UC Irvine

The innate immune system is the first line of defense, providing a rapid, broad-spectrum defense during viral infections. Innate immunity relies on pattern recognition receptors (PRRs) to detect pathogen-associated molecular patterns (PAMPs), the unique molecular patterns found in pathogens, and mount appropriate downstream immune responses. There are multiple PRRs, which allow cells to recognize a wide range of viruses. Upon activation, PRRs initiate a complex cascade of signaling pathways involving numerous adaptor proteins, kinases, and transcription factors to orchestrate the expression of inflammatory genes encoding cytokines, chemokines, and other anti-viral immune proteins important in inhibiting viral replication and propagation. Meanwhile, viruses continuously evolve strategies to evade the host’s innate immune detection, bringing additional pressure to develop multiple layers of protective mechanisms. Moreover, the innate immune system must be tightly regulated to prevent its aberrant activation by distinguishing between self and non-self molecules. Given the complexity of the innate immune system, it is essential to thoroughly investigate the roles and regulatory mechanisms of immune factors. Therefore, the aim of this dissertation is to elucidate novel regulators and their functions within various pathways of innate immunity.

Chapter 1 focuses on identifying a novel regulator in the OAS-RNase L pathway. OAS-RNase L is an important antiviral pathway triggered in response to diverse viral infections. Upon double-stranded RNA binding, OAS enzymes synthesize 2’-5’ linked oligoadenylates (2-5A), leading to the activation of latent endonuclease RNase L. Once activated, RNase L leads to rapid decay of host and viral RNAs and shut down host translation machinery. As many viruses have developed strategies to counteract different steps of the OAS/RNase L pathway, it is crucial to fully characterize the regulators involved in this signaling cascade to understand how viruses evade our innate immunity. To this date, whether there are additional factors other than OAS3 and RNase L is not well known. I developed CRISPR-Translate, a FACS-based genome-wide CRISPR-Cas9 knockout screening method that uses translation levels as a readout and identified IRF2 as a key regulator of OAS3. I demonstrated that IRF2 promotes basal expression of OAS3 in unstressed cells, allowing a rapid activation of RNase L following viral infection. Furthermore, IRF2 cooperates with the interferon response through STAT2 to further enhance OAS3 expression after viral infection. I propose that IRF2-induced RNase L is critical in enabling cells to mount a rapid antiviral response immediately after viral infection, serving as the initial line of defense and giving host cells the necessary time to activate additional antiviral signaling pathways, forming secondary defense waves.

Chapter 2 demonstrates a novel function of APOBEC3B (A3B) in protecting the assemblies of stress granules (SGs) during viral infections. A3B belongs to the family of cytosine deaminase APOBEC3 (A3) family that counteracts viruses, primarily by inducing mutations in the viral genomes. Interestingly, A3s have been shown to restrict viruses independently of their genome editing role. However, the different mechanisms of how A3 members can suppress viral replication without deaminase activity are poorly understood and need further investigation to fill the gap in knowledge on how innate immunity protects cells against viruses. During viral infection, the activation of RNase L is crucial for limiting viral replications by causing widespread RNA decay and shutting down host translation machinery, but this activity of RNase L simultaneously results in SGs disassembly, which appears to be counterproductive for cells. In this study, I demonstrated that A3B is recruited to SGs to counteract RNase L activity and protects the mRNAs associated with SGs from RNase L-induced RNA cleavage during viral infection. This study reveals a novel deaminase-independent function of A3B as a modulator of SGs structures in response to viral infections.

Chapter 3 identifies the regulation of APOBEC3A (A3A) expression during viral infection and cancer mutagenesis. Besides mutating viral genome to inhibit viral replication, A3A is capable of deaminating the human genome and has been shown to be one of the major causes of APOBEC mutational signatures found in patient’s tumors. However, the mechanisms by which viral infection and tumors trigger A3A expression are still poorly understood. In this study, I find that both viral infection and genotoxic stress transiently up-regulate A3A and pro-inflammatory genes using two distinct mechanisms. STAT2-mediated interferon pathway promotes A3A expression in response to foreign nucleic acids through RIG-I/MAVS signaling cascade while DNA damage and replications stress independently activate the NF-κB pathway to induce expression of A3A and other innate immune genes. This study reveals different types of stressors that lead to the expression of A3A and the mechanisms of regulations during viral infections and cancer mutagenesis.