Inhibition of viral replication by ZAP, TRIM25, KHNYN and CpG dinucleotides ZAP is an antiviral RNA binding protein that inhibits a broad range of viruses including retroviruses, alphaviruses, Ebola virus, hepatitis B virus and Japanese encephalitis virus as well as retroelements. TRIM25 and KHNYN are RNA binding proteins that interact with ZAP and are required for its antiviral activity against some viruses. The genomes of many viruses that infect humans, such as HIV, are suppressed in the nucleotide combination of a cytosine followed by guanosine (CpG). This indicates that CpG dinucleotides are detrimental to these viruses. Supporting this hypothesis, when CpGs are introduced into picornaviruses, influenza A virus or HIV, they inhibit viral replication. ZAP binds to regions of viral RNA containing CpGs and can target these RNAs for degradation. It may also have other mechanisms to inhibit viral replication that are poorly understood. Importantly, the introduction of CpG dinucleotides into viral genomes using synthetic biology techniques may be a new way to develop virus vaccines or create oncolytic viruses that preferentially kill cancer cells with dysregulated ZAP expression. A full understanding of how ZAP, TRIM25, KHNYN and CpG dinucleotides inhibit viral replication is necessary to develop these potential therapies. We are using a combination of cellular, molecular and genetic experimental approaches to analyse how ZAP, TRIM25, KHNYN and CpG dinucleotides inhibit viruses and regulate cellular gene expression in health and disease. Specifically, we are characterising: 1. How viral RNA sequence and structure affect ZAP, TRIM25 and KHNYN binding and antiviral activity. 2. The specific mechanisms by which ZAP, TRIM25, KHNYN and CpG dinucleotides inhibit viral gene expression and replication. 3. How cellular proteins that interact with ZAP, TRIM25 and KHNYN regulate their antiviral activity. 4. How ZAP, TRIM25, KHNYN and CpG dinucleotides regulate cellular gene expression in healthy cells and cancer cells. 5. How homologs of ZAP, TRIM25 and KHNYN regulate viral replication and cellular gene expression.