Characterizing novel molecular regulators of antiviral gene expression

Abstract

The intracellular innate immune response to viral infection is among the first lines of defense against these pathogens. For the early establishment of an antiviral cellular state and initiation of inflammatory responses, type I interferons (IFNs) are particularly important, as they potently induce the production of hundreds of IFN-stimulated genes (ISGs), many of which have antiviral functions. The type I IFN response requires tight molecular coordination to achieve both efficient production of antiviral proteins and controlled shutoff of inflammatory responses to avoid tissue damage and autoimmunity. Despite the importance of regulation of this antiviral response, current knowledge of the molecular controls governing its activation and suppression remains incomplete. Further, although ISGs have diverse functions and are induced to differing potencies, our understanding of regulatory controls governing the expression of individual or subclasses of ISGs is limited. Current knowledge of type I IFN response regulation is predominantly centered on transcriptional and post-translational regulatory controls. However, post-transcriptional regulation of antiviral responses has begun to emerge as an important layer of control. An example of these post-transcriptional regulatory controls is the RNA base modification N6-methyladenosine (m6A), which regulates many aspects of mRNA metabolism through transcript-specific effects. m6A deposition is mediated by a cellular complex of proteins including METTL3 and METTL14 (METTL3/14) and other cofactors, and m6A can also be removed from RNA by the demethylase proteins FTO and ALKBH5. The presence of m6A on viral and host RNAs has been shown to influence the outcome of infection by diverse viruses. However, the role of m6A in the response to type I IFNs has not been explored. To investigate the role of m6A in the type I IFN response, we began by manipulating m6A levels in the transcriptome through perturbation of the expression of the cellular m6A machinery and measuring the induction of ISGs after IFN treatment. We found that depletion of the m6A methyltransferase proteins METTL3 and METTL14 (METTL3/14) resulted in less protein production of a subset of ISGs, including the antiviral genes IFITM1 and MX1, after IFN treatment. However, the expression of other ISGs and the overall activation of the IFN responses were unchanged. Using methyl RNA immunoprecipitation and sequencing (meRIP-seq), we found that the transcripts of many ISGs are modified by m6A, and these included the METTL3/14-regulated ISGs IFITM1 and MX1 that we had identified. Using polysome profiling and ribosome profiling, we determined that METTL3/14-regulated ISGs are translationally enhanced by METTL3/14. Additionally, ablation of putative m6A sites within the 3’UTR of IFITM1 decreased the translation of a reporter molecule. Overexpression of the m6A reader protein YTHDF1, which has known roles in promoting translation, enhanced the expression of IFITM1 in an m6A binding-dependent fashion. These experiments characterized METTL3/14 and m6A as novel enhancers of the type I IFN response. To determine whether m6A contributes to type I IFN-mediated viral restriction, we depleted or overexpressed METTL3/14 and pretreated cells with a low dose of IFN-β prior to infection with vesicular stomatitis virus (VSV). Interestingly, METTL3/14 depletion decreased the expression of ISGs and allowed increased VSV infection, while METTL3/14 overexpression had the opposite effect. Together, these studies demonstrate that METTL3/14 and m6A enhance the antiviral effect of type I IFN by promoting the translation of ISGs to support the establishment of an antiviral cellular state. Having discovered a role for m6A in the type I IFN response, we also investigated the role of an m6A demethylase protein, FTO. FTO polymorphisms can have profound effects on human health. Certain polymorphisms are associated with fat mass and obesity, cardiovascular disease, while others can cause growth retardation or embryonic lethality. However, the molecular functions of FTO and the cellular pathways that it affects are still not well characterized. We depleted FTO and measured the production of ISGs following IFN-β treatment and found that the production of m6A-regulated ISGs was increased, as expected. However, unexpectedly, we found that FTO depletion increased the mRNA levels of a subset of ISGs. Pulse labeling of nascent transcripts revealed that FTO suppresses the transcription of these ISGs and that FTO-depleted cells are primed for the production of certain ISGs in response to IFN. We then used cells lacking PCIF1, the writer of 2’-O-N6-dimethyladenosine (m6Am), an RNA modification that FTO can also remove, and found that FTO-mediated regulation of ISGs occurs independently of the m6Am modification. These results identify FTO as a transcriptional regulator of a subset of ISGs, which will add an important dimension to our understanding of the molecular functions of FTO and its contributions to inflammatory disease. Future research revealing the mechanisms by which FTO suppresses ISG transcription will be of great interest. Together, these data identify novel functions of m6A and its related cellular machinery in both positive and negative regulation of the type I IFN response and antiviral gene expression.

Description

Provenance

Citation

Citation

McFadden, Michael J (2020). Characterizing novel molecular regulators of antiviral gene expression. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/22189.

Collections


Dukes student scholarship is made available to the public using a Creative Commons Attribution / Non-commercial / No derivative (CC-BY-NC-ND) license.