIKV rau 36 h, thiab cov protein ntau ntawm ZIKV hnab ntawv, HA-DHCR7 thiab GAPDH tau kuaj pom los ntawm Western blot. H U251 hlwb tau kis nrog HA-vector los yog HA-DHCR7 (1 ug) rau 12 h kis ZIKV rau 36 h, thiab tom qab ntawd incubated nrog nas anti-flavivirus thiab luav anti-HA antibodies, tom qab stained nrog FITC-conjugated antimouse thiab Cy3-conjugated anti-luv cov tshuaj tiv thaiv kab mob thib ob. Nuclei tau stained nrog DAPI. Cov duab tau pom los ntawm confocal microscopy. Scale bar ¼ 100 μm. Cov ntaub ntawv yog los ntawm tsawg kawg peb qhov kev sim ywj pheej (txhais tau tias SD) lossis cov ntaub ntawv sawv cev (C, D, E, G, H). Qhov tseem ceeb ntawm kev txheeb cais tau suav nrog Cov Tub Kawm Ntawv ob-tailed unpaired t-test (F) lossis ib-txoj kev ANOVA nrog Dunnett ntau qhov kev sib piv (A, B). *P <0.05, ***P <0.001. SD, tus qauv sib txawv.

4. Kev sib tham

Hom I IFNs thiab lawv cov ISGs downstream ntsib cov kab mob loj thiab ua lub luag haujlwm tseem ceeb hauv kev tiv thaiv tus tswv tsev tiv thaiv ZIKV (Lazear li al., 2016). Txhawm rau tshem tawm IFN-kev soj ntsuam kev sib kho thiab ua tiav kev rov ua kom txaus, ZIKV hloov kho NS cov proteins tsom mus rau ntau lub ntsiab lus ntawm IFN qhia txoj hauv kev kom khiav tawm ntawm lub cev tiv thaiv kab mob hauv lub cev (Lee li al., 2021). Cov kev tshawb fawb Mounting tau piav qhia tias ZIKV NS1, NS3, NS4A, thiab NS5 inhibit cov qauv sib txawv ntawm RIG-I / IFN-I txoj hauv kev thiab pab txhawb kev kis kab mob siv cov kev ywj pheej thiab ntau haiv neeg (Grant et al., 2016; Kumar et al., 2016 ; Ma et al., 2018; Riedl et al., 2019; Xia et al., 2018; Zheng et al., 2018). Txawm li cas los xij, cov txheej txheem tshwj xeeb ntawm ZIKV NS4B tiv thaiv kev tiv thaiv kab mob tsis zoo yog txhais tau hais tias tsis zoo. ZIKV NS4B yog ib qho protein ntau hydrophobic uas muaj 251 amino acids thiab nyob rau hauv endoplasmic reticulum membrane nrog N-terminal (Zou et al., 2014). NS4B inhibition ntawm IFN-Kuv txoj kev taw qhia tau raug txheeb xyuas hauv cov kab mob sib txawv (Ding li al., 2013; Munoz-Jordan li al., 2003, 2005; Shan et al., 2021; Yi et al., 2016; Zhang et al., 2016; ib., 2021). Ib txoj kev tshawb fawb tsis ntev los no tau tshaj tawm tias ZIKV INMI1 strain cais nyob rau hauv Brazil siv NS4B los tiv thaiv kev poob qis ntawm IFN-I taw qhia txoj hauv kev los ntawm crushing STAT1 phosphorylation thiab thiaj li cuam tshuam STAT1 nuclear thauj (Fanunza li al., 2021). Nyob rau hauv txoj kev tshawb no, peb pom ib tug txawv mechanism nyob rau hauv uas ZIKV NS4B tshem tawm IFN- ntau lawm los ntawm suppressing TBK1 thiab IRF3 phosphorylation, lub upstream tivthaiv ntawm IFN-I signaling pathway. Qhov tseem ceeb, amino acid hloov pauv (G18R) tau pom nyob rau hauv ZIKV NS4B, uas ua rau txo qis IFN- ntau lawm thiab tsis muaj zog ISG qhia, thiab yog li ua kom yooj yim kis kab mob hauv nas, qhia qhov tseem ceeb ntawm NS4B raws li kev txiav txim siab ntawm pathogenesis (Gorman li al. ., 2018). Cov pov thawj tshwm sim tau qhia txog qhov tseem ceeb ntawm cov roj cholesterol metabolism uas koom nrog hauv lub cev tiv thaiv kab mob (Akula li al., 2016; Dang li al., 2017; Reboldi li al., 2014; York et al., 2015). Txawm hais tias ntau qhov nruab nrab hauv cov roj cholesterol metabolism tau raug sau tseg kom muaj cov tshuaj tiv thaiv kab mob dav dav (Blanc li al., 2013; Li et al., 2017; Liu et al., 2013; Xiao et al., 2020), peb txoj kev tshawb fawb qhia txog cov txheej txheem tshiab. tias cov roj cholesterol metabolic enzyme DHCR7 tau koom nrog ZIKV kab mob los ntawm attenuating TBK1 thiab IRF3 activation los pab txhawb kev kis kab mob. Raws li ib qho tseem ceeb enzyme koom nrog desmosterol thiab cov roj cholesterol metabolism, DHCR7 sawv cev rau kev hloov pauv ntawm 7-DHC rau vitamin D nyob rau hauv cov tawv nqaij hlwb raug UVB (Prabhu li al., 2016b). Ntxiv mus, DHCR7 tsis yog nyob rau ntawm lub qhov rooj ntawm txoj kev Bloch nkaus xwb tab sis kuj yog qhov kawg ntawm txoj kev Kandutsch-Russell (Prabhu li al., 2016a, 2016b), uas ua lub luag haujlwm tseem ceeb hauv kev tswj cov roj cholesterol. Kev sib tham ntawm DHCR7 thiab nws cov kev cuam tshuam nrog lwm cov enzymes hauv ntau txoj hauv kev tuaj yeem txhawb nqa ntau lub zog hauv ntau hom cell los yog hauv nruab nrog cev. Cov kev tshawb fawb yav dhau los tau qhia tias thaiv DHCR7 tuaj yeem txo qis desmosterol, qhov ua ntej ntawm cov roj cholesterol biosynthesis, yog li muab kev tiv thaiv txaus tiv thaiv kab mob HCV (Luu li al., 2015; Rodgers li al., 2012). Qhov tseem ceeb, Xiao et al. tau pom tias tus kab mob VSV txo qis DHCR7 kev qhia, ua rau txo cov roj cholesterol, tab sis ntau dua 7-DHC ntau ntxiv hauv macrophages thiab daim siab (Xiao et al., 2020). Macrophages kho nrog 7-DHC ua kom cov tshuaj tiv thaiv kab mob zoo dua; thaum qhov sib ntxiv ntawm cov cholesterol pom kev tiv thaiv me. DHCR7 inhibitors thiab kev tswj hwm ntawm 7-DHC tej zaum yuav muaj txiaj ntsig zoo rau kev tiv thaiv kab mob xws li HIN1, HSV, thiab VSV. Hauv txoj kev tshawb no, peb pom tias DHCR7 inhibitor AY9944 tuaj yeem tiv thaiv tus kab mob ZIKV. Txawm li cas los xij, peb txoj kev tshawb fawb tau qhia tias tus kab mob ZIKV ua rau DHCR7 qhia, tab sis muaj kev cuam tshuam me ntsis ntawm cov roj cholesterol biosynthesis hauv glial hlwb. Peb tsis tau tshuaj xyuas qib ntawm 7-DHC qhia txog kev kis mob ZIKV, txawm li cas los xij, nws tsim nyog tshawb xyuas ntxiv seb 7-DHC thiab AY9944 puas muaj kev sib koom ua ke los kho cov neeg mob uas tau kis tus kab mob ZIKV. kab mob hauv lub hlwb. DHCR7 overexpression txhawb nqa tus kab mob ZIKV, thaum DHCR7 xaiv inhibitor (AY9944) kev kho mob tau nce IFN- ntau lawm tiv thaiv kab mob ZIKV. Kev ua kom TBK1 thiab IRF3 yog qhov tseem ceeb rau priming IFN-I signaling. Kev tshawb nrhiav yav dhau los tau pom tias DHCR7 ntsiag to lossis 7-DHC kev kho mob tau qhib txoj hauv kev PI3K-AKT3 rau phosphorylate IRF3, tab sis tsis yog TBK1 hauv macrophages (Xiao li al., 2020). Txawm li cas los xij, peb qhov kev tshawb pom tau pom tias DHCR7 koob tshuaj-dependently txo qis IRF3 thiab TBK1 qhia, nrog rau IRF3 thiab TBK1 phosphorylation. Cov kev tshawb fawb no tau qhia tias DHCR7 tuaj yeem pab txhawb rau ZIKV kab mob hauv cov roj cholesterol metabolism hauv lub hlwb. Nws tau raug qhia zoo tias ZIKV siv NS cov proteins ncaj qha cuam tshuam nrog cov khoom sib txawv ntawm RIG-I / IFN-I txoj hauv kev kom tsis txhob muaj lub cev tiv thaiv kab mob thiab pab txhawb cov kab mob sib kis. Peb yav dhau los tau tshaj tawm txog kev sib cuam tshuam ntawm ZIKV NS5 nrog RIG-I kom txo qis IFN- ntau lawm (Li et al., 2020). Lwm tus tau pom tias ZIKV NS1 thiab NS2B tsom rau TBK1, NS3 tsom rau RIG-I thiab MDA5 kom txo qis IFN-I thiab ISGs ntau lawm (Lee li al., 2021; Riedl li al., 2019). Txawm li cas los xij, kev ua haujlwm ntawm ZIKV NS proteins tsom rau cov roj cholesterol metabolism tseem tsis tshua pom. Hauv txoj kev tshawb no, peb kuj tau piav qhia txog kev sib cuam tshuam ntawm cov roj cholesterol metabolic enzyme DHCR7 thiab ZIKV NS proteins. Peb pom DHCR7 tshwj xeeb khi rau NS4B, tsis yog rau lwm cov NS proteins. Peb tau muab cov ntaub ntawv pov thawj tseem ceeb uas ZIKV NS4B cuam tshuam nrog DHCR7, thiab tau txhawb nqa nws cov lus qhia, uas zoo ib yam nrog cov txiaj ntsig hauv U251 hlwb thaum kis ZIKV. Qhov tseem ceeb, sib nrug los ntawm kev qhia ncaj qha txog kev koom tes ntawm DHCR7 thiab ZIKV NS4B, peb kuj pom tias ZIKV NS4B nce DHCR7 qhia kom inhibit TBK1 thiab IRF3 phosphorylation thiab pab txhawb ZIKV kab mob. Raws li kev pom zoo nrog peb cov txiaj ntsig, kev tshawb fawb tsis ntev los no tau qhia tias qhov tsis txaus DHCR7 tuaj yeem ua rau IRF3 thiab IFN- signaling inhibit VSV thiab lwm yam kab mob hauv macrophages.

Daim duab 3. Knockdown ntawm DHCR7 txo cov kab mob ZIKV hauv U251 hlwb. U251 hlwb tau hloov pauv nrog cov kab mob lentiviral uas muaj shRNA tawm tsam DHCR7 (shDHCR7-1 thiab sh-DHCR7-2) lossis qhov tsis sib xws (sh-NTC), ua raws li kev xaiv puromycin rau 14 hnub. Qhov kev ua haujlwm poob qis tau txiav txim siab los ntawm RT-PCR. B, C DHCR7-silenceing U251 (sh-DHCR7) thiab sh-NTC U251 hlwb raug thuam los yog ZIKV kab mob (MOI ¼ 1) rau 36 teev. Qib intracellular ZIKV RNA tau kuaj pom los ntawm RT-PCR nrog GAPDH raws li kev tswj hwm sab hauv (B) thiab qib protein ntawm ZIKV lub hnab ntawv, DHCR7, thiab GAPDH tau kuaj pom los ntawm Western blot (C). D, E U251 hlwb raug kho nrog DHCR7 inhibitor AY9944 ntawm qhov qhia tau hais tias concentration rau 2 h, ces kis mob nrog ZIKV (MOI ¼ 1) rau 48 h. Cov hlwb raug xa mus rau RT-PCR txhawm rau kuaj ZIKV RNA (D). Cov qib protein ntawm lub hnab ntawv ZIKV, DHCR7, thiab GAPDH tau kuaj pom los ntawm Western blot (E). F U251 hlwb raug kho nrog DHCR7 inhibitor AY9944 ntawm qhov qhia tau hais tias qhov concentration rau 36 h, thiab DHCR7 thiab GAPDH tau kuaj pom los ntawm Western blot. G, H Vero hlwb tau kis tus kab mob ZIKV-muaj supernatant los ntawm daim duab 3E rau 48 h los txiav txim ZIKV cov ntawv luam los ntawm cov tshuaj plaque. I U251 hlwb raug kho nrog AY9944 ntawm qhov qhia tau hais tias concentration rau 2 h, ces kis mob ZIKV ntawm MOI ¼ 1 rau 36 h. Cov duab tau pom los ntawm confocal microscopy. Scale bar ¼ 100 μm. Cov ntaub ntawv yog los ntawm tsawg kawg peb qhov kev sim ywj pheej (txhais tau tias SD) lossis cov ntaub ntawv sawv cev (C, E, F, G, thiab I). Kev txheeb xyuas qhov tseem ceeb yog suav nrog ib txoj kev ANOVA nrog Dunnett ntau qhov kev sib piv (A, D, H) lossis ob txoj kev ANOVA nrog Sidak ntau qhov kev sib piv (B). ***P <0.001. SD, tus qauv sib txawv.

Fig. 4. DHCR7 inhibits IFN- thiab ISG ntau lawm. A, B U251, thiab Vero hlwb tau kis tus kab mob ZIKV (MOI ¼ 0, 0.5, thiab 1) rau 24 teev thiab cov roj (cholesterol) hauv cellular tau ntsuas los ntawm cov ntaub so ntswg tag nrho cov roj cholesterol. C–H DHCR7-silencing U251 (sh-DHCR7) thiab sh-NTC U251 hlwb tau kis ZIKV (MOI ¼ 1) lossis kho nrog Poly (I: C) (1 ug/mL) rau 6 h. Tag nrho cov cellular RNA raug rho tawm thiab raug rau RT-PCR rau mRNA qhia nrog GAPDH raws li kev tswj hwm sab hauv. Cov kev txheeb ze ntawm IFN- thiab ISGs tau zoo li qub rau cov qauv sh-NTC hauv pawg neeg thuam, thiab cov kev hloov pauv tau pom tias tsis zoo. IFN- (C, D), ISG15 (E, F) thiab ISG56 (G, H). I, J DHCR7-silence U251 (sh-DHCR7) thiab sh-NTC U251 hlwb tau kis tus kab mob ZIKV (MOI ¼ 1) lossis kis nrog Poly(I: C) (1 ug/mL) rau 24 teev, thiab cov protein ntau ntawm ISG15, ISG56 thiab GAPDH tau kuaj pom los ntawm Western blot. K U251 hlwb raug kho nrog cov tshuaj qhia ntawm AY9944 rau 2 h, ces kis ZIKV (MOI ¼ 1) rau 6 h. IFN-RNA qib tau kuaj pom los ntawm RT-PCR nrog GAPDH raws li kev tswj hwm sab hauv. L U251 hlwb raug kho nrog koob tshuaj AY9944 rau 2 teev thiab tom qab ntawd kis tus kab mob ZIKV (MOI ¼ 1) rau 24 teev. Cov qib protein ntawm ISG15, ISG56, thiab GAPDH tau kuaj pom los ntawm Western blot. M U251 hlwb tau hloov nrog HA-vector, HA-DHCR7, lossis HA-DHCR7 (G410S) rau 24 teev thiab kis nrog ZIKV (MOI ¼ 1) rau 6 teev. IFN-RNA qib tau kuaj pom los ntawm RT-PCR nrog GAPDH raws li kev tswj hwm sab hauv. N U251 hlwb tau kis nrog HA-vector, HA-DHCR7, lossis HA-DHCR7 (G410S) rau 24 teev, thiab tom qab ntawd kis nrog ZIKV (MOI ¼ 1) rau 24 teev. Cov qib protein ntawm ISG15, ISG56, thiab GAPDH tau kuaj pom los ntawm Western blot. Cov ntaub ntawv yog los ntawm tsawg kawg peb qhov kev sim ywj pheej (txhais tau tias SD) lossis cov ntaub ntawv sawv cev (I, J, L, N). Kev txheeb xyuas qhov tseem ceeb yog suav nrog ib-txoj kev ANOVA nrog Dunnett ntau qhov kev sib piv (A, B, K, M) lossis ob txoj kev ANOVA nrog Sidak ntau qhov kev sib piv (C–H). ns, P > 0.05, ***P < 0.001. SD, tus qauv sib txawv.

Fig. 5. DHCR7 inhibits TBK1 thiab IRF3 activation. A, B HEK293T hlwb tau koom ua ke nrog TBK1-coding plasmid ua ke nrog DHCR7- qhia plasmid lossis khoob vector (A). HEK293T hlwb tau koom ua ke nrog IRF3-encoding plasmid, RIG-I encoding plasmid, ua ke nrog DHCR7- qhia plasmid lossis khoob vector (B). Cells raug sau tseg tom qab 24 teev tom qab hloov pauv thiab raug rau Western blot nrog cov tshuaj tiv thaiv kab mob [anti-TBK1, anti-P-TBK1(S172), anti-IRF3, anti-PIRF3(S396), anti-HA, thiab anti-GAPDH ]. C U251 hlwb tau hloov pauv nrog cov koob tshuaj ntawm DHCR7 qhia plasmid lossis khoob vector rau 24 teev. Cells tau kis tus kab mob ZIKV (MOI ¼ 1) tua ntawm 6 h tom qab kis kab mob thiab raug rau Western blot nrog cov tshuaj tiv thaiv kab mob [anti-TBK1, anti-P-TBK1(S172), anti-IRF3, anti-P-IRF3( S396), anti-HA, thiab anti-GAPDH]. D U251 hlwb tau hloov nrog HA-vector, HA-DHCR7 lossis HA-DHCR7 (G410S) rau 24 teev. Cells tau kis tus kab mob ZIKV (MOI ¼ 1) tua ntawm 6 h tom qab kis kab mob thiab raug rau Western blot nrog cov tshuaj tiv thaiv kab mob [anti-TBK1, anti-P-TBK1(S172), anti-IRF3, anti-PIRF3(S396) , anti-HA, and anti-GAPDH]. E U251 hlwb raug kho nrog cov tshuaj qhia ntawm AY9944 rau 2 teev thiab kis mob ZIKV (MOI ¼ 1) rau 6 h, tom qab ntawd raug rau Western blot nrog cov tshuaj tiv thaiv [anti-TBK1, anti-P-TBK1(S172), tiv thaiv -IRF3, anti-P-IRF3 (S396), thiab anti-GAPH]. Cov ntaub ntawv yog los ntawm tsawg kawg yog peb qhov kev sim ywj pheej thiab pom tias yog cov ntaub ntawv sawv cev (A–E).

Fig. 6. ZIKV NS4B cuam tshuam nrog DHCR7 los inhibit TBK1 thiab IRF3 phosphorylation. HEK293T hlwb tau sib koom ua ke nrog plasmids encoding HA-DHCR7, thiab plasmids encoding ZIKV non-structural proteins (FLAG-NS1, FLAG-NS3, FLAG-NS4A, FLAG-NS4B, thiab FLAG-NS5) lossis plasmid 3 tswj plasmid. 0 h. Cov cell lysates tau immunoprecipitated nrog anti-Flag antibodies thiab tom qab ntawd soj ntsuam los ntawm immunoblotting nrog cov tshuaj tiv thaiv. B, C HEK293T hlwb tau koom ua ke nrog plasmids encoding HA-DHCR7, plasmids encoding FLAG-NS4B, lossis npliag tswj plasmid rau 30 h. Cov cell lysates tau immunoprecipitated nrog anti-HA antibody (B) los yog anti-FLAG antibody (C) thiab tom qab ntawd soj ntsuam los ntawm immunoblotting nrog cov tshuaj tiv thaiv. D U251 hlwb tau hloov nrog FLAG-NS4B plasmid ua ke nrog plasmids encoding nrog HA-DHCR7. Lub localization ntawm FLAG-NS4B (ntsuab), HA-DHCR7 (liab), nucleus marker DAPI (xiav), thiab kev sib koom ua ke tau soj ntsuam nrog confocal microscopy. Scale bar ¼ 10 μm. E Cell lysates los ntawm HEK293T hlwb hloov nrog HA-DHCR7 tau incubated nrog GST protein los yog GST-NS4B protein uas tau incubated nrog glutathione-Sepharose hlaws. Cov tshuaj sib tov tau kuaj pom los ntawm Western blot nrog anti-HA thiab anti-GST cov tshuaj tiv thaiv (sab saum toj). Lysates los ntawm transfected HEK293T hlwb thiab cov purified proteins tau kuaj pom los ntawm Western blot nrog anti-HA thiab anti-GST cov tshuaj tiv thaiv (hauv qab). F U251 hlwb tau hloov pauv nrog plasmid encoding FLAG-NS4B (0, 1, 2 ug). Cov qib protein txheeb ze ntawm DHCR7, FLAG-NS4B, thiab GAPDH tau kuaj pom los ntawm Western blot. G, HEK293T hlwb tau koom ua ke nrog NS4B-expressing plasmid los yog khoob vector, ua ke nrog TBK1-coding plasmid (F), IRF3-expressing plasmid, thiab plasmids encoding RIG-I (G) . Cells raug sau tseg ntawm 24 teev tom qab hloov pauv thiab raug rau Western blot nrog cov tshuaj tiv thaiv kab mob [anti-TBK1, anti-P-TBK1(S172), anti-IRF3, anti-P-IRF3 (S396), anti-FLAG, thiab antiGAPH ]. I DHCR7-silencing U251 (sh-DHCR7) thiab sh-NTC U251 hlwb tau kis nrog NS4B-expressing plasmid los yog khoob vector. Cells tau sau tseg ntawm 6 h tom qab hloov pauv thiab raug rau Western blot nrog cov tshuaj tiv thaiv kab mob [anti-TBK1, anti-P-TBK1(S172), anti-IRF3, anti-P-IRF3 (S396), anti-FLAG, thiab antiGAPH ]. Cov ntaub ntawv yog tus sawv cev ntawm peb qhov kev sim ywj pheej.

5. Cov lus xaus

Hauv cov ntsiab lus, kev tshawb fawb yav dhau los tau qhia tias cov roj cholesterol metabolism enzyme DHCR7 koom nrog hauv lub cev tiv thaiv kab mob tiv thaiv ntau yam kab mob. Peb txoj kev tshawb fawb tau nthuav tawm tias ZIKV NS4B cuam tshuam nrog DHCR7 thiab ntxias DHCR7 qhia. Qhov nce DHCR7 zoo heev inhibits TBK1 thiab IRF3 ua kom thiab ua rau txo qis IFN- thiab ISG ntau lawm, uas yog li pab txhawb ZIKV kab mob hauv glial hlwb. Cov kev tshawb pom no tau nthuav tawm qhov tshiab kab mob tiv thaiv kab mob tiv thaiv kab mob uas ZIKV kov yeej lub cev tiv thaiv kab mob los ntawm kev tsom ncaj qha DHCR7 nrog NS4B.

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