INTERACTION OF TYPE I INTERFERONS AND MTOR SIGNALING UNDERLYING PRRSV INFECTION QINFANG LIU. B.S., Heilongjiang Bayi Agricultural University, 2012

Transcription

1 INTERACTION OF TYPE I INTERFERONS AND MTOR SIGNALING UNDERLYING PRRSV INFECTION by QINFANG LIU B.S., Heilongjing Byi Agriculturl University, 2012 A THESIS submitted in prtil fulfillment of the requirements for the degree MASTER OF SCIENCE Deprtment of Antomy nd Physiology College of Veterinry Medicine KANSAS STATE UNIVERSITY Mnhttn, Knss 2016 Approved by: Mjor Professor Yongming Sng

2 Copyright QINFANG LIU 2016

3 Abstrct Animl metbolic nd immune systems integrte nd inter-regulte to exert effective immune responses to distinct pthogens. The signling pthwy medited by mechnistic trget of rpmycin (mtor) is criticl in cellulr metbolism nd implicted in host ntivirl responses. Recent studies highlight the significnce of the mtor signling pthwy in the interferon (IFN) response. Type I IFNs medite host defense, prticulrly, ginst virl infections, nd hve myrid roles in ntivirl innte nd dptive immunity. In ddition to their well-known ntivirl properties, type I IFNs lso ffect host metbolism. However, little is known bout how niml type I IFN signling coordintes immunometbolic rections during ntivirl defense. Therefore, understnding the interction of mtor signling nd the type I IFN system becomes incresingly importnt in potentiting ntivirl immunity. Tissue mcrophges (MФs) re primry IFN producer during virl infection, nd their polriztion to different ctivtion sttuses is criticl for regultion of immune nd metbolic homeostsis. Using porcine reproductive nd respirtory syndrome virus (PRRSV) s model, we found tht genes in the mtor signling pthwy were regulted differently in PRRSVinfected porcine lveolr MФs t different ctivtion sttuses. Therefore we hypothesize tht: 1) the mtor signling pthwy involves host nti-prrsv regultion; 2) mtor signling intercts with IFN signling to modulte the ntivirl response; nd 3) different type I IFN subtypes (such s IFN-α1 nd IFN-β) regulte mtor signling differently. We show tht modultion of mtor signling regulted PRRSV infection in MARC-145 cells nd porcine primry cells, in prt, through regulting production nd signling of type I IFNs. In ddition, expression nd phosphoryltion of two key components in the mtor signling pthwy, AKT nd p70 S6 kinse, were regulted by type I IFNs nd PRRSV infection.

4 Tken together, we determined tht the mtor signling pthwy, key pthwy in regultion of cell metbolism, lso medites the type I IFN response, key immune response in PRRSV infection. Our findings revel tht the mtor signling pthwy potentilly hs bidirectionl loop with the type I IFN system nd implies tht some components in the mtor signling pthwy cn serve s trgets for ugmenttion of ntivirl immunity nd therpeutic designs.

5 Tble of Contents List of Figures... vi List of Tbles... vii Acknowledgements... viii Chpter 1 - Generl introduction Type I IFNs mtor signling pthwy Porcine reproductive nd respirtory syndrome virus Potentil interction of mtor nd type I IFN signling in ntivirl regultion... 7 Chpter 2 - Mterils nd Methods Cells nd viruses Cell polriztion, virl infection nd trnscriptomic shotgun sequencing Antivirl nlysis Biossys nd ELISA Rel-Time PCR nd western blotting ssy Gene Silencing bsed on CRISPR/Cs9 system Tbles Chpter 3 - Results Genes in mtor signling pthwy were differentilly expressed in PRRSV-infected MФs t different ctivtion sttuses Regultion of PRRSV infection by the mtor signling pthwy mtor signling modultes the type I IFN response Gene silencing mtor signling regultes PRRSV infection Type I IFNs modulte mtor signling Chpter 4 - Conclusions nd discussion References v

6 List of Figures Figure 1.1 The mtor signling pthwy intercts with the type I IFN system to regulte the ntivirl response Figure 3.1 RNA-Seq nlysis of DEGs in PRRSV-infected porcine lveolr MФs t different ctivtion sttuses Figure 3.2 Regultion of PRRSV infection by mtor inhibitors nd ctivtor in MARC-145 cells Figure 3.3 mtor inhibitors nd ctivtor regulte PRRSV infection in dose- nd timedependent mnner in MARC-145 cells Figure 3.4 Repression of PRRSV infection by mtor inhibitors in porcine lveolr MФs nd mdcs Figure 3.5 mtor inhibitors or n mtor ctivtor modulte expression of type I IFNs nd their receptors in PRRSV-infected porcine lveolr MФs Figure 3.6 Disruption of mtor signling pthwy promotes type I IFN production nd signling Figure 3.7 Blocking mtor signling by down-regulting Rictor or Rptor expression suppresses PRRSV repliction in MARC-145 cells Figure 3.8 Phosphoryltion nd expression of AKT nd p70 S6 kinse, two key components in mtor signling, re differentilly regulted by IFN-α1, IFN-β nd PRRSV infection vi

7 List of Tbles Tble 2.1 RT-PCR primer sequences for porcine type I IFNs nd their receptors [102] Tble 2.2 sgrna primer sequences vii

8 Acknowledgements I would like to sincerely thnk my mjor dvisor Dr. Yongming Sng, for his guidnce nd dediction to my grdute eduction nd reserch progress. Dr. Sng hs lwys given insightful dvice for my projects nd techniclly guided me during whole process. He hs helped me to enhnce my reserch skills, to develop scientific thinking, nd to gretly improve my English speking nd writing. I pprecite the finncil support from his grnts tht offer me n opportunity to study brod nd will be invluble for my future. I would lso like to thnk my dvisor, Dr. Frnk Blech for the gret chnce to work in his lb. He provided me insightful dvice in my reserch nd thesis writing. Thnks to his mentoring nd support. I would lso like to thnk my committee member, Dr. Wenjun M, for his vluble suggestions on my reserch nd thesis writing. My pprecition to our lb mnger, Dr. Brbr Lutjemeier, for her invluble dvice nd ssistnce. I lso wnt to thnk our undergrdute reserch ssistnts, Wytt Brichlli, Joseph Bergkmp nd Jessic Person for their excellent technicl ssistnce. And thnks to Dr. Xiorong Zhng, visiting scholr in our lb, for his suggestions nd help with my reserch. It hs been gret plesure to work with ll our lb members nd I wnt to give my grcious thnks to ll of them for their help in my M.S. study. Finlly, I wnt to express my sincerest thnks to my fmily. Their continuous, unconditionl support nd love encourge me to overcome difficulties nd pursue my drems. viii

9 Chpter 1 - Generl introduction In nimls, the innte immune system cts s the first line in restriction of virl invsion or repliction, nd lunches erly ntivirl response [1]. At the beginning of cell sensing virl infection, cell-ssocited receptors recognize virl components such s single-strnded RNA (ssrna), double-strnded RNA (dsrna), nd virl genomic DNA nd proteins to mount immune responses [2-5]. Through cscde of signling trnsduction, virl infected cells or ctivted cells re elicited to increse production of immune effectors, primrily including interferons (IFNs), other proinflmmtory cytokines/chemokines, nd ntimicrobil peptides [2, 3, 6-8]. For exmple, IFNs, in prticulr, type I IFNs s key ntivirl cytokines, re induced vi interferon regultory fctors (minly IRF3 nd IRF7), nd NF-κB is responsible in modulting most proinflmmtory cytokines nd type I IFNs [6, 7, 9, 10]. Following enggement of virl infection nd induction of these potentil immune effectors, multiple cellulr systems including both immune nd metbolic systems re dpted to effectively control pthogenic invsion [1, 11-14]. In this context, IFN signling is the key in ntivirl immune regultion, nd it hs lso been discovered to hve multiple roles in mediting cellulr growth nd prolifertion rtes [10, 15-20]. Similrly, some key pthwys in regultion of cell growth nd metbolism, such s tht medited by mechnistic trget of rpmycin (mtor), hve been found to be importnt in regultion of immune responses [6, 12, 13, 21]. However, currently there is lck of pltforms to dissect this inter-systemic interction during virl infection. Here, type I IFN- nd mtormedited pthwys were used to decipher the interction between cell immune nd metbolic systems during virl infection. 1

10 1.1 Type I IFNs Over the pst 50 yers IFNs hve been discovered nd used cliniclly in humn nd nimls [15, 16]. IFNs comprise n essentil fmily of cytokines in niml immune systems nd hve pivotl influence on biologicl ctions, especilly those relevnt to ntivirl defense nd crcinogenesis [17, 20]. The interferon fmily is divided into three clsses, type I, II, nd III IFNs [16]. They re distinguished by their distinct receptor complexes, disply different expression pttern nd hve numerous functions in innte nd dptive immune responses [22]. Type I IFNs re trnsduced by their heterodimeric receptor complex (IFNAR1/2), nd form the lrgest subgroup, comprised of more thn 10 subtypes such s IFN-α, IFN-β, IFN-ε, IFN-κ nd IFN-ω generlly displyed in most mmmlin species. Some species-specific subtypes include IFN-δ (pigs nd horses), IFN-τ (cttle), IFN-ζ (mice) nd IFN-αω (pigs, horses nd cttle) [16, 23]. In ddition, IFN-α, IFN-δ nd IFN-ω re subtypes contining multiple genes. The humn genome contins 13 IFN-α functionl genes, nd the swine genome contins 25, 11 nd 8 functionl genes of IFN-α, IFN-δ, nd IFN-ω, respectively [23]. IFN-γ is the only type II IFN, functioning s homodimer nd signling vi its receptor complex (IFNGR1/2) [24, 25]. Type III IFNs, re composed of IFN-λ1, IFN-λ2, IFN-λ3 (lso known s IL-29, IL-28A nd IL-28B, respectively) nd IFN-λ4, which interct with distinct heterodimeric receptors of IFRL1/IL-10R2 to trnsmit signls [26]. Type I IFNs re primrily thought to be ntivirl meditors, re rpidly induced upon virl infection, nd ply n indispensble role in producing rpid innte immune responses nd effective dptive immune responses [10]. Sensing pthogen ssocited moleculr ptterns (PAMPs) of virl components by cellulr pttern recognition receptors (PRRs) is the first step to initite nd regulte type I IFN response 2

11 [19]. The selective fetures of PRRs for virl molecules nd different cell types potentilly induce differentil production of specific IFN subtypes. Cytoplsmic PRRs, such s RIG-I-like receptors (RLRs), re specilized to recognize nucleic cids from RNA viruses. After binding to virl RNA, RLRs undergo conformtionl chnge nd expose their N-terminl cspse ctivtion nd recruitment domins (CARDs) to interct with the dptor molecule, mitochondril ntivirl signling protein (MAVS) for further generting ntivirl signling [10]. Membrne-bound Toll-like receptors (TLRs) lso re criticl to detect virl glycoproteins nd nucleic cids nd re recruited to their respective dptors like myeloid differentition primry response gene 88 (MyD88) for TLR7 nd TLR9, nd tumor necrosis fctor receptor ssocited fctor (TRIF) for TLR3 when virl nucleic cid species re recognized [10, 26]. In ddition to PRRs, cytosolic PAMP sensor, nmed cyclic GMP-AMP (cgamp) synthse (cgas), is initited by sensing foreign DNA nd ctivtes the stimultor of IFN genes (STING) [27]. Upon ctivtion of MAVS, MyD88, TRIF nd STING, TRAF fmily is recruited to trigger mitogen ctivted protein (MAP) kinse, IKK complex nd IKK-relted kinses TBK1/IKKε, resulting in ctivtion nd nucler trnsloction of AP-1, IRF3, IRF7 nd NF-κB to specificlly bind to vrious promoters of type I IFN genes, incresing their production [18]. Once type I IFNs re induced by virl infection, they re secreted by infected cells nd stimulte trget cells to strt type I IFN ction signling [9, 21]. Upon type I IFNs binding to their specific receptors in the surfce of trget cells, lignd-dependent rerrngement, dimeriztion nd trnsphosphoryltion of receptors propgte downstrem signling cscdes [28]. Receptor ggregtion llows utophosphoryltion nd ctivtion of ssocited JAKs/TYKs, IFNAR1 intercting with tyrosine kinse 2 (TYK2), IFNAR2 with Jnus ctivted kinse 1 (JAK1) [29]. Consequently phosphoryltion nd ctivtion of STATs (signl trnsducers nd 3

12 ctivtors of trnscription) re regulted to constitute functionl homodimers or heterodimers which stimulte trnscription of interferon-stimulted genes (ISGs) fter nucler trnsloction to bind their specific promoter sites [30]. Beyond clssic JAKs/STATs signling pthwys, other importnt signling cscdes re lso induced by type I IFNs for optimizing trnscription of diverse ISGs, including MAPKKK (mitogen-ctivted protein kinse kinse kinse) pthwy, PI3K (phosphoinositide 3-kinse)-AKT pthwy, nd mtor (mechnistic trget of rpmycin) signling pthwy [14, 24, 31]. Signling cscdes downstrem re generted through ctions of myrid ISGs including ntivirl effectors (e.g., ISG15 nd Mx1), negtive regultors (e.g., USP18 nd SOC) nd positive regultors (e.g. STAT1/2 nd IRF1, 3, 7, 9) [18, 21]. Following ctivtion of these signling pthwys, wide rnge of protective nd destructive biologicl ctivities re evoked [32, 33]. Therefore, blnced type I IFN response is criticl to induce host immune responses for defense nd survivl (Figure 1.1). 1.2 mtor signling pthwy mtor, n evolutionrily conserved serine/threonine kinse, cts s n overriding node for mintennce of homeostsis in niml cells [34-36]. mtor kinse forms two functionlly distinct multi-protein complexes, mtor complex 1 (mtorc1) nd mtor complex 2 (mtorc1), to exert multiple functions through regultion of vrious elements downstrem [36]. mtorc1 consists of five components: mtor, the ctlytic subunits of the complex; regultory-ssocited protein of mtor (Rptor); mmmlin lethl with Sec13 protein (mlst8); proline-riche AKT substrte 40 kd (PRAS40); nd DEP-domin-contining mtorintercting protein (Deptor) [36]. Except mtor, mlst8 nd Deptor, mtorc2 includes rpmycin-insensitive compnion of mtor (Rictor), mmmlin stress-ctivted protein kinse intercting protein (msin1), nd protein observed with Rictor-1 (Protor-1) [36]. Genetic deletion 4

13 of Rptor nd Rictor, fundmentl subunits for mtorc1 nd mtorc2 respectively, hs provided significnt insight into functions of mtorc1 nd mtorc2 [37]. Over mny yers, diverse mtor inhibitors hve been developed, such s rpmycin, PP242, Everolimus, ARQ 092 nd CC214-2, nd some of them including rpmycin re pproved for clinicl therpy [38, 39]. The two mtor complexes disply different sensitivity to some of these inhibitors. Rpmycin nd its nlogs re the first genertion mtor inhibitors, which ssocite with 12 kd FK506-binding protein (FKBP12) to form complex intercting with FRB (FKBP12- rpmycin binding) domin in mtor kinse to disrupt formtion of mtorc1 [40]. Only mtorc1 ctivity is inhibited by rpmycin in short time period, but prolonged rpmycin tretment lso ffects mtorc2 ctivity [41]. PP242 is non-selective inhibitor, trgets the denosine triphosphte (ATP) binding site of mtor, nd suppresses both mtorc1 nd mtorc2 ctivities [42]. The mtor signling network comprised of mtorc1- nd mtorc2-signling pthwys, senses nd integrtes both intrcellulr nd extrcellulr signls to regulte vrious cellulr processes (e.g. metbolism, growth, prolifertion nd survivl) [36]. In recent studies, it hs been incresingly recognized tht the mtor signling pthwy is lso vitl regultor of innte nd dptive immune responses [12-14]. For exmple, Keting et l. demonstrted tht the mtor signling pthwy engges host ntibody response to produce cross protection ginst lethl influenz infection [43]; mtorc1- nd mtorc2-signling pthwys involves IFN production nd ction signling [25, 31, 44-52]; mtor-medited regultion of interleukin production nd signling hs been shown in humn monocytic cells [53-55]; polrized mcrophges (MФs) re modulted by disrupting mtor signling pthwy [56, 57]. Thus the mtor signling pthwy provides key link between immune responses nd metbolism to 5

14 modulte functions of immune cell popultions, such s monocytes, MФs, T cells, B cells, dendritic cells (DCs) nd neutrl killer (NK) cells [13]. Upon stimultion of externl signls such s cytokines nd growth fctors, the mtor signling pthwy is initited vi upstrem PI3K-AKT (Figure 1.1) [14, 34, 46, 51]. PI3K nd mtorc2 ctivte AKT tht medites inhibitory phosphoryltion of tuberous sclerosis protein 2 (TSC2), which is ssocited with TSC1 to repress the phosphotrnsferse ctivity of mtor through inhibiting RAS-relted smll GTPse RAS homologue enriched in brin (RHEB) [58, 59]. Previous studies found tht PI3K cn be ctivted by severl type I IFNs (IFN-α, IFN-β nd IFN-ω) vi inducing tyrosine phosphoryltion insulin receptor substrte 1 (IRS1) or IRS2 [24, 60, 61]. After IFNs evoking the PI3K signling pthwy downstrem of JAKs, STAT1 is phosphorylted t Ser727 to induce STAT1-medited gene trnscription vi ISREs or GAS (IFNγ ctivtion site) elements [24]. In ddition to JAKs/STATs signling pthwy, IFN signling lso is medited by the PI3K-AKT-mTOR-ULK1-p38 mitogen-ctivted protein kinse (MAPK) pthwy, reveling criticl role for the PI3K-AKT-mTOR signling pthwy in IFN-driven gene trnscription [31, 62, 63]. 1.3 Porcine reproductive nd respirtory syndrome virus Porcine reproductive nd respirtory syndrome virus (PRRSV), the custive gent of most economiclly importnt swine disese tht cuses losses of over $800 million nnully in United Sttes, is smll, enveloped, single-strnded, positive-sense RNA (+ssrna) virus [64, 65]. It belongs to genus Arterivirus, fmily Arteriviride, order Nidovirles, nd ws first identified in erly 1990s in Europe (Lelystd virus, LV) nd the United Sttes (VR2332) [66, 67]. The PRRSV genome hs pproximtely 15kb nd comprises 11 open reding frmes (ORFs) 6

15 encoding 16 nonstructurl proteins: NSP1α, NSPβ, NSP2-6, NSP2TF, NSP2N, NSP7, NSP7b nd NSP8-12 nd eight structurl proteins: glycoprotein (GP) 2-5, GP5, nucleocpsid (N), nonglycosylted membrne (M) nd envelope (E) proteins [68-70]. PRRSV is monocytotropic nd primrily trgets porcine lveolr MФs nd monocyte-derived DCs (mdcs) to cuse infection [71-73]. Except for MФs nd mdcs, cell line generted from kidney of Africn green monkey (MARC-145) is fully permissive to the virus nd routinely used for supporting PRRSV repliction in vitro [74]. PRRSV hs evolved diverse mechnisms to evde nd dysregulte effective host immune responses. Induction nd signling of type I IFNs, group of criticl ntivirl cytokines, re suppressed by PRRSV infection s repetedly observed in cells or pigs [69, 70, 75]. For exmple, PRRSV NSP1α, NSP1β, NSP2, NSP4, NSP11 nd N protein regulte type I IFN production by djusting phosphoryltion nd nucler trnsloction of IRFs or/nd ctivtion of NF-κB signling [68, 76-84]. Induction nd processing of key ntivirl ISGs, such s Mx1, ISG15 nd ISG56, lso re modulted by PRRSV infection [68]. Moreover, previous studies showed tht: ctivtion of PI3K-AKT signling pthwy is regulted by PRRSV infection in time-dependent mnner [85, 86]; PRRSV infection ctivtes mtorc1-signling nd its inhibitor, rpmycin, regultes PRRSV [87]; PI3K inhibition, AKT1 overexpression or mtorc1 inhibitor (rpmycin) tretment modultes virl gene trnscription nd protein synthesis [88]; PI3K-AKT signling pthwy is involved in regultion of PRRSV-medited poptosis nd virus entry [89, 90]. 1.4 Potentil interction of mtor nd type I IFN signling in ntivirl regultion Current studies revel tht the mtor signling pthwy displys incresing importnce in host innte nd dptive immunity [12-14]. However, functions of the mtor signling 7

16 pthwy in ntivirl immunity, in prticulr of regultion of IFN response, remin poorly defined. In this study, we focus on its function in type I IFN production nd signling in ntivirl response. It hs been shown tht both mtorc1- nd mtorc2-signling pthwys re criticl for type I IFN production nd signling [31, 49, 91]. Co et l. report tht PI3K-mTOR-p70 S6 kinse pthwy is required for TLR-induced type I IFN production in plsmcytoid DCs [50]. Type I IFNs coordinte mtor signling to selectively regulte trnscription nd trnsltion of hundreds of ISGs [25, 44, 46, 49]. A recent study further demonstrtes tht ULK1 cts s link between the type I IFN response nd the mtor signling pthwy nd tht the mtor-ulk1 pthwy plys n indispensble role for gene trnscription medited by ISREs nd GAS elements in type I IFN signling [62]. In ddition, the mtor signling pthwy lso cn be ctivted/regulted by type I IFN response [25, 49, 51]. The function of the mtor signling pthwy in nti-prrsv innte immune response remins unknown, prticulrly in nti-prrsv type I IFN response, lbeit few studies hve shown tht PI3K-AKT-mTOR signling pthwy involves regultion of PRRSV repliction [85-90]. Furthermore, mmmlin type I IFNs consists of multiple subtypes with different ntivirl ctivity [15, 16]. Antivirl ctivities of humn nd porcine type I IFNs hve been well nlyzed, nd porcine type I IFNs show different nti- PRRSV ctivity tht lso is cell type-dependent [23, 92]. However, it hs not been chrcterized how distinct type I IFNs regulte the mtor signling pthwy. Therefore, PRRSV ws chosen s model to decipher the potentil interction of mtor nd IFN signling in ntivirl responses. The ims of this study were to: 1) identify functions of mtor signling in nti- PRRSV regultion; 2) investigte regultion of nti-prrsv type I IFN response by the mtor 8

17 signling pthwy; nd 3) explore modultion of the mtor signling pthwy by diverse type I IFNs (Figure 1.1). 9

18 Growth fctors, Cytokines, etc. PRRSV etc. PI3K TLRs mtorc2 AKT RIG-I mtorc1 TSC1/2 Metbolism Antivirl defense? NF-kB IRFs STATs IFNs ILs AMPs IFNs JAKs/TYKs IFNs Figure 1.1 The mtor signling pthwy intercts with the type I IFN system to regulte the ntivirl response. Virl infection ctivtes IFN signling to produce type I IFNs nd other proteins, exerting ntivirl defense minly vi the JAKs/TYKs pthwy. The mtor signling network, consisting of mtorc1- nd mtorc2-signling pthwys, senses externl signls primrily to modulte metbolism, cell growth nd differentition through the PI3K-AKT signl trnsduction pthwy. AKT indirectly regultes mtorc1 ctivity vi ctions of TSC1/2. In our study, PRRSV ws used s model to decipher potentil interction of the mtor signling pthwy nd the type I IFN system in ntivirl immunity. We hypothesize tht 1) mtor signling pthwy is involved in nti-prrsv defense; 2) mtor signling intercts with type I IFN signling to regulte ntivirl responses; nd 3) type I IFNs modulte mtor signling pthwy differently. 10

19 Chpter 2 - Mterils nd Methods 2.1 Cells nd viruses Experiments involved in nimls nd viruses were pproved by the Knss Stte University Institutionl Animl Cre nd Use nd Biosfety Committees. Animl procedures nd isoltion of porcine lveolr MФs nd peripherl blood mononucler cells (PBMCs) were previously described [93, 94]. In brief, 5-week-old cliniclly helthy pigs from herd without virl infection history were used for collection of primry cells. PBMCs were isolted from blood collected by jugulr venipuncture from nesthetized pigs, using Histopque-1077 (Sigm, Sint Louis, MO). Immeditely fter euthnsi, MФs were obtined by lvging lungs with 1 PBS (ph7.4, Sigm), then wshing cells three times with RPMI (Roswell Prk Memoril Institute)-1640 medium (Gibco, Crlsbd, CA). Isolted primry cells were used immeditely or cryopreserved in Recovery Cell Culture Freezing Medium (Gibco). Africn green monkey kidney (MARC-145, ATCC) cells were grown in Modified Egle s medium (MEM, Gibco) contining 8% fetl bovine serum (FBS, Gibco) nd 1 Antibiotic-Antimycotic (Gibco), 293FT cells (Invitrogen, Crlsbd, CA) in Dulbecco's Modified Egle Medium (DMEM, Gibco) supplemented with 10% fetl bovine serum (FBS, Gibco), 1 MEM Non-Essentil Amino Acids Solution (NEAA, GIBCO). MФs nd PBMCs were mintined in RPMI-1640 medium contining 10% FBS nd 1 Antibiotic-Antimycotic. mdcs were generted from PBMCs stimulted with IL-4 (2ng/ml) nd GM-CSF (5ng/ml) (R&D Systems, Minnepolis, MN) nd mintined in RPIM-1640 medium contining 10% FBS nd 1 Antibiotic-Antimycotic [95]. P129-GFP nd DsRed-lbelled PRRSV were used in this study [93, 94, 96]. 11

20 2.2 Cell polriztion, virl infection nd trnscriptomic shotgun sequencing MФs hve long been considered s one of most importnt immune effector nd regultor cells, re distributed widely throughout the body, disply multiple roles in both innte nd dptive immune responses, nd lso serve n indispensble role in inflmmtion nd its resolution [97-99]. To mintin its vrious functions, MФs undergo phenotypicl polriztion in response to diverse environmentl stimulnts [100]. Typicl ctivtion sttues chrcterized in MФ polriztion include clssicl (M1) nd lterntive (M2) sttes [93, 94, 100, 101]. M1 sttus is induced in response to IFN-γ nd bcteril products, such s lipopolyscchrides (LPS) [93]. M2 sttus is further ctegorized into three subclsses: M2, induced by type II cytokines IL-4 or IL-13; M2b, obtined by triggering Fcγ receptors plus TLR stimulus; nd M2c, ctivted by glucocorticoid (GC), IL-10 nd/or TGF-β [93]. Procedures of MФ polriztion were performed s previously described [93, 94]. Briefly, porcine lveolr MФs were stimulted with LPS, IFN-α1, IFN-γ, IL-4 nd IL-10 t 20 ng/ml for 30 h (R&D Systems), followed by infection of P129-GFP t multiplicity of infection (MOI) of 0.1 for 5 h. After polriztion nd infection, cells were wshed twice with fresh culture medium, nd then totl RNA ws extrcted from cells of ech tretment using RNA/DNA/protein purifiction kit (Norgen Biotek, Ontrio, Cnd). To qulify for constructing RNA-Seq librries, RNA concentrtion nd qulity were evluted with Nno-Drop 8000 spectrometer (NnoDrop, Wilmington, DE) nd Agilent 2100 Bionlyzer (Agilent Technologies, Snt Clr, CA) to insure RNA smples with A260/A280>1.8 nd RNA integrity number (RIN)>7.0. All trnscriptomic shotgun sequencing ws conducted following the procedures of Illumin Pipeline (BGI Americs, Cmbridge, MA). Genome-wide trnscriptomic nlysis ws performed using M clen reds per smple. Dt nlyses were conducted s described [93, 94]. 12

21 2.3 Antivirl nlysis MARC-145 cells were treted with mtor inhibitors, rpmycin nd PP242, or mtor ctivtor, MHY1485 t different concentrtion for 12, 24, 36 or 48 h (Sigm), nd then infected with DsRed-lbelled PRRSV t MOI of 1.0 for h. All imges were collected using Nikon fluorescent microscopy (Shinjuku, Tokyo, Jpn) t mgnifiction of 20, nd virl infection ws quntified with SpectrMx i3 (Moleculr Devices, Sunnyvle, CA). Porcine primry cells were infected with DsRed-lbelled PRRSV t MOI of 0.5 long with mtor inhibitors or ctivtor for h, visulized with fluorescent microscopy nd quntified using SpectrMx i3. All chemicls used were dissolved in dimethyl sulfoxide (DMSO, cell culture grde, Sigm). 2.4 Biossys nd ELISA MARC-145 cells were treted with mtor inhibitors or ctivtor for 24 h, nd infected with DsRed-lbelled PRRSV t MOI of 1.0 for nother 24 h. Superntnts from ech cell culture smple were inctivted with UV light for 1 h, nd used to mesure IFNs with biossy in MARC-145 cells stbly trnsformed with IRF3-, IRF7- or Mx1-promoter driven luciferse reporter system, or n ELISA kit (R&D system) for detecting IFN-α subtypes [102]. In brief, MARC-145 (IRF3, IRF7, or Mx1) cells were treted with inctivted superntnts for 24 h, lysed with Glo lysis buffer nd quntified by Stedy-Glo Luciferse Assy System (Promeg, Mdison, WI). 2.5 Rel-Time PCR nd western blotting ssy Porcine lveolr MФs were infected by DsRed-lbelled PRRSV t MOI of 0.5 long with mtor inhibitors or ctivtor tretment for 12 h, nd totl RNA ws extrcted from cells of ech tretment using RNA/DNA/protein purifiction kit. Rel-Time PCR (RT-PCR) ssy 13

22 ws performed using GoTq 2-Step RT-PCR System (Promeg). Totl cdna ws reversely trnscribed from RNA pools (2 μg RNA in 20-μl rection mixture) using rndom primers. RT- PCR nlysis ws conducted using StepOnePlus RT-PCR system (Applied Biosystems, Grnd Islnd, NY). Rections were run with 1 μl cdna in 20-μl rection mixture, nd set t 95 C for 2 min followed by 40 mplifiction cycle of 95 C for 15s, 60 C for 1 min with melting curve, 95 C for 15s, 60 C for 1 min, rmp from 60 C to 95 C t n 1% rte, nd 95 C for 15s. Criticl threshold (Ct) vlues nd melt curves were monitored nd collected with enclosed softwre. Reltive gene expression dt were first normlized ginst Ct vlues of the housekeeping gene (β-actin), nd the reltive expression index (2 -ΔΔCt ) ws determined compred with expression levels of control smple for stimulted regultion. MARC-145 cells were treted with porcine IFN-α1 nd IFN-β (R&D system), or infected with DsRed-lbelled PRRSV for 24 h, then lysed using 100 μl CelLytic M (Sigm) for cells supplemented with protese inhibitors (Sigm) t 4 C for 30 min. Soluble proteins were collected by centrifugtion t g, 4 C for 15 min. After combining with 4 LDS smple buffer, proteins were resolved on 4 12% grdient Bis-Tris gel nd trnsferred to PVDF membrne using NuPAGE electrophoresis system (NuPAGE Novex, Invitrogen). After blocking with 5% non-ft dry milk (Bio-Rd, Hercules, CA), filters were probed with primry ntibodies ginst AKT, Phospho-AKT (Ser473), p70 S6 kinse, Phospho-p70 S6 kinse (Thr389) or β-actin t 1:1000 dilution, followed by blotting with 1:2000 fold dilution of HRPlinked secondry ntibody (Cell Signling Technology, Dnvers, MA). Blotted filters were imged with Kodk Imge Sttion 4000 (Rochester, NY). 14

23 2.6 Gene Silencing bsed on CRISPR/Cs9 system The clustered regulrly interspced short plindromic repet (CRISPR), short nd repetitive segment of prokryotic DNA, ws first identified in microbilly dptive immune system to confer resistnce to foreign viruses/phges nd plsmids [103]. The CRISPR/Cs9 (CRISPR-ssocited nuclese 9) system hs been widely utilized for sequence-specific regultion of gene expression [104]. Over the pst severl yers, this RNA-guided genomic editing technique hs been developed extensively, involved in trnscription repression nd ctivtion, specific sequence replcement or insertion, nd gene muttion or knockout [105, 106]. The trget genes in mmmlin cells cn be quickly, esily nd efficiently down-regulted by using Cs9 with one or few single guide RNAs (sgrnas) [107]. In this study, we estblished stbly Cs9-expressing MARC-145 cell line nd down-regulted Rictor nd Rptor expression by combining 3 or 5 sgrnas, using lentivirus-bsed pltform [ ]. phr-cs9-2a-puro nd phu6/bb-gfp plsmids were constructed on the bsis of phr- SFFV-dCs9-BFP (Addgene, #46910), pspcs9(bb)-2a-gfp (Addgene, #48138) nd pgrnahumnized (Addgene, #44248) [ ]. The frgment of Cs9-2A ws mplified from pspcs9(bb)-2a-gfp nd inserted into phr-sffv-dcs9-bfp using MluI nd BmHI, followed by puromycin frgment mplified from pgrna-humnized with BmHI nd SbfI. phu6/bb-gfp ws chieved by replcing murine U6 promoter in pgrna-humnized with humn U6 promoter from pspcs9(bb)-2a-gfp using XbI nd NotI, nd ll BbsI sites were mutted in pgrna-humnized vector bckbone. All sgrna expression constructs were obtined with BbsI by inserting n nneled oligo pir encoding 20-nt guide sequences [107]. All restrictive enzymes, Quick Ligtion Kit, Quick-Lod Tq 2 Mster Mix nd Phusion High-Fidelity PCR Kit were purchsed from New Englnd Biolbs (Ipswich, MA). 15

24 Lentivirl constructs for efficient trnsfection nd expression of Cs9 nd sgrna in mmmlin cells were produced using 2 nd genertion lentivirl system with pmd2.g (Addgene, #12259) nd pspax2 (Addgene, #12260). 293FT cells were trnsfected with envelop plsmid (pmd2.g), pckging plsmid (pspax2) nd trnsfer plsmid (phr-cs9-2a-puro or phu6/bb-xx-gfp) t rtio of 0.9:1.5:2.1 or 0.9:1.5:1.5 (μg). Briefly, 293FT cells were grown in 24 well plte overnight to rech 70% confluence nd trnsfected with totlly 1 μg DNA using XtremeGene9 trnsfection regent (Roche, Indinpolis, IN) t trnsfection regent:dna rtio of 2.5:1. After incubtion for 8 h, growth medium ws refreshed, nd lentivirus-contining superntnts were collected for trnsducing trget cells t 48 h post-trnsfection. The MARC- 145 cell line tht stbly expresses Cs9-2A-puro ws estblished by lentivirl trnsduction, nd selected with complete culture medium including 7 μg/ml puromycin (InvivoGen, Sn Diego, CA). The pure cell line ws cquired by seril dilution. After ttining MARC-145-Cs9 cells, they were trnsduced with sgrna expressing lentiviruses to down-regulte trget genes. 16

25 2.7 Tbles Tble 2.1 RT-PCR primer sequences for porcine type I IFNs nd their receptors [102]. Primer nme Sequence (5 to 3 ) Product size (bp) IFN-α1 Sense Antisense GGC TCT GGT GCA TGA GAT GT GCC TTC TTC CTG AAT CTG TCT TA 337 IFN-α5/6 Sense Antisense GCA CAA ATG AGG AGA ATA TCT CCT CCT GAG TCT GTC TTG 437 IFN-α7/11 Sense Antisense GGG ACT TTG GAT CCC CTC AT GTG GAG GAA GAG AAG GAT G 369 IFN-α9 Sense Antisense GTG CTG CTC AGC TGC AAG AGT CCT CCT CCA GCA GGG GC 384 IFN-α12 Sense Antisense CCT CAG CCT TCC TCA CGG T CTC ATG ACT TCT GCC CTG AT 509 IFN-αω Sense Antisense AGA TCT TCC GCC TCT TCA GCA CAA TTC TGG TTT CCA CCC TGA CAA CCT 261 IFN-β Sense Antisense ATG TCA GAA GCT CCT GGG ACA GTT AGG TCA TCC ATC TGC CCA TCA AGT 246 IFN-δ1 Sense Antisense TAT AAG CTT CTG GCA GGA GT AGC CTT GAG TCA TCT TGT

26 IFN-δ3/4/5 Sense Antisense AGA ACT TGT CTG CTG TCC ATT TTT GGA GAA GAC ACC GGA 209 IFN-δ6/7 Sense Antisense CAA TGG CCC ACA TCC ATT TGC T AGA TGT GTC ACA AGT GTG CCT 214 IFN-δ8/9 Sense Antisense ATG CTC TGC TCC ACT CCT GC GTG CCT TGA GTC ATC TGG ATT GG 194 IFN-ε Sense Antisense TTG GTA CTG CTG GCT TCT TCC ACT AAC TGC CCT GAA GAG GCT GAA GAT 255 IFN-κ Sense Antisense GCA GAA TGA GCC ATT CGT TTC CCA TCC TCT TCC TCC TGC AAG CAT TGT 259 IFN-ω1 Sense Antisense TGG TGC TTC TGC GTC AGA TG CTC ACC TGC ACC AAG CAG GAC 265 IFN-ω2 Sense Antisense TTC GTG CTC TCT CTA CCG ATG CAG AGA TGG CCT GGA CCT 225 IFN-ω4 Sense Antisense TCT GCA TCA GAT GAG GAG AC CAA ATG TCT GCT CTT CCA TCT 278 IFN-ω5 Sense Antisense TCA TGC TCT CTC TAC TGA CAG C TGG AGC TTG TCC AGG AGG A 300 IFNAR1 Sense Antisense ACC ACA GTG AAA CAT CAC CTG CCT TGT TGA TGA CGG GAG GAA ACA GGA

27 IFNAR2 Sense Antisense TCA ACG GGA ATC AGA GTC GTC AGA TCA GGA AAT ACC CAG GCG GAC AAT 180 β-actin Sense Antisense TCG CCG ACA GGA TGC AGA AGG A AGG TGG ACA GCG AGG CCA GGA T

28 Tble 2.2 sgrna primer sequences Primer nme Sequence (5 to 3 ) sgrictor-1 Sense Antisense CAC CGC CGA TCG CCG CCA TAT TGA AAA CTC AAT ATG GCG GCG ATC GGC sgrictor-2 Sense Antisense CAC CGA TCT GAC CCG AGG TAA CGC G AAA CCG CGT TAC CTC GGG TCA GAT C sgrictor-3 Sense Antisense CAC CGA CAA GAC CTC CAG TTC CAG A AAA CTC TGG AAC TGG AGG TCT TGT C sgrictor-4 Sense Antisense CAC CGT AGC AGT GAT CCA AAA GGA AAA CTC CTT TTG GAT CAC TGC TAC sgrictor-5 Sense Antisense CAC CGT CTT TCA GGT TTC ATC CCA G AAA CCT GGG ATG AAA CCT GAA AGA C sgrptor-1 Sense Antisense CAC CGG TCC TGG CCT TCA GCC CCG AAA CCG GGG CTG AAG GCC AGG ACC sgrptor-2 Sense Antisense CAC CGC ATT TCG GAC TCC ATC AGT G AAA CCA CTG ATG GAG TCC GAA ATG C sgrptor-3 Sense Antisense CAC CGG GAA ACT ACC AAG TTC AAG AAA CCT TGA ACT TGG TAG TTT CCC 20

29 Chpter 3 - Results 3.1 Genes in mtor signling pthwy were differentilly expressed in PRRSV-infected MФs t different ctivtion sttuses Genome-wide nlysis of gene regultion ws conducted in PRRSV-infected porcine lveolr MФs t different ctivtion sttes s described [93, 94]. The differentilly expressed genes (DEGs) involved in mtor signling pthwy including mtor complexes, their upstrem regultors nd downstrem effectors, were extrcted for further nlysis (Figure 3.1A). Some of them displyed significntly differentil expression nd were selected s cndidte trgets to nlyze ntivirl regultion of MФs by mtor signling (Figures 3.1B nd 3.1C). mtor kinse, the pivotl component in both mtorc1 nd mtorc2, ws up-regulted in IFN-γ (M1) nd IL- 4 (M2) stimultions nd down-regulted in LPS (M1) nd IL-10 (M2c) tretments; however, mtor kinse ws less regulted by ntivirl IFN-α1, indicting tht mtor is more reltive to MФ ctivtion sttus, or ntivirl regultion through cell polriztion [110]. Two downstrem effectors of mtorc1 nd mtorc2, RPS6KB2 (lso clled p70 S6 kinse) nd AKT3, were differentilly regulted by MФ polriztion. The key subunit of mtorc2, Rictor, ws gretly up-regulted by IFN-α1 [47, 111]. ULK1, cting s crosslink for IFN-mTOR, ws downregulted by IFN-γ, LPS nd IFN-α1, nd up-regulted by IL-4 nd IL-10 [62]. Genes relted to the mtor pthwy included lrge group of DEGs in PRRSV-infection MФs t different ctivtion sttuses, which suggests tht the mtor signling pthwy is closely involved in nti- PRRSV regultion. 21

30 3.2 Regultion of PRRSV infection by the mtor signling pthwy The involvement of mtor signling in regultion of nti-prrsv response ws first investigted using phrmceuticl pproch. Two mtor inhibitors, rpmycin nd PP242, nd n mtor ctivtor, MHY1485, were selected to modulte the mtor signling pthwy [42, 112, 113]. Compred with rpmycin, which shows selective suppression primrily on mtorc1, PP242 is non-selective inhibitor cting on both mtorc1- nd mtorc2-signling pthwys. PRRSV infection ws substntilly repressed by mtor inhibitors; especilly, the non-selective inhibitor, PP242, which showed much better effect thn rpmycin in MARC- 145 cells (Figures. 3.2A, 3.2B nd 3.3A). Cells treted by both rpmycin nd PP242 resulted in n dditive effect on virl repression. In contrst, the ctivtor, MHY1485 reversed the inhibition of mtor ctivity by PP242. The ddition of MHY1485 t physiologicl doses to PRRSVinfected cells, which were pre-treted with PP242, displyed reverse effect on PP242- suppression of PRRSV repliction (Figures 3.2C nd 3.3B) [114]. However, MHY1485 t high concentrtion of 8 M inhibited PRRSV infection modertely, which could be relevnt to its inhibitory effect on cell utophgy (Figures 3.2B nd 3.3A) [113, 115]. All three tested inhibitors nd the ctivtor ffect virl infection in dose- nd time-dependent mnner (Figure 3.3A). In prticulr, prolonging rpmycin tretment significntly reduced PRRSV infection in MARC- 145 cells. Although primrily trgeting mtorc1, it ws shown tht long-term exposure of cells to rpmycin lso led to suppression of mtorc2 ctivity [116]. In ddition, we used porcine primry cells to confirm the results from MARC-145 cells, n Africn kidney cell line frequently used for culture of PRRSV in vitro. PP242 displyed n inhibitory effect on PRRSV infection in both MФs nd mdcs with dose-dependence s shown in MARC-145 cells (Figures 3.4A nd 3.4B). Rpmycin showed inhibition on PRRSV infection 22

31 only in mdcs. The ctivtor, MHY1485, conferred undetected effect on PRRSV infection in both MФs nd mdcs. No cytotoxic effect ws shown in MARC-145 cells nd porcine primry cells treted with mtor inhibitors or ctivtor. Results suggest tht inhibition of mtor signling provides potentil route to regulte nti-prrsv response nd host ntivirl immunity is significntly modulted by mtor signling pthwy. 3.3 mtor signling modultes the type I IFN response IFNs serve s criticl meditors in regultion of overll ntivirl response. Through interction with their specific receptors, type I IFNs induce expression of myrid of ISGs to exert ntivirl nd other biologicl functions [1, 18, 19]. It is implied tht mtor ctivity is closely relted to type I IFN production nd signl ugmenttion, but its involvement in PRRSV infection hs not been studied [14, 31, 45-47, 49, 91, 117]. To test if mtor signling is involved in IFN production upon PRRSV infection, we fmily-wide nlyzed porcine type I IFN gene expression in virus-infected porcine cells with phrmceuticl modultion of mtor signling. Porcine lveolr MФs were infected with PRRSV long with mtor inhibitors or ctivtor, nd the expression of type I IFNs nd their receptors ws exmined using vlidted primers [102]. We observed tht PRRSV infection repressed expression of most type I IFN subtypes, including IFN-α1/α5/6/α9/12, IFN-δ1/3/4/5 nd IFN-ω1/2/5, which re effective nti-prrsv IFNs (Figure 3.5A) [92, 102]. PP242 significntly reversed suppression of ll nlyzed type I IFN subtypes except for IFN-αω nd IFN-β (Figure 3.5B). In comprison to PP242, rpmycin showed much less effect on PRRSV-induced inhibition of type I IFNs, nd the ctivtor MHY1485 only enhnced production of IFN-α9, IFN-δ6/7/8/9 nd IFN-ω1/2 significntly. In ddition, PP242 tretment conferred high expression levels of type I IFN receptors. However, mtor ctivtor MHY1485 suppressed expression of receptor genes of IFNAR1/IFNAR2. We mesured IFN-α 23

32 proteins secreted in culture superntnts collected from MARC-145 cells treted with mtor inhibitors/ctivtor. We showed tht mtor ctlytic inhibitor PP242 ws ble to heighten IFN-α production, the most effective IFN subtype tht cts ginst PRRSV (Figure 3.6A) [92]. ISGs re collection of hundreds of genes up-regulted in response to IFN production, nd ply multiple roles in ntivirl regultion. Typicl ISGs, including PRRs, IRFs, nd other signl trnsducing proteins, work in mgnifying IFN signling or directly inctivting viruses [18]. IRFs, prticulrly IRF3 nd IRF7, re criticl trnscription fctors in medition of both IFN production nd ction. Using the promoter-reporter system constructed with centrl promoter elements of humn IRF3, IRF7 nd Mx1 genes [102], we showed tht PRRSV infection suppressed promoter ctivity of IRF3, IRF7 nd Mx1, nd the suppression ws successfully reversed with tretment of mtor inhibitors, rpmycin nd PP242 t physiologicl concentrtion (Figures 3.6B, 3.6C nd 3.6D). In contrst, mtor ctivtor, MHY1485, showed little effect on PRRSV-suppression on promoter ctivity of IRF3, IRF7 nd Mx1 genes. These findings indicte tht PRRSV repliction nd ntivirl response in cells re significntly regulted through modultion of mtor signling, which in turn ffects both cell metbolic nd immune sttuses. Furthermore, through detection of type I IFN production t mrna nd protein levels s well s using bio-ssys to monitor ISG-stimulting ctivity, we showed tht regultion of mtor signling in ntivirl response functions, t lest in prt, through chnging production or ction of type I IFNs. 3.4 Gene silencing mtor signling regultes PRRSV infection Using phrmceuticl pproch, we hve demonstrted tht mtor signling regultes cell ntivirl responses prtly through modultion of the type I IFN system [25, 44, 45, 47, 51]. Here, using gene silencing, we further confirmed this observtion. Rptor nd Rictor re two key 24

33 subunits of mtorc1 nd mtorc2, respectively. Studies showed tht knockout of Rptor nd Rictor completely blocked mtorc1- or mtorc2-pthwy ctivity, respectively [111, 118, 119]. Use of newly developed genome editing system, CRISRP/Cs9, we suppressed trnscription of either Rictor or Rptor ner 60% in MARC-145 cells (Figures 3.7A nd 3.7B). Consistent with our observtion using mtor inhibitors, genetic suppression of Rictor, the essentil fctor for mtorc2, significntly suppressed PRRSV infection in MARC-145 cells (Figures 3.7C nd 3.7D). In contrst, PRRSV infection ws only slightly inhibited by silencing Rptor expression. Our tests repetedly showed tht inhibition of mtorc2 ctivity by inhibitors (such s PP242 or prolonging tretment with rpmycin) or gene silencing of Rictor provided better protection ginst PRRSV infection. Biossys of culture superntnts in stimultion of ISG promoter ctivity verified tht silencing the Rictor gene enhnced the cellulr cpcity to up-regulte promoter ctivity of ISG genes including IRF3, IRF7, nd Mx1, plusibly through incresing type I IFN production in Rictor-silent cells (Figures 3.7E, 3.7F nd 3.7G). 3.5 Type I IFNs modulte mtor signling Previous studies hve reveled tht the mtor signling pthwy intercts nd ffects IFN signling trnsduction [25, 44, 45, 47, 51, 52, 117, 120]. For exmple, ctivtion of AKTmTOR pthwy promoted mrna trnsltion of IFN-stimulted genes [44]. However the regultion of mtor cscde by different type I IFN subtypes nd viruses hs not been well investigted. In this study, we found tht phosphoryltion nd expression of p70 S6 kinse nd AKT, the key downstrem effectors of mtorc1 nd mtorc2 respectively, were remrkbly regulted by porcine IFN-α1 nd IFN-β, nd PRRSV infection in MARC-145 cells (Figure 3.8). IFN-α1 nd PRRSV infection stimulted phosphoryltion of p70 S6 kinse t T389, which phosphoryltes ribosoml protein S6 nd trnsltion initition fctor, eif4b, nd cts 25

34 downstrem of mtor signling; however, only IFN-α1 elevted p70 S6 kinse expression [121, 122]. In ddition, IFN tretments nd PRRSV infection incresed expression of AKT nd decresed its phosphoryltion t S473, indicting tht IFN-signling my interct with mtorsignling through regultion of AKT t different levels. In summry, type I IFNs could interct with the mtor-signling cscde t severl pthwy components. Modultion of the mtor signling pthwy provides novel trgets to regulte virus-host interction nd to potentite ntivirl responses. 26

35 A mtorc mtor Upstrem Regultors mtor Downstrem Effectors MV-IFN-α1 M2-IL10 M1-LPS M2-IL4 M1-IFNγ M0-PBS B Significnt DEGs in IFN tretments M0-PBS M1-IFN-γ M2-IL-4 M1-LPS M2-IL-10 MV-IFN-α C Log 2 (Fold chnge to the DPBS control) Reltive fold chnge mtor RICTOR AKT3 IKBKB PIK3CD PIK3CG RHEBL1 PRKAB2 STRADB TP53 EIF4E RPS6KB2 EIF4EBP2 IFN- IL-4 LPS IL-10 IFN- 1 ULK1 ULK2 Figure 3.1 RNA-Seq nlysis of DEGs in PRRSV-infected porcine lveolr MФs t different ctivtion sttuses. Subsets of genes involved in mtor signling pthwy were extrcted nd displyed using hetmp to show reltive gene expression normlized s reds per kilobse of trnscript per million reds mpped (RPKM) (A). Some DEGs, which were significntly regulted in cytokine tretments compred with the control, served s cndidte genes for regultion of ntivirl response, nd were further emphsized using hetmp (RPKM) (B) nd br chrt (log of fold chnges to the control) t the bottom (C). =p (FDR, flse discovery rte)<0.001 to the control of PBS. All gene symbols re from NCBI Gene dtbse ( 27

36 A Control Rpmycin PP242 MHY1485 DsRed-PRRSV Bright field Merge R e l tiv e In fe c tio n ( % ) B C o n tro l R p (0.0 1 M ) R p (0.1 M ) R p (1 M ) P P ( M ) D s R e d -P R R S V b b P P (0.5 M ) P P (2 M ) P P (8 M ) M H Y ( M ) M H Y (0.5 M ) M H Y (2 M ) M H Y (8 M ) C R e l tiv e In fe c tio n ( % ) C o n tro l D s R e d -P R R S V b c C o n tro l M H Y (1 M ) M H Y (2 M ) M H Y (4 M ) R p (0.0 1 M ) P P (2 M ) R p (0.1 M ) R p (1 M ) Figure 3.2 Regultion of PRRSV infection by mtor inhibitors nd ctivtor in MARC- 145 cells. Cells were treted with 1 µm rpmycin, 2 µm PP242 or 2 μm MHY1485 for 24 h, then infected with DsRed-lbelled PRRSV (MOI of 1), visulized nd imged with fluorescent microscopy (A) t 36 h post infection (hpi); or treted with seril dilution of mtor inhibitors, rpmycin nd PP242, nd ctivtor MHY1485, nd quntified using SpectrMx i3 t 36 hpi (B); or pretreted with 2 μm PP242 for 24h, then infected with DsRed-PRRSV long with MHY1485 or rpmycin t different concentrtion, nd quntified with SpectrMx i3 t 36 hpi (C). n=3, =p<0.001, b=p<0.01, c=p<0.05 to the control. Rp: rpmycin, MHY: MHY

37 R e l tiv e In fe c tio n ( % ) A C o n tro l R p (0.0 1 M ) R p (0.1 M ) R p (1 M ) D s R e d -P R R S V P P (0.5 M ) P P ( M ) 1 2 h 2 4 h P P (2 M ) P P (8 M ) M H Y ( M ) 3 6 h 4 8 h M H Y (0.5 M ) M H Y (2 M ) M H Y (8 M ) R e l tiv e In fe c tio n ( % ) B 1.5 D s R e d -P R R S V C o n tro l C o n tro l M H Y (1 M ) M H Y (2 M ) M H Y (4 M ) R p (0.0 1 M ) P P (2 M ) R p (0.1 M ) 1 2 h 2 4 h 3 6 h 4 8 h R p (1 M ) Figure 3.3 mtor inhibitors nd ctivtor regulte PRRSV infection in dose- nd timedependent mnner in MARC-145 cells. Cells were pre-treted with seril dilution of mtor inhibitors, rpmycin nd PP242, or ctivtor MHY1485 for 12, 24, 36 or 48 h, then infected with DsRed-lbelled PRRSV (MOI of 1), nd quntified with SpectrMx i3 t 36 hpi (A); or pre-treted with 2 μm PP242 for 12, 24, 36 or 48 h, then infected with DsRed-PRRSV long with MHY1485 or rpmycin t different concentrtion, nd quntified with SpectrMx i3 t 36 hpi (B). Rp: rpmycin, MHY: MHY

38 R e l tiv e In fe c tio n ( % ) A 1.2 D s R e d -P R R S V (M s ) C o n tro l R p (0.0 1 M ) c R p (0.1 M ) R p (1 M ) P P (0.1 M ) P P (0.5 M ) P P (2 M ) M H Y (0.1 M ) M H Y (0.5 M ) M H Y (2 M ) R e l tiv e In fe c tio n ( % ) B 1.2 D s R e d -P R R S V (m D C s ) C o n tro l R p (0.0 1 M ) b c R p (0.1 M ) R p (1 M ) P P (0.1 M ) P P (0.5 M ) P P (2 M ) M H Y (0.1 M ) M H Y (0.5 M ) M H Y (2 M ) Figure 3.4 Repression of PRRSV infection by mtor inhibitors in porcine lveolr MФs nd mdcs. Cells were incubted with DsRed-lbelled PRRSV (MOI of 0.5) for 1 h, nd then treted with mtor inhibitors (rpmycin or PP242) or ctivtor (MHY1485) t indicted concentrtions for nother 20 h. Infected MФs (A) nd mdcs (B) were quntified using SpectrMx i3 t 20 hpi. n=4, =p<0.001, b=p<0.01, c=p<0.05 to the control. Rp: rpmycin, MHY: MHY

39 B Reltive fold chnge Reltive fold chnge A c b b c IFN- 1 IFN- 5/6 IFN- 7/11 IFN- 9 c b b IFN- 1 IFN- 5/6 IFN- 7/11 IFN- 9 IFN- 12 b c b IFN- IFN- IFN- 1 b IFN- 12 IFN- IFN- IFN- 1 IFN- 3/4/5 c IFN- 3/4/5 IFN- 6/7 IFN- 8/9 b c c IFN- 6/7 IFN- 8/9 IFN- IFN- IFN- 1 IFN- IFN- IFN- 1 IFN- 2 IFN- 4 IFN- 5 IFNAR1 IFN- 2 IFN- 4 IFN- 5 IFNAR1 IFNAR2 Figure 3.5 mtor inhibitors or n mtor ctivtor modulte expression of type I IFNs nd their receptors in PRRSV-infected porcine lveolr MФs. Cells were incubted with DsRed-lbelled PRRSV (MOI of 0.5) for 1 h, nd then treted with mtor inhibitors (Rpmycin 1 μm, or PP242 2 μm) or ctivtor (MHY μm) for 12 h. Totl RNAs were extrcted to nlyze expression of type I IFNs nd their receptors in cells infected with PRRSV or not (A) nd PRRSV-infected cells w/wo inhibitor or ctivtor tretments (B) by specific primers using two-step RT-PCR. Control ws normlized to 0, n=3, =p<0.001, b=p<0.01, c=p<0.05 to the control. Rp: rpmycin, MHY: MHY1485. b c b c b c c b c b IFNAR2 Rpmycin PP242 MHY

40 A 80 IFN- production B 3000 IRF3-promoter ctivity IFN- (ng/ml) luciferse ctivity C luciferse ctivity Control Rpmycin PP242 MHY1485 Control Rpmycin PRRSV IRF7-promoter ctivity b PP242 MHY1485 D luciferse ctivity 0 PRRSV 1000 Mx1-promoter ctivity 500 Control Rpmycin PP242 MHY1485 Control Rpmycin PP242 MHY1485 c c 0 Control Rpmycin PP242 MHY1485 Control Rpmycin PP242 MHY Control Rpmycin PP242 MHY1485 Control Rpmycin PP242 MHY1485 PRRSV PRRSV Figure 3.6 Disruption of mtor signling pthwy promotes type I IFN production nd signling. MARC-145 cells were treted with mtor inhibitors (rpmycin nd PP242) nd ctivtor (MHY1485) for 24h, nd then infected with DsRed-lbelled PRRSV (MOI of 1) for nother 24h. Cell culture superntnts were collected nd PRRSV ws inctivted with UV illumintion. Superntnts were used to mesure IFNs with n ELISA for detection of IFN-α subtypes (A), or biossy in MARC-145 cells stbly trnsformed with n IRF3-, IRF7- or Mx1-promoter driven luciferse reporter system (B, C nd D). n=3, =p<0.001, b=p<0.01, c=p<0.05 to the control. 32

41 A Reltive fold chnge 1.2 Rictor b sicontrol sirictor B Reltive fold chnge 1.2 Rptor sicontrol sirptor Rictor β-actin Rptor β-actin C sicontrol DsRed-PRRSV sgrna Bright field Merge sirictor sirptor 33