Biology subjects

Amato, K. A.

Publications and source records attributed to Amato, K. A..

2 recordsLinked to original sources

bioRxiv2023

Pathogen-driven CRISPR screens identify TREX1 as a regulator of DNA self-sensing during influenza virus infection

Intracellular pathogens interact with host factors, exploiting those that enhance replication while countering those that suppress it. Genetic screens have begun to define the host:pathogen interface and establish a mechanistic basis for host-directed therapies. Yet, limitations of current approaches leave large regions of this interface unexplored. To uncover host factors with pro-pathogen functions, we developed a novel fitness-based screen that queries factors important during the middle-to-late stages of infection. This was achieved by engineering influenza virus to direct the screen by programing dCas9 to modulate host gene expression. A genome-wide screen identified the cytoplasmic DNA exonuclease TREX1 as a potent pro-viral factor. TREX1 normally degrades cytoplasmic DNA to prevent inappropriate innate immune activation by self DNA. Our mechanistic studies revealed that this same process functions during influenza virus infection to enhance replication. Infection triggered release of mitochondrial DNA into the cytoplasm, activating antiviral signaling via cGAS and STING. TREX1 metabolized the mitochondrial DNA preventing its sensing. Collectively, these data show that self-DNA is deployed to amplify host innate sensing during RNA virus infection, a process tempered by TREX1. Moreover, they demonstrate the power and generality of pathogen driven fitness-based screens to pinpoint key host regulators of intracellular pathogens.

microbiology↗

bioRxiv2021

Influenza A virus undergoes compartmentalized replication in vivo dominated by stochastic bottlenecks

Transmission of influenza A viruses (IAV) between hosts is subject to numerous physical and biological barriers that impose genetic bottlenecks, constraining viral diversity and adaptation. The presence of bottlenecks within individual hosts and their potential impacts on evolutionary pathways taken during infection and subsequent transmission are poorly understood. To address this knowledge gap, we created highly diverse IAV libraries bearing molecular barcodes on two independent gene segments, enabling high-resolution tracking and quantification of unique virus lineages within hosts. Here we show that IAV infection in lungs is characterized by multiple within-host bottlenecks that result in "islands" of infection in lung lobes, each with genetically distinct populations. We performed site-specific inoculation of barcoded IAV in the upper respiratory tract of ferrets and tracked viral diversity as infection spread to the trachea and lungs. We observed compartmentalized replication of discrete barcoded populations within the lobes of the lung. Bottlenecks stochastically sampled individual viruses from the upper respiratory tract or the trachea that became the dominant genotype in a particular lobe. These populations are shaped strongly by founder effects, with no evidence for positive selection. The segregated sites of replication highlight the jackpot-style events that contribute to within-host influenza virus evolution and may account for low rates of intrahost adaptation.

microbiology↗