DOI: 10.5061/dryad.gf1vhhmzd
title: Data from: Seoul orthohantavirus evades innate immune activation by reservoir endothelial cells

description: Pathogenic hantaviruses are maintained world-wide within wild,
 asymptomatic rodent reservoir hosts, with increasingly frequent human
 spillover infections resulting in severe hemorrhagic fever or
 cardio-pulmonary disease. With no approved therapeutics or vaccines,
 research has, until recently, focused on understanding the drivers of
 immune-mediated pathogenesis. An emerging body of work is now
 investigating the mechanisms that allow for asymptomatic, persistent
 infections of mammalian reservoir hosts with highly pathogenic RNA
 viruses. Despite limited experimental data, several hypotheses have arisen
 to explain limited or absent disease pathology in reservoir hosts. In this
 study, we directly tested two leading hypotheses: 1) that reservoir host
 cells induce a generally muted response to viral insults, and 2) that
 these viruses employ host-specific mechanisms of innate antiviral
 antagonism to limit immune activation in reservoir cells. We demonstrate
 that, in contrast to human endothelial cells which mount a robust
 antiviral and inflammatory response to pathogenic hantaviruses, primary
 Norway rat endothelial cells do not induce antiviral gene expression in
 response to infection with their endemic hantavirus, Seoul orthohantavirus
 (SEOV). Reservoir rat cells do, however, induce strong innate immune
 responses to exogenous stimulatory RNAs, type I interferon, and infection
 with Hantaan virus, a closely related hantavirus for which the rat is not
 a natural reservoir. We also find that SEOV-infected rat endothelial cells
 remain competent for immune activation induced by exogenous stimuli or
 subsequent viral infection. Importantly, these findings support an
 alternative model for asymptomatic persistence within hantavirus reservoir
 hosts: that efficient viral replication within reservoir host cells may
 prevent the exposure of critical motifs for cellular antiviral recognition
 and thus limits immune activation that would otherwise result in viral
 clearance and/or immune-mediated disease. Defining the mechanisms that
 allow for infection tolerance and persistence within reservoir hosts will
 reveal novel strategies for viral countermeasures against these highly
 pathogenic zoonotic threats.
description: The data collected here were generated from in vitro (cell
 culture) studies of virus replication and innate immune activation in rat
 and human endothelial cells. We collected microscopy images (tif, jpeg),
 western blotting images (tif, png), and RT-PCR analyses (excel,
 prism).  <strong>Viruses and <em>in
 vitro</em> infections</strong> Seoul virus
 strain SR11 and Hantaan virus strain 76-118 were propagated on Vero E6
 cells (ATCC, CRL-1586) for 12 days. Infectious virus was isolated by
 harvesting supernatant and centrifuging at 1000rcf for 10 minutes to
 remove cellular debris. Rat-passaged SEOV was generated using SR11 stock
 and propagated over three passages (MOI 0.01) in RLMVECs for ten days
 each. SEOV Baltimore strain, kindly provided by Dr. Steven Bradfute (UNM),
 was generated in Vero E6 cells and harvested 12 days post-infection.
 Sendai virus/52 was purchased from ATCC (Sendai/52, VR-105). For virus
 infections, cells were seeded in cell culture vessels 18-24 hours prior to
 infection at a target density of 70%. Virus stock was diluted to the
 target, cell-specific MOI/HAU using serum-free Dulbecco’s modified Eagle’s
 medium (DMEM, VWR 45000-304) supplemented with 1x pen/strep, 1%
 nonessential amino acids, 2.5% HEPES, and cells were infected for one hour
 at 37ºC. Cells were washed twice with sterile PBS solution
 (FisherScientific, SH30264FS), and appropriate culture medium was added
 for the duration of the experiment. For infected cell RNA experiments,
 Vero E6 cells were infected with SEOV for five days at MOI 0.5 prior to
 harvest in TRIzol reagent and subsequent RNA extraction. Percent SEOV N
 positive and infectious unit quantification experiment was performed using
 SEOV stock virus derived from Vero E6 cells, 1:2 dilution series in
 serum-free media was performed and cells were infected in a 96-well plate
 (Agilent, 204624-100) for 1 hour at 37ºC followed by addition of
 methylcellulose (Sigma M0512-500G) overlay with supplemented at 2x
 concentration DMEM (Gibco, 12100-046) supplemented with 2% FBS, 1x
 PenStrep, 1% HEPES. UV inactivated virus was treated with UV radiation
 (FisherBrand UV crosslinker, 13-245-221) until specific absorption of
 103.8 mJ/cm<sup>2</sup>. <strong>Cell
 Culture</strong> Vero E6 cells (ATCC, CRL-1586)
 and HEK293T (ATCC, CRL-3216) cells were cultured in Dulbecco’s modified
 Eagle’s medium (DMEM) supplemented with 10% heat-inactivated FBS, 1%
 pen/strep, 1% nonessential amino acids, 2.5% HEPES. Primary rat
 microvascular endothelial cells (RLMVEC, VEC Technologies) were cultured
 in MCDB-131 base medium (Corning) supplemented with EGMTM-2 Endothelial
 SingleQuots Bullet Kit (Lonza, CC-4176) and 10% heat-inactivated FBS.
 Human umbilical endothelial cells (HUVEC-C; ATCC, CRL-1730) were cultured
 in Lonza EGM-Plus (Lonza, CC-4542) supplemented with bullet kit (Lonza,
 CC-5036) and 10% heat-inactivated FBS. All endothelial cells were cultured
 in tissue culture-treated plastics coated with rat tail collagen (VWR,
 47747-218). HUVEC RLR CRISPR knockout lines were generated and validated
 previously (69). Recombinant rat IFNβ was purchased from R&D
 Systems (13400-1) and used at 10-150U/milliliter. RLMVEC knockout cells
 were generated using LentiCrispr V2 plasmid system (88) with gRNAs
 targeting RIG-I, MDA5, or Cas9 Scramble as control (Table 1). Briefly,
 HEK293T cells were transfected using ProFection Mammalian Transfection
 System (Promega, PAE1200) with targeted LentiCrispr V2 plasmid (Addgene
 Plasmid #98290), pSPAX (Addgene #12260), and p-VSVG (Addgene #138479). One
 day post-transfection, fresh DMEM was added to HEK293T cells. Virus was
 harvested two days post-transfection from HEK293T supernatant, filtered
 through a 0.45μm filter, and added to RLMVECs for 24 hours. Single
 knockout cells were placed under puromycin (VWR, AAJ67236-8EQ) selection
 at 1μg/milliliter for one week. RLR knockout cells were generated using
 the opposite RLR gRNA (e.g., RLMVEC RIG-I<sup>-/-</sup> with
 puromycin resistance, then treated with MDA5<sup>-/-</sup>
 gRNA with blastocydin resistance) in a LectiCrispr V2 plasmid encoding
 blastocydin selection and placed under selection (10μg/milliliter) for 1
 week. Knockout efficacy was verified via treatment with rat recombinant
 IFNβ and immunoblot (<strong>Supplemental</strong>
 <strong>Fig S5</strong>).   
 
 <strong>Target</strong> 
 <strong>Domain</strong>
 <strong>gDNA
 Sequence</strong> 
 Non-target 
 N/A 
 GACGGAGGCTAAGCGTCGCAA 
 RIG-I 
 Exon 1 
 CAGCTATATGAGTTCCTGGC 
 MDA5 Exon
 2 TCCTGGATGTTCTTCGCCAA
 Table 1.
 gDNA sequences for RLMVEC CRISPR knockout lines.
 <strong>Focus Forming Unit Assay</strong>
 Infectious virus was quantified using immunostaining as
 previously described (60). Briefly, Vero E6 cells were infected with 100μL
 of culture supernatants and incubated for two hours at 37ºC. After
 incubation, a 2% carboxymethylcellulose overlay containing supplemented
 DMEM (2% pen/strep, 2% nonessential amino acids, 2% HEPES, 4%
 heat-inactivated FBS). Cells were incubated for seven days at 37ºC. After
 incubation, cells were fixed with 95% EtOH:5% Acetic Acid for 10 minutes
 at -20ºC. Cells were then probed for SEOV N (α SEOV nucleocapsid, custom
 generated with Genscript) or HTNV N (anti-HTNV nucleocapsid 76–118, BEI
 resources NR-12152) at room temperature for 2 hours or 4ºC overnight. HRP
 conjugated secondary antibodies (donkey α rabbit for α HTNV, Jackson
 Immunoresearch 711-035-152; or goat α mouse for α SEOV, Jackson
 Immunoresearch 115-035-003) were incubated for 2 hours at room
 temperature. Foci were then stained using Vector VIP Substrate Kit (Vector
 Laboratories, SK-4600) and counted under a light microscope to calculate
 titer. <strong>Immunofluorescence and Microscopy
 Analysis</strong> To calculate SEOV N positivity
 and cell-specific titers, Vero E6, HUVEC, and RLMVEC were cultured in
 96-well plates and infected with serial dilutions of SEOV as described in
 infection methods above. 24 hours post-infection, cells were fixed with
 95% EtOH:5% Acetic Acid solution for ten minutes at -20ºC then blocked for
 one hour in 3% FCS in PBS. SEOV N positive RLMVECs at 0.05, 0.1, and 0.25
 MOI timecourse were cultured in 6-well plates and fixed and blocked in the
 same manner as the 96-well plate assay. Cells were probed overnight at 4°C
 for SEOV N protein (custom, Genscript) at dilution 1:400 in 1xPBS and with
 secondary antibody AlexaFluor 555 goat a mouse (Thermo Fisher Scientific,
 A-31570) at dilution 1:400 for two hours at room temperature. Nuclear
 stain DAPI was used at 1:10,000 for ten minutes at room temperature
 (SeraCare, 5930-0006). High-content imaging was acquired on CellInsight
 CX7 High-content Analysis platform (Thermo Fisher Scienitific, CX7A1110)
 at 10x objective magnification using iDev software. SEOV N positive cells
 were quantified using the Spot Detector Applet in the iDev software.
 Regions of interest (ROI) were defined based on DAPI nuclear stain, and
 SEOV N positive cells were defined as having at least one spot within the
 ROI as thresholded relative to each cell type. Data were collected on a
 total of 25 fields per well for all cell types for 96-well plate, or 250
 fields per well for 6-well plates. Representative images were acquired on
 the EVOS FL Auto imaging system (Thermo Fisher Scientific, AMF7000) at 10x
 magnification for 96-well plate, or 20x magnification for MOI series
 6-well plate.<strong> </strong>RLMVECs assayed for Mx1/2/3
 expression during SEOV infection were fixed using 4% paraformaldehyde
 (VWR, 97064-606) solution in sterile PBS for 30 minutes at room
 temperature, then permeabilized with 0.01% Triton X-100 (VWR, 97062-208)
 and blocked for one hour in 3% FCS in PBS. Cells were treated with DAPI
 nuclear stain 1:1000, SEOV N nucleocapsid 1:400, or Mx 1/2/3 1:400 in PBS
 (Santa Cruz Biotechnology, sc-166412 AF488). Representative images were
 acquired via the EVOS FL Auto imaging system at 20x magnification (Thermo
 Fisher Scientific). <strong>Cell-specific Titer
 Calculations</strong> Cell-specific titers were
 calculated by determining the number of SEOV N-expressing cells for each
 cell type using a serial dilution of SEOV stock. This method is adapted
 from a recent publication by Menke et al. (61). Briefly, total number of N
 expressing cells was quantified as described above. 
 Titer was calculated for all replicate wells in each dilution for
 which the number of N-positive cells decreased two-fold, to match the
 inoculum dilutions. Replicates were averaged and then the calculated
 titers for each dilution condition were averaged to determine the titer on
 each cell type. Four replicates per dilution and &gt;3 independent
 experiments were performed for each cell type.
 <strong>RNA Methods</strong> Cells
 were lysed for RNA analysis using TRIzol Reagent (ThermoFisher Scientific,
 15596026). RNA used for infected cell RNA transfection was isolated using
 phenol:chloroform extraction following manufacturer protocol. RNA used for
 gene expression analysis was purified using Zymo Research Direct-zol RNA
 Miniprep Plus kit (VWR 76211-340) according to manufacturer’s
 instructions. RNA concentrations were quantified using a NanoDrop UV-Vis
 Spectrophotometer (ND-1000). cDNA was synthesized using Applied Biosystems
 High-Capacity cDNA Reverse Transcription Kit (FisherScientific, 43-688-14)
 by adding 400ng total RNA to the reaction mixture containing random
 primers following manufacturer guidelines. Real-time PCR and
 quantitative-Real-time PCR was performed using the SYBR Green PCR Master
 Mix (ThermoFisher Scientific, 4364346) and the primers described in Table
 1. In-house designed primers were purchased from Integrated DNA
 Technologies and TaqMan primer assays were purchased from ThermoFisher.
 Host antiviral ISG expression and SeV gene expression was normalized to
 rat Chmp2a mRNA, and fold change calculated as ddCt over mock infection.
 Viral RNA copies for SEOV were calculated using a DNA plasmid standard
 encoding the N gene and the noted primers during qRT-PCR. Results were
 visualized and statistically analyzed using GraphPad Prism software (Prism
 10). Synthetic polyinosinic:polycytidylic acid (poly(I:C)) was purchased
 from Sigma-Aldrich (MilliporeSigma, P0913-50MG). The hepatitis C virus
 (HCV) xRNA was <em>in vitro</em> transcribed from synthetic
 DNA oligonucleotide templates (Integrated DNA Technologies) using the T7
 MEGAshortscript kit (Ambion) as previously described (89-91). Following
 manufacturer protocol, transfections were completed using JetPRIME
 transfection reagent (VWR, 89129-924). 
 <strong>Protein Methods</strong>
 Cell lysates to be analyzed for protein expression by immunoblot
 analysis were harvested in protein lysis buffer and prepared as previously
 described (92). Briefly, protein was harvested in RIPA buffer and
 clarified through 25,000rcf centrifugation for 15 minutes at 4ºC. Protein
 was quantified via Pierce BCA Protein Assay Kit (ThermoFisher Scientific,
 23225). 15μg-30ug of protein was loaded per sample into a 10%
 polyacrylamide gel and, after denaturing electrophoresis, transferred to a
 0.45µm nitrocellulose membrane (VWR, 10120-006). Membranes were blocked at
 room temperature in 10% FCS in PBS-T. Primary antibodies (table 2) were
 incubated at 4º overnight. HRP-conjugated secondary antibodies against
 primary antibody species were incubated for 1 hour at room temperature.
 Blots were imaged on BioRad Chemidoc MP Imaging System using
 chemiluminescence Pierce Substrate for Western Blotting (VWR,
 PI80196). 
 <strong>Target Species</strong> 
 <strong>Protein</strong>
 
 <strong>Manufacturer</strong> 
 Rat 
 Mx 1/2/3 
 SantaCruz Biotech (sc-166412) 
 Rat 
 Mx 1/2/3 AF488 
 SantaCruz Biotech (sc-166412 AF488) 
 Rat 
 MDA5 GeneTex
 (GTX103138) 
 Rat RIG-I
 Cell Signaling Technologies
 (3743T) 
 Human, Rat 
 βActin SantaCruz
 Biotech (sc-47778) 
 Human Mx
 Cell Signaling (37849S)
 Human
 IFIT1 
 Abcam  (ab236256) 
 SEOV SEOV
 Nucleocapsid Custom,
 Genscript 
 Table 3. Primary antibodies used for protein detection in
 immunoblot and immunofluorescence assays.
 <strong>Densitometry Quantification of Immunoblots Using
 ImageJ</strong><br>Densitometry analysis of immunoblots was
 performed using ImageJ (NIH) to quantify protein band intensities.
 Immunoblot images were first converted to 8-bit grayscale. The rectangular
 selection tool was used to draw a box around each band, ensuring
 consistent box sizes across all bands. The area under the curve (AUC) for
 each band was measured using the “Analyze → Gels → Plot Lanes” function,
 which generates intensity profiles of the bands. The peak areas
 corresponding to each band were selected using the wand tool, and
 background intensity was subtracted from each lane. The relative band
 intensities were normalized to the loading control (β-actin) to correct
 for variations in protein loading. Data from presented experiments were
 reported as relative protein expression levels compared to
 β-Actin. <strong>Secreted cytokine
 analysis</strong> Tissue culture supernatant from
 HUVEC (Cas9 and RLR<sup>-/-</sup>) was collected four days
 post SEOV infection (MOI 0.01) and clarified for cellular debris by
 centrifugation (1000 rcf for 10 minutes). Clarified supernatants were
 UV-inactivated (specific absorption of 103.8
 mJ/cm<sup>2</sup>) and shipped to Eve Technologies for
 interrogation on the Human Cytokine/Chemokine Panel A 48-Plex Discovery
 Assay Array (HD48A). One experiment was performed with three biological
 replicates and two technical assay replicates.
 <strong>Statistical analyses</strong>
 Statistical significance was assess using GraphPad Prism software
 version 10. Where noted in figure legends, ANOVA and student’s T-test
 analyses were applied, with * representing adjusted p&lt;0.05. All
 comparisons reaching statistical significance are displayed on
 graphs.
description: # Seoul orthohantavirus evades innate immune activation by reservoir
 endothelial cells
 [https://doi.org/10.5061/dryad.gf1vhhmzd](https://doi.org/10.5061/dryad.gf1vhhmzd) ## Description of the data and file structure The data here represent the results of data collected to study the impact of hantavirus infection of cellular gene expression and protein abundance. This study was published in the scientific journal PLOS Pathogens. We collected data through real-time PCR analysis, western blotting, secreted cytokine analysis, and immunofluorescence. These represent the outcome of in vitro infections of human and rat endothelial cells with seoul orthohantavirus or hantaan orthohantavirus and are the foundation of the conclusions outlined in the published article. Files are organized into folders in accordance with published figures for the manuscript (Figures 1-7) and supplemental files (S1-S8). Within each Figure folder, the data are subfolders according to the data used to generate the figure panels (A-E) for each figure, as ordered in the manuscript. Finally, within those subfolders are further subfolders containing the raw data generated from each replicate experiment performed and presented in the figure panels, as presented in the article. These sub-subfolders are often named SDK or AMK followed by several numbers. Each initial-number combination represents an independent experiment completed by the authors which are numbered in the laboratory organization scheme. Abbreviations: CX7 – high-content microscopy imaging platform to analyze virus infections EVOS – fluorescence imaging microscope IFA – immunofluorescence RT-PCR – real-time PCR analyses to measure gene transcription WB – western blotting to measure protein expression     KO – crispr knockout cells ### Files and variables #### File: Data\_repository.zip **Description:** The folder contains subfolders of raw data used for analysis as presented in the publication. Subfolders are categorized by figure and panel within each figure as presented in the publication. ## Code/software ImageJ software can be used to quantify the density of bands for western blots to measure densitometry ## Access information Other publicly accessible locations of the data: * n/a Data was derived from the following sources: * n/a