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Activation of RNase L by murine coronavirus in myeloid cells is 1 dependent on basal Oas gene expression and independent of virus 2 induced interferon 3 4 L Dillon Birdwell 1 Zachary B Zalinger 1 Yize Li 1 Patrick W Wright 2 Ruth Elliott 1 Kristine M 5 Rose 1 Robert H Silverman 3 and Susan R Weiss 1 6 7 1 Department of Microbiology Perelman School of Medicine University of Pennsylvania 8 Philadelphia PA 2 Department of Biomedical Engineering Washington University in St Louis St 9 Louis MO and 3 Department of Cancer Biology Cleveland Clinic Cleveland OH 10 11 Running title RNase L activation depends on basal Oas expression 12 13 present address 14 PATH 15 455 Massachusetts Avenue NW Suite 1000 16 Washington DC 20001 17 18 19 Address correspondence to 20 Susan R Weiss 21 Department of Microbiology 22 University of Pennsylvania 23 School of Medicine 24 203A Johnson Pavilion 25 36th Street and Hamilton Walk 26 Philadelphia PA 19104 6076 27 Phone 215 898 8013 28 Email weisssr upenn edu 29 30 Abstract number of words 238 31 Text number of words 6320 32 33 JVI Accepted Manuscript Posted Online 6 January 2016 J Virol doi 10 1128 JVI 03036 15 Copyright 2016 American Society for Microbiology All Rights Reserved 2 ABSTRACT 34 The oligoadenylate synthetase ribonuclease L OAS RNase L pathway is a potent interferon IFN 35 induced antiviral activity Upon sensing double stranded RNA OAS produces 2 5 oligoadenylates 36 2 5A which activate RNase L Murine coronavirus MHV non structural protein 2 ns2 is a 2 5 37 phosphodiesterase PDE that cleaves 2 5A thereby antagonizing RNase L activation PDE activity 38 is required for robust replication in myeloid cells as a mutant of MHV ns2 H126R encoding an inactive 39 PDE fails to antagonize RNase L activation and replicates poorly in bone marrow derived 40 macrophages BMM while ns2 H126R replicates to high titer in several types of non myeloid cells as 41 well as in IFN receptor deficient Ifnar1 BMM We reported previously that myeloid cells express 42 significantly higher basal levels of Oas transcripts than non myeloid cells Here we investigated the 43 contributions of Oas gene expression basal IFN signaling and virus induced IFN to RNase L 44 activation Infection with ns2 H126R activated RNase L in Ifih1 BMM to a similar extent as in WT BMM 45 despite the lack of IFN induction in the absence of MDA5 expression However ns2 H126R failed to 46 induce RNase L activation in BMM treated with IFNAR1 blocking antibody as well as in Ifnar1 47 BMM both expressing low basal levels of Oas genes Thus activation of RNase L does not require 48 virus induced IFN but rather correlates with adequate levels of basal Oas gene expression 49 maintained by basal IFN signaling Finally overexpression of RNase L is not sufficient to 50 compensate for inadequate basal OAS levels 51 52 3 IMPORTANCE 53 The oligoadenylate ribonuclease L OAS RNase L pathway is a potent antiviral activity Activation 54 of RNase L during murine coronavirus MHV infection of myeloid cells correlates with high basal 55 Oas gene expression and is independent of virus induced interferon secretion Thus our data 56 suggest that cells with high basal Oas gene expression levels can activate RNase L and thereby 57 inhibit virus replication early in infection upon exposure to viral dsRNA before the induction of 58 interferon and prior to transcription of interferon stimulated antiviral genes These findings challenge 59 the notion that activation of the OAS RNase L pathway requires virus to induce type I IFN which in 60 turn upregulates OAS gene expression as well as to provide dsRNA to activate OAS Our data 61 further suggest that myeloid cells may serve as sentinels to restrict viral replication thus protecting 62 other cell types from infection 63 64 4 INTRODUCTION 65 The coronavirus mouse hepatitis virus MHV strain A59 A59 causes moderate hepatitis 66 and mild encephalitis followed by chronic demyelinating disease in susceptible C57BL 6 B6 mice 67 1 3 A59 is cleared from the liver and central nervous system primarily by the T cell response 68 seven to ten days post infection 4 5 However type I interferon IFN production an early innate 69 immune response is crucial for early control of MHV infection as mice deficient in type I IFN 70 receptor expression Ifnar1 uniformly die by two days after infection 6 8 Interestingly A59 fails 71 to induce IFN in most cell types with the notable exception of myeloid cells 7 Induction of 72 IFN in macrophages and brain resident microglia during MHV infection is dependent on sensing 73 of viral dsRNA by the cytosolic RNA helicase melanoma differentiation associated gene 5 MDA5 74 encoded by Ifih1 7 9 10 IFN induces a large array of interferon stimulated genes ISGs which 75 include pattern recognition receptors PRRs signaling molecules transcription factors and antiviral 76 effectors 11 16 Figure 1 left side diagrams IFN synthesis and signaling in MHV infected 77 macrophages The only other source of type I IFN during A59 infection primarily IFN is induced 78 through a TLR7 dependent pathway in plasmacytoid dendritic cells pDC 17 79 Among the ISGs are several Oas genes encoding proteins that function as nucleic acid 80 sensors to synthesize 2 5 oligoadenylates 2 5A in response to viral dsRNA in the host cytosol 81 18 Mice express several OAS proteins that produce 2 5A including OAS1a g OAS2 OAS3 as 82 well as OASL2 19 21 The 2 5A binds to and activates latent ribonuclease L RNase L by inducing 83 conformational changes and subsequent dimerization 11 13 22 RNase L activation leads to 84 restriction of virus replication through the degradation of host and viral single stranded RNAs 85 inhibition of protein synthesis and finally apoptosis 14 23 24 Figure 1 right side diagrams 86 activation of RNase L 87 Interactions of viruses with the OAS RNase L pathway are complex Many viruses encode 88 proteins that inhibit this pathway to various extents underscoring the significance of this system in 89 5 restricting viral propagation 13 25 28 Among the most potent of these inhibitors is the A59 90 accessory protein non structural protein 2 ns2 a 2 5 phosphodiesterase PDE that cleaves 2 5A 91 thereby preventing RNase L activation 25 An A59 mutant ns2 H126R expressing an inactive PDE 92 due to an H126R substitution of a catalytic histidine residue fails to effectively antagonize RNase L 93 and consequently is attenuated for replication in myeloid cells and in the livers of mice 25 94 We have found that RNase L activation is most robust in myeloid lineage cells where basal 95 Oas gene expression levels are highest compared with several other types of non myeloid primary 96 cells including astrocytes neurons and oligodendrocytes as well as transformed cell lines 29 97 Constitutive low level type I IFN production in the absence of infection maintains basal levels of 98 expression of ISGs including OAS 30 31 It has been generally accepted that RNase L activation 99 requires viral infection to both induce type I IFN production which in turns upregulates OAS gene 100 expression and to provide dsRNA for activation of OAS to produce 2 5A 13 Thus unlike most 101 other IFN induced activities which can be stimulated in uninfected cells by paracrine IFN exposure 102 RNase L can be activated only in infected cells 103 To further elucidate the cell type dependent determinants of RNase L activation we 104 investigated basal Oas mRNA expression basal IFN signaling and viral induction of IFN as three 105 potential contributors Our data indicate that RNase L activation depends on relatively high basal 106 mRNA expression levels of Oas genes typical of myeloid cells and overexpression of RNase L is not 107 sufficient to overcome insufficient levels of OAS Furthermore RNase L activation requires adequate 108 basal IFN signaling to maintain basal Oas mRNA expression However in contrast to the current 109 paradigm RNase L activation does not require IFN induction during virus infection of macrophages 110 These data suggests that myeloid cells can activate RNase L early during infection and before the 111 induction of IFN and thereby limit viral spread to other cell types 112 113 114 MATERIALS AND METHODS 115 6 Viruses cell lines and mice Murine L2 fibroblasts were maintained in Dulbecco s modified 116 Eagle s medium DMEM supplemented with 10 fetal bovine serum FBS HEPES 10 mM and 117 1 penicillin streptomycin Plaque assays were performed on L2 cells as described previously 32 118 Murine 3T3 pLZ fibroblasts and control 3T3 neo 33 were grown in DMEM containing 10 FBS 119 supplemented with G418 350 g mL and 1 penicillin streptomycin The recombinant 120 coronaviruses inf MHV A59 wild type A59 referred to as A59 here and inf ns2 H126R referred to 121 as ns2 H126R here were obtained from Dr Stuart Siddell University of Bristol Bristol United 122 Kingdom and have been described previously 2 34 Newcastle disease virus expressing green 123 fluorescent protein NDV GFP 7 29 35 was obtained from Dr Luis Martinez Sobrido University of 124 Rochester School of Medicine C57BL 6 B6 mice were purchased from the National Cancer 125 Institute Frederick MD Rnasel mice bred for 10 generations to obtain a B6 background were 126 described previously 14 Ifih1 36 and Ifnar1 37 mice both with B6 background were obtained 127 from Dr Michael S Diamond Washington University in St Louis St Louis MO Both strains were 128 further bred and maintained in the animal facility at the University of Pennsylvania 129 130 qRT PCR RNA was isolated with an RNeasy minikit Qiagen Valencia CA Quantitative real time 131 reverse transcriptase PCR qRT PCR was performed as described previously 2 Briefly 200 ng 132 cells or 350 ng tissue of total RNA was reverse transcribed into cDNA using reverse transcriptase 133 Superscript III Invitrogen in a total volume of 20 L Then 2 L of cDNA was combined with 12 5 134 L of iQ5 SYBR green mix Bio Rad Hercules CA 6 5 L diethyl pyrocarbonate DEPC treated 135 water and 4 L primer mix 5 M each DNA was amplified using an iQ5 iCycler Bio Rad and 136 cycle threshold CT values were recorded Expression levels of mRNA were quantified as C T 137 values relative to actin mRNA with the equation 2 CT where C T C T Target Gene C T actin 138 Quantitative RT PCR primer sequences are available upon request 139 140 Primary cell cultures i Bone marrow derived macrophages BMM were generated from the hind 141 7 limbs of B6 WT Ifih1 or Ifnar1 mice as described previously 25 38 39 and cultured in DMEM 142 supplemented with 10 FBS and 30 L929 cell conditioned medium for 6 days before infection 143 Cultures were routinely 99 pure as assessed by positive staining for expression of CD11b and 144 negative staining for expression of CD11c ii Bone marrow derived dendritic cells BMDC were 145 generated from the hind limbs of WT mice as described by 40 41 and cultured in RPMI 1640 146 Sigma Aldrich supplemented with 10 FBS 2 mM L glutamine 1 penicillin streptomycin 50 M 147 mercaptoethanol and 20 ng mL of GM CSF Peprotech The cells were fed on day 3 6 and 8 148 with fresh media and supplements On day 10 cells were harvested and replated at 200 000 149 cells well using growth media with 5 ng mL GM CSF on 24 well non tissue culture treated plates and 150 used for infections Cells were routinely 95 CD11c and 80 to 90 immature MHCII lo iii 151 Hippocampal neurons were prepared from embryonic day 15 5 E15 to E16 mouse embryos as 152 described previously 42 Briefly cells were seeded onto poly L lysine coated tissue culture plates 153 cultured in neurobasal medium containing B 27 supplement Invitrogen 1 penicillin streptomycin 154 2 mM L glutamine and 4 g mL glutamate for 4 days in the absence of an astrocyte feeder layer 155 and then used for infections Neuron cultures were routinely 95 to 98 pure as determined by 156 positive immunostaining for MAP2 and negative immunostaining for CD11b microglia specific 157 marker glial fibrillary acidic protein GFAP astrocyte specific marker and OLIG2 158 oligodendrocyte specific marker 42 iv Mixed glial cultures consisting of astrocytes and 159 microglia were generated from the brains of 1 to 3 day old neonatal mice as describe previously 160 25 Briefly tissue was dissociated by mechanical disruption through a 70 m nylon mesh filter and 161 plated in complete medium consisting of DMEM supplemented with 10 FBS 1 nonessential 162 amino acid solution 2 mM L glutamine 1 penicillin streptomycin and 10 mM HEPES and cultured 163 for 9 to 11 days These cells were lifted from their culture vessel using the enzyme free Hank s 164 based balanced salt solution cell disassociation buffer Gibco v Astrocyte cultures were 165 generated as described in iv and after 9 to 11 days in culture the flasks were shaken to remove 166 nonadherent microglial cells and the remaining adherent cells were 95 pure astrocytes as 167 8 determined by positive immunostaining for GFAP 25 and used for infection The protocols were 168 approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania 169 170 Infections of cell cultures Virus was added to cells at a multiplicity of infection MOI of 1 PFU cell 171 and allowed to adsorb for 1 hour at 37 C Cultures were washed with PBS 3 times and fed with 172 medium as described for each cell type The culture supernatants were harvested at the times 173 indicated for the specific experiments and the titers were determined by plaque assay on L2 cells 174 175 Treatment with IFN and 2 5A Cells were treated with 100 units mL universal IFN for four hours 176 Cells were transfected with HPLC purified 10 M 2 5A p3A3 in 3 ug mL Lipofectamine 2000 177 Invitrogen or Lipofectamine alone and four hours later cell lysates harvested and RNA isolated 178 179 IFNAR1 blocking antibody treatment BMM cultures were treated with 0 2 or 5 g mL of 180 IFNAR1blocking mAb clone MAR1 5A3 BD Sciences or an isotype control purified NA LE mouse 181 IgG 1 Clone107 3 BD Sciences for one hour at room temperature with gentle agitation before virus 182 infection or mock infection 43 183 184 Bioassay for antiviral activity Supernatants recovered from cells that were infected with MHV 185 strains at an MOI of 1 PFU cell were exposed to 600 mJoules cm 2 UVA light in a Stratalinker 1800 186 Stratagene to inactivate the virus L2 mouse fibroblasts were treated with the UV inactivated 187 supernatants for 24 hours and then infected with NDV GFP at an MOI of 1 PFU cell as described 188 previously 35 Control cells were treated with 100 U mL universal IFN Quansys Biosciences UT 189 for 24 hours before NDV GFP infection At 12 and 24 hours post infection cells were fixed in 190 Dulbecco s phosphate buffered saline Gibco containing 4 paraformaldehyde and examined for 191 enhanced GFP EGFP expression under an Eclipse TE2000 U fluorescence microscope Nikon 192 9 Instruments Inc Images were acquired using NIS Elements Basic Research microscope imaging 193 software Nikon Instruments Inc 194 195 IFN quantification IFN protein in supernatants of MHV infected BMM was quantified with a 196 commercial capture enzyme linked immunosorbent assay kit VeriKine Mouse Interferon Beta ELISA 197 Kit PBL Laboratories Piscataway NJ according to the manufacturer s instructions 198 199 Ribosomal RNA degradation assay For quantification of rRNA cleavage total RNA from virus 200 infected cells was isolated using an RNeasy kit Qiagen and quantified using a Nanodrop analyzer 201 Equal amounts of RNA were separated on RNA chips and analyzed with an Agilent 2100 202 Bioanalyzer Agilent Technologies as described previously 25 44 RNA integrity numbers referred 203 to as RIN values 45 a measurement of RNA integrity produced by the bioanalyzer are also 204 indicated 205 206 Immunoblotting Cells were treated with 0 or 100 units mL of universal IFN for four hours and 207 then lysed in nonidet P 40 NP 40 buffer 1 NP 40 2 mM EDTA 10 glycerol 150 mM NaCl and 208 50 mM Tris pH 8 0 containing protease inhibitors Roche Protein concentrations were measured 209 using a DC protein assay kit Bio Rad Supernatants were mixed 1 1 with 2X SDS PAGE sample 210 buffer Samples were boiled separated by 10 SDS PAGE and transferred to polyvinylidene 211 difluoride PVDF membranes Blots were blocked with 5 nonfat milk and probed with the following 212 antibodies directed against OAS1A clone E 2 Santa Cruz 1 200 OAS2 clone G 9 Santa Cruz 213 1 200 OAS3 clone D 7 Santa Cruz 1 200 mouse RNase L goat polyclonal T 16 Santa Cruz 214 1 200 mouse RNase L rabbit polyclonal 1 1000 46 human RNase L mouse monoclonal 215 against human RNase L 1 1000 11 as well as anti GAPDH HRP Abcam 1 4000 anti tubulin 216 HRP Abcam 1 4000 Goat anti mouse IgG 2a HRP Santa Cruz 1 4000 goat anti mouse HRP 217 10 Santa Cruz 1 5000 donkey anti goat HRP Santa Cruz 1 5000 and donkey anti rabbit IgG HRP 218 GE Healthcare 1 10000 secondary antibodies were used to detect the primary antibodies of the 219 appropriate species The blots were visualized using Super Signal West Dura Extended Duration 220 Substrate Thermo Scientific In Figures 4F and 5D blots were probed sequentially with antibodies 221 directed against OAS1 OAS2 OAS3 RNase L and GAPDH with blots being stripped between 222 antibody treatments In Figures 2D and 3B parallel gels were run and blotted with individual 223 antibodies All immunoblots were performed at least twice 224 225 Quantifying IFNAR1 surface expression BMM and mixed glial cultures were stained with 226 antibodies against GFAP BD clone 1B4 CD11b eBioscience clone M1 70 F4 80 Biolegend 227 clone BM8 and the type I interferon receptor IFNAR1 Biolegend clone MAR1 5A3 or an isotype 228 control Biolegend clone MOPC 21 Staining for GFAP was conducted following permeabilization 229 with the Cytofix cytoperm Plus Fixation Permeabilization kit BD Cells were analyzed with an LSR II 230 Becton Dickinson and resulting data was analyzed using FlowJo Software Treestar Astrocytes 231 GFAP CD11b F4 80 and microglia GFAP CD11b F4 80 from mixed cultures and macrophages 232 GFAP CD11b F4 80 from bone marrow derived cultures were assessed for surface expression of 233 IFNAR1 Biolegend clone MAR1 5A3 or an isotype control Biolegend clone MOPC 21 234 Fluorescence intensity dependent on both surface IFNAR1 density and cell surface area 235 was normalized by cell size using previously described methods 47 This allowed us to compare 236 receptor density between cells of different sizes Briefly forward scatter FSC a measure of cell 237 volume and side scatter SSC a measure of cell granularity were used in a linear least squares 238 regression model to determine fluorescence intensities corrected for cell size and shape Residuals 239 from the model represent the variability in fluorescence that is not due to cell size and cell 240 granularity These residuals were offset by the sample specific average fluorescence intensity to 241 calculate the final values Calculations and analysis were performed with MATLAB Mathworks 242 Natick MD 243 11 244 Statistical analysis Plotting of data and statistical analysis were performed using GraphPad Prism 245 software GraphPad Software Inc CA Statistical significance was determined by the unpaired two 246 tailed Student s t test 247 248 RESULTS 249 Activation of RNase L during ns2 H126R infection of dendritic cells correlates with high basal 250 Oas gene expression 251 We showed previously that while A59 antagonizes the OAS RNase L pathway in myeloid 252 lineage cells including brain resident microglia and liver resident Kupffer cells as well as bone 253 marrow derived macrophages BMM 2 25 29 34 ns2 H126R a mutant expressing an inactive 254 phosphodiesterase PDE fails to antagonize RNase L and consequently has severely restricted 255 replication in these cell types In contrast replication of ns2 H126R in primary cell types including 256 neurons astrocytes and hepatocytes as well as in immortalized cell lines was as robust as A59 257