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1 Antagonism of RNase L is required for murine coronavirus replication in Kupffer cells and liver 2 sinusoidal endothelial cells but not in hepatocytes 3 Yize Li and Susan R Weiss 4 Department of Microbiology Perelman School of Medicine University of Pennsylvania Philadelphia 5 PA 19104 6 7 8 Running title KC and LSEC limit MHV NS2 mutant liver replication 9 Key words Murine coronavirus MHV phosphodiesterase hepatocytes Kupffer cells liver 10 sinusoidal endothelial cell hepatitis 11 Corresponding Author Susan R Weiss Department of Microbiology University of Pennsylvania Perelman School of Medicine 203A Johnson Pavilion 36th Street and Hamilton Walk Philadelphia PA 19104 6076 PHONE 215 898 8013 FAX 215 573 4858 Email weisssr upenn edu Abstract 250 words Text 3544 words ABSTRACT 12 JVI Accepted Manuscript Posted Online 24 August 2016 J Virol doi 10 1128 JVI 01423 16 Copyright 2016 American Society for Microbiology All Rights Reserved on September 29 2016 by CORNELL UNIVERSITY http jvi asm org Downloaded from 2 Mouse hepatitis virus strain A59 infection of mice is a useful tool for studying virus host interaction 13 during hepatitis development The NS2 H126R mutant is attenuated in liver replication due to loss of 14 phosphodiesterase activity which the wild type virus uses to block the 2 5 oligoadenylate 15 synthetase OAS ribonuclease L RNase L antiviral pathway The activation of RNase L by 16 NS2 H126R is cell type dependent and correlates with high basal expression levels of OAS as found in 17 myeloid cells We tested the hypothesis that resident liver macrophages Kupffer cells KC are the 18 most likely cell type to restrict NS2 H126R and prevent hepatitis As found previously A59 and NS2 H126R 19 replicate similarly in hepatocytes and neither activates RNase L as assessed by an rRNA 20 degradation assay In contrast in KCs A59 exhibited a 100 fold higher titer than NS2 H126R and 21 NS2 H126R induced rRNA degradation Interestingly in liver sinusoidal endothelial cells LSEC the 22 cells that form a barrier between blood and liver parenchymal cells NS2 H126R activates RNase L 23 which limits viral replication Similar growth kinetics were observed for both viruses in KC and LSEC 24 from RNase L mice demonstrating that both use RNase L to limit NS2 H126R replication Depletion of 25 KC by gadolinium III chloride or LSEC by cyclophosphamide partially restores liver replication of 26 NS2 H126R leading to hepatitis Thus during MHV infection hepatitis which damages the 27 parenchyma is prevented by RNase L activity in both KC and LSEC but not in hepatocytes This 28 may be explained by the undetectable levels of RNase L as well as OASs expressed in hepatocytes 29 30 31 32 33 34 35 36 37 IMPORTANCE 38 on September 29 2016 by CORNELL UNIVERSITY http jvi asm org Downloaded from 3 Mouse hepatitis virus infection of mice provides a useful tool for studying virus host interactions 39 during hepatitis development The NS2 H126R mutant is attenuated in liver replication due to loss of 40 phosphodiesterase activity with which the wild type virus blocks the potent OAS RNase L antiviral 41 pathway RNase L activation by NS2 H126R is cell type dependent and correlates with high basal 42 expression levels of OAS as found in myeloid cells We showed that hepatocytes that comprise the 43 liver parenchyma do not activate RNase L when infected with NS2 H126R nor do they restrict 44 replication However both Kupffer cells KC liver resident macrophages and liver sinusoidal 45 endothelial cells LSEC which line the sinusoids activate RNase L in response to NS2 H126R These 46 data suggest that KC and LSEC prevent viral spread into the parenchyma preventing hepatitis 47 Furthermore hepatocytes express undetectable levels of OASs and RNase L which likely explains 48 the lack of RNase L activation during NS2 H126R infection 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 INTRODUCTION 64 on September 29 2016 by CORNELL UNIVERSITY http jvi asm org Downloaded from 4 Mouse hepatitis virus MHV belongs to the order Nidovirales family coronaviridae and genus 65 Betacoronavirus Coronaviruses are enveloped nonsegmented positive strand RNA viruses 1 66 MHV strain A59 induces moderate to severe hepatitis in infection of mice 2 replication and the 67 consequent liver pathogenesis are dependent on the 2 5 phosphodiesterase PDE activity of the 68 viral accessary protein NS2 3 69 The 2 5 oligoadenylate 2 5A synthetase OAS ribonuclease RNase L pathway is a potent IFN 70 induced antiviral activity 4 Upon infection by diverse viruses including the human liver pathogenic 71 hepatitis C virus 5 6 dsRNA is detected by OAS proteins Four mouse OAS species OAS1a g 72 OAS2 OAS3 and OASL2 and 3 human OAS species OAS1 OAS2 OAS3 can bind dsRNA in 73 vitro and be activated to generate 2 5A 7 the latter binding to and promoting activation and 74 dimerization of RNase L which results in both cellular and viral RNA degradation and thus inhibition 75 of viral replication 7 8 76 The PDE NS2 of MHV cleaves 2 5A and inhibits the activation of RNase L 3 An A59 mutant 77 NS2 H126R which encodes a catalytically inactive PDE replicates to a minimal extent in the mouse 78 liver and causes minor to no liver damage during infection 3 Previous studies have illuminated that 79 the activation of the OAS RNase L antiviral pathway is cell type dependent Bone marrow derived 80 macrophages BMM and bone marrow derived dendritic cells BMDC as well as microglia brain 81 resident macrophages limit NS2 H126R replication by activating the OAS RNase L pathway The basal 82 mRNA expression levels of OASs vary among the cell types and high levels of OAS correlate with 83 activation of the pathway 9 10 84 While the liver is known as a digestive organ it also serves as an immunological organ 11 13 The 85 liver is composed of two populations of cells including approximately 80 liver parenchymal cells 86 hepatocytes and 20 nonparenchymal cells NPC About 10 NPC are Kupffer cells KC or 87 on September 29 2016 by CORNELL UNIVERSITY http jvi asm org Downloaded from 5 liver macrophages and 50 of NPC are liver sinusoidal endothelial cells LSEC 13 We found 88 previously that NS2 H126R infection failed to induce RNase L activation in hepatocytes and that 89 NS2 H126R replicated to similar extent as A59 in that cell type 9 However little is known about the 90 activation of the OAS RNase L pathway in the other liver cell types and specifically in which cell type 91 RNase L activation will limit NS2 H126R replication although previous studies implicated a protective 92 role of KC 3 Here we isolated the various primary liver cells and infected them with WT A59 and 93 NS2 H126R to assess the activation of RNase L as well as quantify viral replication from these cells 94 We found KC and LSEC but not hepatocytes can limit NS2 H126R replication indicating they have an 95 active OAS RNase L pathway and this is likely due to differential levels of expression of RNase L as 96 well as OAS among these liver cell types To further study the protective role of KC and LSEC we 97 performed in vivo cell depletion in mice and showed depletion of KC or LSEC enhance NS2 H126R liver 98 replication and pathogenicity 99 MATERIAL AND METHODS 100 Cells lines mice and viruses Mouse L2 fibroblasts were cultured as described previously 14 101 Recombinant coronaviruses inf MHV A59 A59 and inf NS2 H126R NS2 H126R have been described 102 previously 15 16 C57BL 6 B6 mice were purchased from the National Cancer Institute 103 Frederick MD and bred in the University of Pennsylvania animal facility All procedures were 104 approved by the University of Pennsylvania Institutional Animal Care and Use Committee IACUC 105 Isolation of primary liver cells Mice were euthanized with CO 2 and the livers were perfused 106 Briefly the animal was opened to expose the inferior vena cava IVC and portal vein a catheter 107 was inserted into the IVC and connected to a pump filled with dissociation buffer NaCl 8g L KCl 108 0 4g L NaH 2 PO 4 H2O 78mg L Na 2 HPO 4 120 45mg ml HEPES 2380mg L Na 2 CO 3 350mg L 109 ethylene glycol tetraacetic acid EGTA 190mg L glucose 900mg L pH 7 4 The portal vein was 110 on September 29 2016 by CORNELL UNIVERSITY http jvi asm org Downloaded from 6 cut immediately to allow the buffer 20 30 ml to flow through the liver 111 For hepatocytes the liver was digested with collagenase IV 0 05 Sigma Aldrich in liver 112 digestion buffer NaCl 8g L KCl 0 4g L NaH 2 PO 4 H2O 78mg L Na 2 HPO 4 120 45mg ml HEPES 113 2380mg L Na 2 CO 3 350mg L phenol red 6mg L CaCl 2 2H2O 560mg L pH 7 4 After digestion 114 the liver was removed from the mouse and cells were dissociated from Glisson s capsule and were 115 filtered through a 100 m cell strainer Becton Dickinson BD The cells were centrifuged at 40 g for 116 3min at 4 C and the pellet was re suspended in digestion buffer containing 1mg ml DNase I 117 Roche The cells were centrifuged and washed with MEM minimum essential medium Eagle 118 Sigma Aldrich three times and were re suspended in William s E media Gibco supplemented with 119 10 FBS 100U ml of penicillin and 100mg ml streptomycin 2mM L Glutamine The cells were 120 placed on plates coated with rat tail collagen Sigma Aldrich Four hours after plating the cells were 121 washed with phosphate buffer saline PBS and used for experimentation 122 Kupffer cells KC were isolated from mixed primary liver cell cultures using previously described 123 methods 17 Briefly after perfusion with dissociation buffer the liver was digested with collagenase 124 I 0 05 Sigma Aldrich in digestion buffer supplemented with 50 g ml Trypsin inhibitor Sigma 125 Aldrich Cells were harvested and washed three times with MEM and cultured in DMEM Gibco 126 10566 supplemented with 10 FBS 100U ml of penicillin and 100mg ml streptomycin 100 M 127 mercaptoethanol 10 g ml insulin 10mM HEPES and 50 g ml gentamicin in a flask coated with rat 128 tail collagen 1X10 7 cells per T175 flask The next day the flasks were shaken to remove the dead 129 cells and on days 4 and day 7 fresh medium was added and the KC were harvested at days 9 to 12 130 post plating by shaking and then selected by binding to a petri dish BD KC were recovered by 131 TrpLE select enzyme Invitrogen digestion and plated for experiments 132 The liver sinusoidal endothelial cell LSEC isolation protocol was adapted from previously described 133 on September 29 2016 by CORNELL UNIVERSITY http jvi asm org Downloaded from 7 methods 18 Briefly after perfusion with dissociation buffer the liver was digested with collagenase 134 I 0 025 and collagenase II 0 025 Sigma Aldrich in digestion buffer supplemented with 135 50 g ml Trypsin inhibitor Sigma Aldrich After digestion the liver was removed and cells were 136 dissociated from Glisson s capsule in preservation buffer NaCl 4 15g L 0 25g L KCl 1 12g L 137 HEPES 0 24g L NaOH bovine serum albumin BSA 10g L and filtered through a 100 m cell 138 strainer BD The supernatant was centrifuged at 60 g for 2 min three times to remove 139 parenchymal cells The supernatant was further centrifuged at 1350 g 4 C for 10min to harvest the 140 non parenchymal cells NPC The NPC were re suspended in preservation buffer and were applied 141 on top of a 25 50 Percoll GE Amersham bilayer The cells were centrifuged at 1350 g 4 C for 142 10min LSEC and KC were collected from the interface between the two density layers Cells were 143 washed with preservation buffer and resuspended in RPMI 1640 medium Gibco supplemented 144 with100U ml of penicillin and 100mg ml streptomycin Cells then were plated onto a petri dish and 145 incubated for 8 min to remove KC which attach to the surface the removal of KC was repeated once 146 more The supernatant containing the LSEC was harvested and centrifuged at 1350 g 4 C for 147 10min The pellet was resuspended in RPMI 1640 medium Gibco supplemented with 100U ml of 148 penicillin and 100mg ml streptomycin and placed onto rat tail collagen coated plates Two hours 149 after plating the cells were washed vigorously with ice cold PBS twice The cells remaining on the 150 plates were used for further experiments 151 Antibodies Mouse anti human HNF 4 cross reactive with mouse HNF 4 monoclonal antibody 152 mAb 5 g ml R rat anti mouse CD68 1 100 154 Serotech mAb conjugated to Alexa Fluor 488 were used to detect CD68 by IFA OAS1A clone E 155 2 Santa Cruz 1 200 mAb OAS2 clone G 9 Santa Cruz 1 200 OAS3 clone D 7 Santa Cruz 156 1 200 goat anti RNase L clone T 16 Santa Cruz 1 200 as well as anti GAPDH Thermo Fisher 157 1 1000 goat anti mouse IgG HRP Santa Cruz 1 5000 and donkey anti goat IgG HRP 1 5000 158 on September 29 2016 by CORNELL UNIVERSITY http jvi asm org Downloaded from 8 secondary antibodies were used to detect the primary antibodies of the appropriate species 159 Immunolabeling Cells were fixed in PBS containing 4 paraformaldehyde Bio Rad blocked with 160 2 BSA and immunolabeling with anti HNF 4 antibodies was used to detect hepatocytes 161 immunolabeling with anti CD68 antibodies conjugated with Alexa Fluor 488 was used to distinguish 162 KC CD68 positive and LSEC CD68 negative Primary anti HNF 4 antibody was detected with 163 goat anti mouse Alexa Fluor 488 Invitrogen DAPI 4 6 Diamidino 2 Phenylindole Dihydrochloride 164 Invitrogen was used to stain the nucleus Fluorescence was visualized with a Nikon Eclipse 2000E 165 U fluorescence microscope and images were acquired using Nikon NIS Element BR software 166 Nikon 167 Low density lipoprotein LDL uptake assay of LSEC After isolation cells were cultured in RPMI 168 1640 without FBS medium containing 5 g ml of LDL conjugated with Alexa Fluor 488 Invitrogen 169 was added the cells were incubated at 37 C for 4 hours and at 30 minutes before imaging 1 g ml 170 of Hoechst Sigma Aldrich was add to the medium Fluorescence was visualized with a Nikon 171 Eclipse 2000E Ufluorescence microscope and images were acquired using Nikon NIS Element BR 172 software Nikon 173 Virus growth kinetics Hepatocytes 1 5X10 5 cell per well in 24 well plates KC 1X10 5 cell in 48 174 well plates or LSEC 5 0X10 5 cell per well in 48 well plates were infected with A59 or NS2 H126R 175 MOI 1 PFU cell and at the indicated time points cells and supernatants were harvested together 176 and then frozen 80 C and thawed three times and finally viruses were titrated by L2 cell plaque 177 assay 19 178 KC and LSEC depletion and MHV infection Gadolinium III chloride was used deplete KC from 179 the livers of mice B6 mice were intravenously injected with 20mg kg of Gadolinium III chloride in 180 100 l of saline 20 Cyclophosphamide was used to deplete LSEC from the livers of mice B6 mice 181 on September 29 2016 by CORNELL UNIVERSITY http jvi asm org Downloaded from 9 were intraperitoneally i p injected with 300mg kg of cyclophosphamide in 200 l saline Twenty four 182 hours post injection mice were infected with 200 pfu of virus intrahepatically i h On day 5 post 183 infection mice were sacrificed and livers were harvested for virus titration 19 and histological 184 analysis 3 185 Western blotting Cells were treated or mock treated with 1000U of IFN PBL overnight 186 harvested and washed in PBS and lysed with NP40 buffer 1 NP 40 2 mM EDTA 10 glycerol 187 150 mM NaCl 50 mM Tris pH 8 0 with protease inhibitor cocktail Roche Cell lysates were mixed 188 with 4X Laemmli buffer and boiled at 95 C for 5 minutes and analyzed by electrophoresis on 4 15 189 gradient SDS gels Proteins were transferred to polyvinylidene difluoride PVDF membranes which 190 were treated with 5 nonfat milk in TBST Tris HCl buffer saline with 0 5 Tween 20 blocking 191 buffer for one hour followed by incubation overnight at 4 C with antibodies diluted into TBST 192 Membranes were then washed three times with TBST and incubated with secondary antibodies for 1 193 hour at room temperature washed three times with TBST and then incubated with SuperSignal 194 West Dura Extended Duration substrate Thermo and the signal was detected using an Amersham 195 Imager 600 GE 196 197 Histology Livers were isolated and fixed in 4 paraformaldehyde embedded in paraffin and 198 sectioned Sections were stained with hematoxylin and eosin as described previously 3 199 200 Statistical analysis Two tailed student t test was performed to determine statistically significant 201 differences for in vitro experiments The Mann Whitney test was used to analyze differences in virus 202 titer in different mouse tissues Any undetectable titers from in vitro and in vivo infections were 203 entered as the limit of detection value for each experiment All data were analyzed by using Prism 204 software GraphPad Software Inc CA 205 RESULTS 206 on September 29 2016 by CORNELL UNIVERSITY http jvi asm org Downloaded from 10 Isolation and purification of Kupffer cells KC liver sinusoidal endothelial cells LSEC and 207 hepatocytes from mouse liver 208 To study the role of individual liver cell types in limiting NS2 H126R replication we purified each of the 209 three major cell types representing 95 of liver cells and cultured them in vitro Specifically we 210 focused on the parenchymal cells the hepatocytes and the non parenchymal Kupffer cells KC 211 and liver sinusoidal endothelial cells LSEC using established isolation and culture methods as 212 described elsewhere 17 18 To assess the purity of the liver cell preparations we used the 213 nonspecific stain DAPI in all the tests to identify the nuclei To identify hepatocytes we stained with 214 an antibody that recognizes hepatocyte nuclear factor 4 HNF4 a marker for hepatocytes green 215 21 Over 99 of the cells in the hepatocyte preparation were HNF 4 positive as seen in Fig 1A 216 C which show the merged blue and green stains KC cells the resident liver macrophages were 217 detected by immunostaining for CD68 Fig 1D a KC specific marker 22 In the KC purified 218 preparation we found that 95 the cells were CD68 positive cells LSEC were isolated from total 219 liver non parenchymal cells LSEC readily take up LDL low density lipoprotein 23 Therefore we 220 incubated the LSEC with Alexa Fluor 488 conjugated LDL for 2 hours and then monitored the cells 221 Over 95 of the cells were Alexa Fluor 488 positive Fig 1F To confirm there was little to no KC 222 cell contamination the LSEC were stained with anti CD68 antibodies less than 1 of cells were 223 CD68 positive cells Fig 1E These results indicate that our isolation methods for hepatocytes KC 224 and LSEC yielded highly purified products 225 226 Kupffer cells KC and liver sinusoidal endothelial cells LSEC but not hepatocytes limit 227 NS2 H126R replication in vitro through activation of RNase L To determine which cells limit 228 NS2 H126R replication hepatocyte KC and LSEC cells derived from B6 WT and RNase L KO mice 229 were infected in vitro with WT A59 and mutant NS2 H126R As observed previously in B6 hepatocytes 230 A59 and NS2 H126R replicated similarly 9 However in B6 KC and LSEC A59 showed 10 100 fold 231 on September 29 2016 by CORNELL UNIVERSITY http jvi asm org Downloaded from 11 higher titer than NS2 H126R at 15 and 24 hours post infection When KC and LSEC from RNase L KO 232 mice were infected with NS2 H126R the virus replicated to a similar titer as A59 These results suggest 233 that both KC and LSEC limit NS2 H126R virus replication by activation of the OAS RNase L pathway 234 as evidenced by the fact that replication in both these cell types was robust in cells from RNase L 235 KO mice and minimal in cells from B6 animals To directly determine whether RNase L is activated 236 in KC and LSEC cultures of each liver cell type as well as BMM were infected with A59 and 237 NS2 H126R and at 12 hour KC and BMM 13 hour LSEC 15 hour hepatocytes post infection cells 238 were lysed total cellular RNA was harvested and analyzed for degradation by a Bioanalyzer as 239 shown in Fig 3 Degradation of ribosomal RNA rRNA was observed in KC LSEC and BMM but not 2