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