The mechanistic basis for the ZAP antiviral system targeting viral and cellular RNAs

ZAP is an antiviral protein found in most vertebrates that restricts a diverse range of viruses by binding viral RNA to inhibit its translation into viral proteins and target it for degradation. A full understanding of how ZAP inhibits viruses can be used to develop new therapeutic applications. Introducing ZAP binding sites in viral RNA could increase ZAP-mediated inhibition to create new live attenuated virus vaccines. Another application is to introduce ZAP binding sites into viruses which selectively replicate in tumours to kill the cancer cells and trigger immune responses. Several types of cancer, including liver, colon and bladder, have lower ZAP expression than adjacent non-cancer tissue and this is associated with poor disease progression and survival. ZAP-restricted oncolytic viruses may be a good treatment for these types of cancer since they will replicate in the cancer cells more efficiently than the surrounding healthy tissue.

However, to exploit these possibilities, we need a better understanding of how ZAP inhibits viral replication. First, it is unclear how ZAP selects which viral RNAs to bind to. Therefore, we will characterise how ZAP specifically binds target RNA and use this understanding to introduce optimal ZAP binding sites into viral RNA to increase their sensitivity to ZAP-mediated inhibition. Second, while ZAP interacts with many cellular proteins, only a few proteins are known to regulate its antiviral activity. To identify proteins that promote or inhibit its activity, we will use genetic screens based on a comprehensive list of its interacting partners. Third, it is unclear where in the cell ZAP acts to inhibit virus gene expression. We will use cutting edge microscopy techniques to visualise how ZAP inhibits a virus.

While ZAP inhibits a broad range of viruses, for the purposes of this grant we will use three medically relevant viruses to characterise the ZAP antiviral pathway. We will analyse how ZAP inhibits HIV-1, whose worldwide spread has caused the AIDS pandemic and represents a straightforward system to analyse ZAP function; SARS-CoV-2, which causes COVID-19 and for which novel therapeutics are needed; and influenza A virus, which causes seasonal respiratory disease and periodic pandemics with severe disease and requires new vaccination strategies to be developed. Overall, understanding how ZAP inhibits viral replication will allow us to potentially develop these new therapies.

ZAP is an antiviral protein that inhibits a broad range of viruses. It binds viral RNAs to inhibit their translation and target them for degradation. One application of the ZAP antiviral system is to introduce ZAP-response elements through synonymous genome recoding into viral RNA. This has at least two potential applications. First, it could be used to develop live attenuated virus vaccines by introducing ZAP-response elements into viruses that have evolved to supress these sequences in the viral RNA, which is common in many human RNA viruses. Second, several types of cancer have decreased ZAP expression and ZAP-sensitive oncolytic viruses could be a novel treatment for these cancers since they would preferentially replicate in the cancer cells compared to the healthy adjacent tissue.

However, there are several knowledge gaps for how ZAP inhibits viral replication and these need to be overcome to fulfil the therapeutic potential of this antiviral system. While ZAP directly binds a CpG dinucleotide, other unclear sequence characteristics are also required for ZAP to target a specific RNA. We will use a combination of in vitro and in vivo binding experiments to define optimal ZAP-response elements and test them using synonymous genome recoding with a focus on influenza A virus. While ZAP interacts with a large number of proteins, only a few have been functionally validated to promote or inhibit ZAP antiviral activity. We will identify and characterise novel ZAP cofactors or regulatory proteins. Finally, how ZAP localises to specific cytoplasmic membranes or compartments to mediate antiviral activity is unclear and we will use live cell imaging technologies to characterise how ZAP traffics and localises in the context of a viral infection. Overall, we will analyse how ZAP selects specific target RNA, which proteins it interacts with to inhibit viral replication and how subcellular localisation controls ZAP antiviral activity.