Distinct Antiviral Signatures Revealed by the Magnitude and Round of Influenza Virus Replication in Vivo

Distinct antiviral signatures revealed by the magnitude and round of influenza virus replication in vivo

Louisa E. Sjaastada,b,1, Elizabeth J. Fayb,c,1, Jessica K. Fiegea,b, Marissa G. Macchiettod, Ian A. Stonea,b, Matthew W. Markmana,b, Steven Shend, and Ryan A. Langloisa,b,c,2

aDepartment of Microbiology and Immunology, University of Minnesota, Minneapolis, MN 55455; bCenter for Immunology, University of Minnesota, Minneapolis, MN 55455; cBiochemistry, Molecular Biology and Biophysics Graduate Program, University of Minnesota, Minneapolis, MN 55455; and dInstitute for Health Informatics, University of Minnesota, Minneapolis, MN 55455

Edited by Michael B. A. Oldstone, The Scripps Research Institute, La Jolla, CA, and approved August 8, 2018 (received for review May 9, 2018) Influenza virus has a broad cellular tropism in the respiratory tract. virus cannot spread; therefore, any differences in viral abun- Infected epithelial cells sense the infection and initiate an antiviral dance will be a direct result of replication intensity. Infection of response. To define the antiviral response at the earliest stages of mice revealed uninfected cells and cells with both low and high infection we used a series of single-cycle reporter viruses. These levels of virus replication. These populations exhibited unique viral probes demonstrated cells in vivo harbor a range in magni- ISG signatures, and this finding was corroborated through the tude of virus replication. Transcriptional profiling of cells support- use of a reporter virus capable of specifically detecting active ing different levels of replication revealed tiers of IFN-stimulated replication. This suggests that the antiviral response is tuned to gene expression. Uninfected cells and cells with blunted replica- the level of virus replication to generate a response appropriate tion expressed a distinct and potentially protective antiviral to the level of threat. To understand how the antiviral response signature, while cells with high replication expressed a unique and tropism change from the first to second wave of replication reserve set of antiviral genes. Finally, we used these single-cycle we sequentially infected mice with viruses incapable of spread- reporter viruses to determine the antiviral landscape during virus ing. This strategy uncovered differential protection of ciliated spread, which unveiled disparate protection of epithelial cell epithelial cells mediated by IFN. These data demonstrate that subsets mediated by IFN in vivo. Together these results highlight epithelial cells supporting high or low levels of replication in vivo

the complexity of virus–host interactions within the infected lung display tailored antiviral responses and that protection afforded MICROBIOLOGY and suggest that magnitude and round of replication tune the by IFN is not equal among all cell types during virus spread. antiviral response. Together these findings demonstrate the complexity of virus– host interactions in vivo and illustrate how the cellular response influenza virus | interferon-stimulated gene | viral tropism is tuned to the level and round of replication. Results nfluenza A virus (IAV) drives significant morbidity and mor- Itality worldwide each year. IAV has a broad cellular tropism in Single-Cycle Infection Reveals IAV Replication Heterogeneity in Vivo. the respiratory tract with the ability to infect many epithelial cell To determine the infected cell landscape during the first round types (1). Rig-I–like receptors detect virus in epithelial cells, of IAV replication in vivo we engineered a reporter virus resulting in the production of type I and III interferons (IFNs) incapable of disseminating. The hemagglutinin (HA) ORF of and other proinflammatory cytokines (2). IFNs act through autocrine and paracrine signaling pathways to induce the pro- Significance duction of IFN-stimulated genes (ISGs), which promote a general antiviral state. Several individual ISGs have been identified that Influenza A virus has a broad cellular tropism in the respiratory perturb IAV at multiple stages of the viral life cycle. For example, tract. Infected epithelial cells sense the infection and initiate an IFITM3 blocks entry, Mx disrupts the IAV polymerase, and PKR antiviral response. Here, we used single-cycle replication re- inhibits viral protein synthesis (3–7). The induction of an antiviral porter viruses to analyze the early cellular response to in- state can also be driven directly by virus replication, independent fluenza infection in vivo. This approach revealed distinct tiers of IFN signaling (8–10). It is unknown how the level of virus of antiviral responses that were associated with the magnitude replication within a single cell affects the induction of global of virus replication. We also unveiled disparate protection of cellular responses. Even in the presence of a robust antiviral re- epithelial cell types mediated by interferon during virus spread. sponse, some infected cells continue manufacturing new viruses These results demonstrate the early landscape of virus–host and naive cells still become infected. How the antiviral response interactions in vivo with the magnitude and round of replica- alters tropism during virus spread has not been determined. tion revealing distinct antiviral signatures and responses. Studies aimed at determining the cellular response to IAV infection have been performed by exploiting powerful genetic systems Author contributions: L.E.S., E.J.F., and R.A.L. designed research; L.E.S., E.J.F., J.K.F., M.G.M., and I.A.S. performed research; M.G.M. and S.S. contributed new reagents/analytic (CRISPR, RNAi, yeast two hybrid, etc.) in vitro or by assessing tools; L.E.S., E.J.F., I.A.S., M.W.M., S.S., and R.A.L. analyzed data; and L.E.S., E.J.F., and bulk infected tissue in vivo (7, 11–13). While these analyses have R.A.L. wrote the paper. been critical for evaluating host factors that support or inhibit IAV, The authors declare no conflict of interest. the understanding of the complex interplay between different cell This article is a PNAS Direct Submission. types, anatomical locations, and immune responses in the context Published under the PNAS license. of virus infection in vivo is still incomplete. Single-cell analyses can Data deposition: The data reported in this paper have been deposited in the Gene Ex- help bridge this gap and have demonstrated the heterogeneity in pression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. IAV replication and the antiviral response in vitro (14–16). Un- GSE112794). fortunately, current single-cell mRNA-seq strategies using WT vi- 1L.E.S. and E.J.F. contributed equally to this work. rus cannot distinguish between newly infected cells and cells in 2To whom correspondence should be addressed. Email: [email protected]. which replication has been controlled in vivo. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. To overcome these limitations, we engineered a reporter virus 1073/pnas.1807516115/-/DCSupplemental. to specifically label cells in the first round of replication. This

www.pnas.org/cgi/doi/10.1073/pnas.1807516115 PNAS Latest Articles | 1of6 Downloaded by guest on October 2, 2021 IAV was replaced by mCherry while preserving complete HA found similar ratios of mCherry low to high cells in both cell vRNA 3′ and 5′ packaging signals and virus grown in a HA- types (SI Appendix, Fig. S1 E–H), suggesting that heterogeneity complementing cell line (17, 18). The virus cannot produce de novo in early virus replication levels is not driven by cell type. HA protein and assemble new virions that can infect other cells Multiple IAV particles can infect a single cell, which could [thus termed single-cycle IAV (scIAV)]. Infection of mice revealed drive differences in fluorescence intensity between cells. To ad- three distinct populations of lung epithelial cells: those with no, low, dress this, mice were infected with a mixture of scIAV-mCherry or high mCherry fluorescence (Fig. 1A). The heterogeneity in and scIAV-GFP. At 24 hpi there was only a small percentage of mCherry expression suggests that virus polymerase activity dur- mCherry-GFP double positive cells (Fig. 1C). The difference in ing the early stages of scIAV infection varies from cell to cell. fluorescence intensity between the low and high populations is ∼ SI Appendix I Both low and high mCherry populations were observed at similar 25-fold ( , Fig. S1 ), further suggesting that infection ratios at both 12 and 24 hours postinfection (hpi) (Fig. 1A) and with multiple particles is not driving the disparity. To determine required de novo virus polymerase activity (SI Appendix, Fig. S1 whether the range of replication is dependent on virus dose, mice + A and B). Both mCherry low and high CD24high and podoplanin were infected with a 10-fold lower inoculum of scIAV-mCherry and cells supporting low and high replication were still observed (pdpn) [ciliated epithelial cells and type I alveolar (TIA) cells, SI Appendix J respectively] were identified, suggesting cell type does not drive ( , Fig. S1 ). Prolonged presence of infectious particles in the lungs could replication heterogeneity (SI Appendix, Fig. S1C). result in varied time of infection and lead to replication disparity. Replication disparity could be driven by anatomical location This is unlikely, given the speed of IAV entry in vitro and that virus and/or proximity to other infected cells. To address this, mice dose and duration of infection did not impact heterogeneity in were infected with scIAV-mCherry and lungs were analyzed by single-cell analyses (15, 19, 20). However, the half-life of an in- fluorescence microscopy. We detected mCherry low and high fectious particle in vivo has never been experimentally determined. cells in both large and small airways and did not observe any We exploited the scIAV system to address this question. Because restriction based on proximity to other infected cells (Fig. 1B and SI Appendix D scIAVs cannot spread, only viruses that have not yet entered a cell , Fig. S1 ). To further define tropism during the first can be detected. Mice were infected with scIAV-mCherry and vi- round of infection, we determined the number of mCherry low rus titer from the lungs was determined at 6, 12, and 24 hpi. By and high club cells (CC10) and type II alveolar cells (SPC). We 6 hpi, only ∼6% of the virions delivered in the initial dose were detectable in the extracellular lung environment with a half-life of ∼1.7 h (Fig. 1D). This is consistent with mathematical modeling scIAV-ctrl 24 hpi scIAV-mCherry 12 hpi scIAV-mCherry 24 hpi experiments, which predicted a half-life between 0.6 and 3 h (21, A 22). Therefore, the vast majority of virions will have entered cells before the induction of the early innate immune responses.

SSC High Levels of Replication Reveal a Distinct Antiviral Gene Signature. To investigate the intracellular responses to variable levels of − − 0.005 1.86 2.19 IAV replication, we profiled the transcriptomes of CD45 CD31 mCherry negative, low, and high cells by mRNA-seq. Reads that B mCherry mapped to the IAV genome were markedly higher in the Naive scIAV mCherry 1 scIAV mCherry 2 mCherry high population compared with low or negative (Fig. 2A), validating the use of mCherry expression level as a surro- Dapi gate for virus replication. Failure to package or express all eight Low segments or internal truncations of segments can result in at- High tenuated replication (23–25). Normalized read counts between segments and read abundances across segments were similar between mCherry low and high cells (SI Appendix, Fig. S2 A and 6 B), suggesting that we are detecting bona fide infected cells. 60 CD0.64 0.087 10 Multidimensional scaling (MDS) of mouse transcripts demon- 50 5 10 40 strated significant differences between mCherry low and high cells across the first two dimensions (Fig. 2B). Importantly, the 30 + 4 GFP 10 20 negative population was derived from the same lung as mCherry 10 cells and was subjected to the same inflammatory environment,

# of reporter+ cells 3 97.5 1.73 10 0 making it an effective control for determining virus replication- % of starting inoculum 0 6 1218 24 specific gene signatures. Global changes in the transcriptional mCherry Low High hpi mCherry response of mCherry low and high compared with mCherry neg- total mCherry+ GFP+ ative were analyzed by gene ontology (GO) enrichment. mCherry low and high cells down-regulated many of the same pathways, Fig. 1. Heterogeneity in replication levels of IAV in epithelial cells in vivo. (A) Mice were infected with 105 pfu of scIAV-ctrl (Left) or scIAV-mCherry, and live primarily those involved in cell adhesion, extracellular matrix, and − − SI Appendix C CD45 CD31 cells were analyzed for mCherry expression at 12 (Middle)or24 development ( ,Fig.S2 ). Cell death pathways were (Right) hpi. Data are representative of 2 (12 hpi) or 10 (24 hpi) experiments increased in mCherry high cells, while DNA replication and cell with three to four mice per group. (B) Naive mice (Left)ormiceinfectedwith cycle pathways were up-regulated in mCherry low cells (SI Ap- scIAV-mCherry (Middle and Right) and analyzed for mCherry low and high pendix, Fig. S2C). To determine whether cell cycle was associated cells by histocytometry. Images are representative of two to three experi- with lower amounts of replication, we pulsed mice with BrdU, ments with three mice per group and two sections per lung. (C) Mice were infected them with scIAV-mCherry, and analyzed them at 24 hpi. coinfected with 105 pfu of scIAV-mCherry and 105 pfu of scIAV-GFP. Live + − − There was no increase in BrdU cells in the infected cell pop- CD45 CD31 cells were analyzed for GFP and mCherry expression 24 hpi + + + + ulation, and BrdU cells made up only 0.6% of the mCherry low (Left). Total mCherry , mCherry-high, mCherry-low, and GFP mCherry cells SI Appendix D were quantified (Right). Data are representative of three experiments with population ( ,Fig.S2 ), suggesting that entry into the n = 3–4 mice per group. (D)Micewereinfectedwith105 pfu of scIAV-mCherry cell cycle is not driving restriction of viral replication. and virus from lungs was titered on Madin-Darby canine kidney-HA cells at Cells with low or high levels of virus replication may differ- the indicated hpi. entially activate antiviral pathways. To determine the antiviral

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1807516115 Sjaastad et al. Downloaded by guest on October 2, 2021 + enhanced GFP (28). Due to this rapid degradation, any GFP cells A 7 B 10 naive detected in vivo must be the result of active virus replication (Fig. 1 3A). Mice were infected with scIAV-GFP or scIAV-destGFP, and 6 negative − − 10 low CD45 CD31 lung epithelial cells were analyzed for GFP ex- 5 high pression. Less than 15% of the total scIAV-GFP–infected epi- 10 thelial cells were detected by scIAV-destGFP, suggesting that + 4 0 many WT GFP cells are no longer supporting active replication 10 + (Fig. 3B and SI Appendix,Fig.S3A). GFP lung epithelial cells IAV reads (cpm) reads IAV 3 10 from mice infected with WT GFP- or destGFP-expressing viruses logFC dimension 2 were isolated and their transcriptomes profiled by mRNA-seq. As 2 + 10 -1 expected, destGFP cells contained more IAV mRNA than WT + GFP cells (Fig. 3C). Cellular transcripts were analyzed by MDS neg low high -2 -1 012 + naive and revealed significant differences in destGFP compared with mCherry logFC dimension 1 + WT GFP cells, primarily across the second dimension (Fig. 3D). + relative expression (logCPM) GO analysis of down-regulated pathways in the destGFP and WT C + GFP populations revealed the same cell adhesion, extracellular naive mCherry high mCherry low mCherry neg matrix, and development pathways that were down-regulated in 1 mCherry low and high cells (SI Appendix,Fig.S3B). This concordance suggests that cells specifically decrease certain sets of genes in response to IAV infection. To assess the antiviral response generated in cells supporting + active replication, we analyzed ISG expression in destGFP cells 2 − − 2 compared with destGFP , WT GFP , and naive cells (Fig. 3E). − 0 Cluster 1 ISGs were specifically induced in WT GFP and -2 − -4 destGFP cells. This expression pattern was similar to cluster 2 in the mCherry expression analyses (Fig. 2). We compared these

3 expression relative two clusters and found several overlapping genes (Fig. 3F), many

of which have been demonstrated to have direct antiviral activity MICROBIOLOGY against IAV (3, 26, 27, 29). There was also a cluster of ISGs that were only expressed in actively replicating cells (Fig. 3E, cluster 4 4b), analogous to cluster 4 in Fig. 3. We compared cluster 4b to the unique ISGs expressed in mCherry high cells and found a Fig. 2. Unique transcriptional signatures in cells supporting low and high high level of concordance (Fig. 3G). These data demonstrate levels of replication. Mice were infected with 105 pfu of scIAV-mCherry and live − − that cells supporting high levels of active virus replication express CD45 CD31 mCherry negative, low, and high cells were profiled by mRNA- a distinct set of ISGs that is not expressed in other infected cells. seq. (A) IAV cpm. (B) MDS of naive and mCherry negative, low, and high cells based on host mRNA reads. (C) Heatmap of 221 ISGs differentially expressed in Overall, our analyses using scIAV-destGFP recapitulated results the indicated populations. Cutoff of false discovery rate (FDR) is <0.05. obtained using scIAV-mCherry revealing distinct antiviral signatures in cells supporting active virus replication.

response, we analyzed the 221 differentially expressed ISGs and Tropism Is Altered by IFN During Virus Spread. During an infection, revealed several distinct groups of ISGs that varied with the level IAV spreads and new cells are infected despite a local antiviral of virus replication (Fig. 2C). Cluster 2 ISGs were highly induced and proinflammatory response. To label cells infected after the in mCherry negative and low cells but not in mCherry high cells. initiation of inflammation, we employed a sequential infection strategy using scIAVs with distinct fluorophores to model virus This cluster included several genes with known antiviral activity A against IAV, including Eif2ak2 (PKR), Trim56, and Pml (3, 26, spread in vivo (Fig. 4 ). Lung epithelial cells were analyzed for mCherry expression 24 h after the second infection. There was a 27). Cluster 4 was induced only in mCherry high cells (Fig. 2C), + significant decrease in the overall number of mCherry cells in suggesting that high levels of virus replication may produce a dis- the sequential infection, indicating that the first infection in- tinct antiviral response. Levels of Ifnar1 were similar in mCherry duced an immune response that conferred some protection (Fig. low and high cells (SI Appendix, Fig. S2E). Additionally, Mx1, 4 B and C). Interestingly, mCherry low and high cells were ob- which is IFN signaling dependent (8), was induced to a similar SI Appendix E served at similar proportions in single and sequential infection. degree in mCherry low and high cells ( ,Fig.S2 ), To determine whether there is a change in tropism during the + suggesting that differential extracellular IFN signaling alone may second round of replication, mCherry cells were analyzed for Irf7 not drive the disparity in ISG expression. Expression of was markers of ciliated epithelial cells and TIA cells (CD24high and Irf3 + + + similar in all infected cells while was decreased in mCherry pdpn , respectively). The frequency of mCherry pdpn cells was high cells, suggesting that induction of these critical sensors is not similar in single and sequential infection. However, there was a + driving the ISG disparity (SI Appendix,Fig.S2E). However, Ifnb significant decrease in the frequency of mCherry CD24high cells was higher in the mCherry high cells than in other cell populations in the sequentially infected animals compared with mice infected (SI Appendix,Fig.S2E). Overall, our data show distinct ISG sig- with scIAV-mCherry alone (Fig. 4D). To elucidate the mecha- natures within cells with low or high virus replication. nisms of protection during the second round of infection, we − − sorted CD45 CD31 mCherry negative, low, and high cells from Active Virus Replication Imparts Specific Antiviral Responses. We sequentially infected mice for mRNA-seq analysis. These data hypothesized that the ISGs uniquely expressed in mCherry high were compared with the single-infection data shown in Fig. 2. cells are specific to conditions of unchecked viral replication and MDS analysis of the host transcripts demonstrated significant may represent a “last resort” antiviral effort by the host. To differences between single and sequentially infected cells along identify cells harboring actively replicating virus, we developed a the second dimension (Fig. 4E). GO analysis of the genes sig- scIAV encoding destabilized GFP (destGFP). The half-life of nificantly down-regulated in both mCherry low and high se- this protein is only 2 h compared with over 24 h for the standard quentially infected cells demonstrated enrichment in several

Sjaastad et al. PNAS Latest Articles | 3of6 Downloaded by guest on October 2, 2021 A B scIAV GFP scIAV destGFP virus 24hrs GFP replication status

GFP had/has replication

dest SSC GFP no active replication

dest GFP active replication 0.73 0.10 GFP CD GFP cluster 1 7 Cd9 Ext1 10 1 6 Gak Fndc3b 10 F Rbm25 Map3k5 5 0.5 Wars Stat3 10 Optn destGFP+ Prkd2 4 destGFP- Rtcb 10 0 Irf2 Pi4k2b GFP+ Whamm 3 GFP- Trim56 Stard5 10 -0.5 Ifi27 naive Unc93b1 Bag1 IAV reads (CPM) reads IAV 2 Slfn5

LogFC Dimension 2 LogFC Rnf114 10 Lgmn Rab27a 1 -1 Isg20 Trim5 10 Nmi Sqle Birc3 Epas1 -1.5 Uba7 0 Spats2l Fam46a Gca Sp110 Fbxo6 -2 -1 0 1 2 Pml GFP- Oas3 Mov10 Ankfy1 naive GFP+ LogFC Dimension 1 Ddx60 destGFP- Tdrd7 Tmem51 destGFP+ Stat1 Ncf1 Herc6 Eif2ak2 Glrx Rnf213 Helz2 Sptlc2 Ddx58 Adar E relative expression (logCPM) Parp12 Lamp3 Fig. 3. Virus expressing destabilized GFP labels ac- Stat2 Pnpt1 Max Commd3 tively infected cells revealing distinct transcriptional

mCherry 2 cluster Steap4 Trim25 N4bp1 Ehd4 responses. (A) Model for the use of scIAV-destGFP to Irf9 Glipr2 identify cells with actively replicating virus. (B) Mice naive destGFP+ destGFP- GFP- Ifit1bl1 Eif3l infected with 105 pfu of scIAV-ctrl, -GFP, or -destGFP 1 Ifi211 Ccnd3 Gk and live CD45−CD31− cells analyzed for GFP expres- Chmp5 Alyref Ptma B4galt5 sion 24 hpi. Data are representative of three experiments with n = 3–4 mice per group. (C and D) Live 2 G − − + − GFP CD45 CD31 GFP and GFP cells were sorted and cluster 4b 2 Fam134b mRNA was mapped to the mouse and IAV genome. 1 Psmb8 0 Csrnp1 Ripk2 (C) Normalized IAV reads in each of the sorted cell 3 Gpx2 -1 Serpine1 Glipr2 populations. (D) MDS plot of the indicated pop- -2 Maff Cx3cl1 Arg2 Atf3 ulations based on mRNA reads. (E) Heatmap of the 4a -3 Sat1 Slc1a1 relative expression relative Lgals3 197 differentially expressed ISGs in the indicated Tnfaip3 Gem Ccl5 Lrg1 populations. FDR < 0.05 was used as a cutoff. (F) Rnf19b Cd74 Nup50 Marcksl1 Venn diagram of genes from mCherry cluster 2 in Fig.

mCherry 4 cluster Junb Pim3 2 and GFP cluster 1 in Fig. 3. (G) Venn diagram of Cdkn1a Max 4b Ddit4 Slc16a1 genes from mCherry cluster 4 in Fig. 2 and GFP Mcl1 Odc1 Ifngr1 Slc25a30 cluster 4b in Fig. 3. Only genes induced to >10 cpm in Atp10d at least one condition are shown.

pathways involved in ciliated epithelial cell maintenance and cell in vivo analysis also demonstrated heterogeneity in virus rep- development (Fig. 4F), further supporting the data in Fig. 4D. lication and antiviral responses, although the number of epithelial No pathways were significantly up-regulated between single and cells analyzed was low (33). Consistent with previous findings, our sequentially infected cells. We hypothesized that the first round data reveal heterogeneity in virus replication levels and the cellular of replication drives an IFN response that enhances the pro- response to IAV during the early stages of infection in vivo. While tection of ciliated epithelial cells, but not TIA cells. To test this, we cannot determine whether these cellular changes are a cause or mice were treated intranasally (i.n.) with IFNβ or IFNλ and 16 h a consequence of replication disparity, they reveal that the antiviral later challenged with scIAV-mCherry. TIA cells and ciliated signatures are not equal in all infected cells. epithelial cells were analyzed for mCherry expression. Both IFNβ IFN-I and -III drive the expression of hundreds of ISGs, which and IFNλ treatment led to a significant decrease in the frequency can vary by cell type (34). A landmark series of studies demon- of infected ciliated epithelial cells but did not protect TIA cells strated a paucity of individual ISGs that inhibit a diverse array of (Fig. 4G and SI Appendix, Fig. S4). These data suggest that viruses (35, 36). IRF1, Mx, and IFITM2/3 were among the few tropism is altered during virus spread through differential IFN- ISGs that were able to significantly blunt IAV infection and mediated protection in vivo. replication (35). Additionally, IAV grown in absence of ISGs tolerates more mutations, suggesting that multiple ISGs normally Discussion constrain the virus (37). We found a constellation of known anti- Multiple studies using single-cell sequencing have revealed het- IAV ISGs expressed in mCherry low and negative cells that were erogeneity in IAV replication and the antiviral response in vitro absent in cells supporting active or high levels of replication. (14, 15). These data are consistent with previous reports demon- While only correlative, it is interesting to speculate that these may strating stochasticity in single-cell responses to infection (16, 30– be protective combinations of ISGs, and failure to induce these 32). Importantly, single-cell sequencing following synchronized and genes permits high levels of replication. In addition to host factors, unsynchronized IAV infection revealed that duration and in- replication disparity could be in part due to mutations in the viral fectious dose are not responsible for the heterogeneity (15). Single- genome that enhance resistance or susceptibility to IFN. As such,

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1807516115 Sjaastad et al. Downloaded by guest on October 2, 2021 AB single single sequential 0.0027 0.0005 0.513 0.015 analysis 0 24 48

sequential GFP

analysis 0 24 48 hours post infection 98.1 1.92 98.1 1.33 C 6 10 mCherry p=0.0018 p=0.002 D 5 type-I alveolar cells ciliated cells 10 p=0.005 60 15 p=0.0006

4 10 40 10 # of mCherry+ cells 3 20 5 10

single single single 0 % CD24high of mCherry+ 0

sequential sequential sequential % podoplanin+ of mCherry+ mCherry mCherry mCherry single single total low high sequential E sequential single F mCherry low naive microtubule-based movement control microtubule-based process 1 negative cell projection organization Fig. 4. Tropism is altered by IFN during virus spread. low 051015 high fold enrichment (A) Model demonstrating sequential infection strategy. (B) Representative flow plot of A.(C) Numbers + MICROBIOLOGY 0 of total mCherry , mCherry low, and mCherry high sequential mCherry high cells in single and sequential infection. (D) Frequency

logFC dimension 2 control cilium morphogenesis + negative cilium assembly of total and mCherry CD24high ciliated cells and -1 low microtubule-based movement + high cell projection assembly pdpn type I alveolar cells. (E) MDS plot of single microtubule-based process infected mCherry negative, low, and high cells from cell projection organization Fig. 3 and mCherry negative, low, and high cells from 12-3 012 051015 logFC dimension 1 fold enrichment sequential infection. (F) Gene ontology enrichment G analysis (DAVID, biological process) of the down- 20 type-I 20 type-I 15 ciliated cells 15 ciliated cells regulated mCherry low and high genes that were significantly different (FDR < 0.05) from single to se- alveolar cells alveolar cells p=0.001 15 15 quential infection. Red indicates pathways overlapping 10 10 − − in mCherry low and high cells. (G)CD45CD31 cells 10 10 from mice treated with IFNβ and infected with scIAV- 5 5 mCherry analyzed for total (black) or infected (red) 5 5 + pdpn and CD24high cells. (B–D) Representative of five % total CD24high % total

% total podoplanin+ % total = – 0 0 0 0 experiments with n 3 4 mice per group. (G)Rep-  % CD24high of mCherry+ resentative of three experiments with n = 3–4mice % podoplanin+ of mCherry+    single IFN single IFN single IFN single IFN per group.

Du et al. (38) recently identified several IAV mutations that lead and MDA5 and could act as an additional level of regulation to to IFN sensitivity. Moreover, the basal expression of one or more prevent aberrant activation of proinflammatory ISGs. ISGs alone, or in combination with induced ISGs, could be driving Both immune and epithelial cells sense IAV in vivo (40), and the disparate levels of virus replication. mCherry high cells also responses can be dependent on the cell type. We have previously have increased levels of Ifnb expression compared with other demonstrated that club cells, which survive virus replication, populations of cells in the infected lung (SI Appendix,Fig.S2E). exhibit prolonged ISG signatures (41, 42). Our data demonstrate An alternative hypothesis is that autocrine IFN signaling alone, in that IAV tropism changes over the course of infection. Ciliated combination with high levels of replication or basal ISG levels, cells were afforded greater protection from secondary infection could drive the distinct ISG responses. compared with TIA cells. Importantly, this could only be dis- Our data reveal a distinct set of ISGs expressed in cells with covered through an in vivo scIAV sequential infection strategy. high or active virus replication. These ISGs may represent a re- While IFNβ and IFNλ can drive this selective protection, it is serve set of genes that are only turned on in cells that fail to blunt unknown whether IFN-mediated protection is direct or indirect replication. Interestingly, some of these genes are chemokines and through other epithelial or innate immune cells. other inflammatory mediators, which may help to orchestrate an Here we demonstrate heterogeneity in the levels of virus inflammatory response to control virus spread from these cells. It replication in vivo. Using two different virus reporters we reveal may be important to only express these genes when control of that distinct sets of antiviral genes are expressed in cells har- replication has failed to prevent immunopathology. In addition, boring low and high levels of virus replication. Through a se- pattern recognition receptor usage may be an underlying mecha- quential infection strategy we demonstrate that virus tropism is nism of this ISG signature. While RIG-I is thought to be the altered during virus spread where ciliated epithelial cells have primary sensor for IAV in epithelial cells, recent evidence dem- augmented protection from infection. These results demonstrate onstrates that MDA5 is important for the cellular response (39). a dynamic environment within tissues that is driven by both virus High levels of replication might be needed to activate both RIG-I replication levels and the infected cell type.

Sjaastad et al. PNAS Latest Articles | 5of6 Downloaded by guest on October 2, 2021 Materials and Methods and -N 1 parameters (46). To obtain significant differentially expressed Mice and Virus Infection. C57BL/6J mice were purchased from The Jackson genes, the experimental groups by design were compared with control Laboratory. Mice were infected i.n. with the indicated doses of scIAV. Ex- group (naive or negative) and the edgeR bioconductor package was used for periments involving mice were performed as dictated by the University of statistical analysis (47, 48). Sequencing data were deposited under GEO se- Minnesota Institutional Animal Care and Use Committee. ries accession no. GSE112794. Further details are provided in SI Appendix, SI Materials and Methods. Virus Rescue. Viruses were rescued in 293T cells by plasmid-based transfection with IAV PR8 in the pDZ vector (43, 44). The 5′ (106 bp) and 3′ (156 bp) packaging Microscopy and Histocytometry. Lungs were sectioned and stained with the signals were preserved, except AUG codons in the 5′ packaging signal which indicated antibodies. Images were obtained on a Leica DM6000B EPI fluo- were mutated. The designed gene of interest [mCherry, GFP, destGFP (pCAG- rescent microscope and analyzed using Imaris software. Further details are GFPd2) was a gift from Connie Cepko, Harvard University, Boston, Addgene provided in SI Appendix, SI Materials and Methods. plasmid no. 14760 (45), or Cre] was cloned in between the HA packaging signals. Further details are provided in SI Appendix, SI Materials and Methods. Statistics. Statistical analysis was executed using GraphPad Prism 7 software. Comparisons between two groups were performed using a two-tailed Stu- Flow Cytometry. Lungs were processed as described in SI Appendix, SI Ma- dent t test, and P < 0.05 was considered statistically significant. Error bars are terials and Methods. Single-cell suspensions were stained with a viability calculated using SEM. stain and with the indicated directly conjugated antibodies. Further details are provided in SI Appendix, SI Materials and Methods. ACKNOWLEDGMENTS. We thank Drs. Luis Martinez-Sobrido and Adolfo García-Sastre for reagents and Dr. Jason Mitchell and the Center for Immu- Next-Generation mRNA Sequencing Analysis. RNA was obtained and se- nology Imaging, the University of Minnesota Flow Cytometry Facility, and quenced as described in SI Appendix, SI Materials and Methods. Sequencing the Genomics Center for technical assistance. This work was supported by reads were mapped to the mouse (mm10) and influenza A/PR/8/34(H1N1) NIH K22 AI110581 and NIH R01 AI132962 (to R.A.L.), NIH T32 AI007313 (to genomes using Bowtie aligner (bowtie2 version 2.2.4) with local mode, -L 22 E.J.F.), and NIH T32 HL007741 (to J.K.F.).

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