Cell-to-Cell Variation in Defective Virus Expression and Effects on Host Responses during Influenza Virus Infection

Abstract

Virus and host factors contribute to cell-to-cell variation in viral infections and determine the outcome of the overall infection. However, the extent of the variability at the single-cell level and how it impacts virus-host interactions at a system level are not well understood. To characterize the dynamics of viral transcription and host responses, we used single-cell RNA sequencing to quantify at multiple time points the host and viral transcriptomes of human A549 cells and primary bronchial epithelial cells infected with influenza A virus. We observed substantial variability in viral transcription between cells, including the accumulation of defective viral genomes (DVGs) that impact viral replication. We show (i) a correlation between DVGs and virus-induced variation of the host transcriptional program and (ii) an association between differential inductions of innate immune response genes and attenuated viral transcription in subpopulations of cells. These observations at the single-cell level improve our understanding of the complex virus-host interplay during influenza virus infection. IMPORTANCE Defective influenza virus particles generated during viral replication carry incomplete viral genomes and can interfere with the replication of competent viruses. These defective genomes are thought to modulate the disease severity and pathogenicity of an influenza virus infection. Different defective viral genomes also introduce another source of variation across a heterogeneous cell population. Evaluating the impact of defective virus genomes on host cell responses cannot be fully resolved at the population level, requiring single-cell transcriptional profiling. Here, we characterized virus and host transcriptomes in individual influenza virus-infected cells, including those of defective viruses that arise during influenza A virus infection. We established an association between defective virus transcription and host responses and validated interfering and immunostimulatory functions of identified dominant defective viral genome species in vitro. This study demonstrates the intricate effects of defective viral genomes on host transcriptional responses and highlights the importance of capturing host-virus interactions at the single-cell level.

Viral and host heterogeneity and their effects on the viral life cycle

“…The IAV genome consists of eight viral RNA segments that are not always expressed in an infected cell 13 , 58 , 59 . Failure to express all eight segments impairs viral mRNA transcription and the productivity of infection 12 , 13 , 17 , 60 , 61 . More recently, one study coupled single-cell transcriptomics with long-read PacBio sequencing, determining that two-thirds of cells were infected with IAV variants with mutations and deletions that lead to higher levels of immune activation than the wild-type variant 8 .…”

“…Yet DVG production decreases infectivity by competing with full-length genomes and robustly triggering the host antiviral response. DVGs also affect temporal variation of the host cell transcriptome 61 . The role of DVGs during infection remains unclear but may lie in providing diversity to viral populations with high recombination or reassortment rates, or in offering a link to the development of persistent infections.…”

Cell type- and replication stage-specific influenza virus responses in vivo

“…We have previously used a single-cycle influenza A virus (scIAV) expressing mCherry in place of the coding sequence for the hemagglutinin (HA) segment (ΔHA-mCherry) to uncover replication heterogeneity during the early stages of IAV infection in mice [14]. Other groups have found similar heterogeneity at early timepoints in vitro [15][16][17][18], as well as heterogeneity in the ability to induce IFN production in infected cells [18][19][20][21]. Our previous analyses were unable to distinguish genes induced directly by virus infection from those driven by IFN and inflammation.…”

Single cell heterogeneity in influenza A virus gene expression shapes the innate antiviral response to infection

“…Numerous studies have leveraged recent advances in single cell analysis methods to assess the extent of cellular heterogeneity present during infection by a variety of viruses, including IAV [8][9][10][11][12][13][14][15][16][17]. These studies connect to a larger body of work that has begun to explore single cell heterogeneity within different host cell populations and tissues [18][19][20].…”