Taylor Lab | Elucidating structure and mechanism ZAP antiviral response

A 2027 Crick PhD project with Ian Taylor.

Zinc finger antiviral protein (ZAP) is an interferon-stimulated gene that restricts a wide range of viruses including retroviruses, Ebola virus, hepatitis B virus and SARS-CoV-2[1]. It binds CpG dinucleotide arrays in viral RNA, constituting a ZAP-response element (ZRE), to target it for degradation. Many RNA viruses that cause human disease have supressed CpG abundance, partly due to selective pressure from ZAP.

By contrast, CpG suppression is absent in most insect-specific RNA viruses and ZAP may protect humans from some arthropod-borne viruses.

ZAP has three structural domains required for its antiviral activity: a zinc finger RNA-binding domain (RBD), a central ADP ribose binding domain, and a catalytically inactive ADP-ribosyl transferase-like domain[1]. The RBD binds a CpG in RNA through a pocket formed by a single zinc finger. However, RNA specificity may be more complex than CpG recognition alone, as local RNA sequence and structure may modulate ZAP binding together with an additional requirement for the E3 ubiquitin ligase Trim
25[1]. ZAP does not have enzymatic activity and must recruit the endoribonuclease KHNYN to degrade viral RNA[2]. KHNYN contains a di-KH domain of unknown function [3], an Mn-dependent RNA endonuclease exPIN domain[4], a CUE-like domain that binds ubiquitin and NEDD8, and a C-terminal nuclear export signal[2, 5]. Nevertheless, it remains unknown how ZAP, KHNYN and Trim
25 form an antiviral complex and interact with viral RNA to induce degradation.

Many therapeutic applications would benefit from a molecular understanding of ZAP restriction of viral replication including gene therapy and mRNA vaccines that would benefit from eliminating ZREs that inhibit their expression. Conversely, engineering ZREs into viral genomes not targeted by ZAP has been used to develop potential live attenuated virus vaccines. Additionally, ZAP is down regulated in certain cancers, at least in part because it controls the expression of apoptosis regulatory proteins.

Therefore, understanding how ZAP mediates selective RNA degradation may allow new cancer treatments to be developed.

In this project, the student will apply structural, biochemical and enzymological approaches to characterise KHNYN-ZAP-Trim
25 molecular complexes to analyse the regulation of nuclease activity, how ZAP recruits KHNYN and Trim
25 and determine how the ZAP-KHNYN-Trim
25complexes assemble on RNA. The student will purify ZAP, KHNYN and Trim
25 domains and full-length proteins to analyse complex formation, RNA-binding and RNA degradation. In addition, the student will also determine cryo-EM structures of ZAP-KHNYN, ZAP-KHNYN-RNA and ZAP-KHNYN-Trim
25 complexes. The structural and biochemical data from these studies will be used to make mutations in ZAP and KHNYN predicted to abrogate complex formation or nuclease activity that can be tested for their effects on inhibition of retrovirus replication.

This project would suit candidates with a background in molecular biology or biochemistry and an interest in structural biology and virology.

Please describe any experience you have in structural biology and how this can contribute to the understanding of cellular inhibition of viral infection.

1. Ficarelli, M., Neil, S.J.D. and Swanson, C.M. (2021)

Targeted Restriction of Viral Gene Expression and Replication by the ZAP Antiviral System.

2. Ficarelli, M., Wilson, H., Pedro Galao, R., Mazzon, M., Antzin-Anduetza, I., Marsh, M., . . Swanson, C.M. (2019)

KHNYN is essential for the zinc finger antiviral protein (ZAP) to restrict HIV-1 containing clustered CpG dinucleotides.

Structural and functional characterization of the extended-diKH domain from the antiviral endoribonuclease KHNYN.

KHNYN is a manganese-dependent endoribonuclease required for ZAP-mediated antiviral restriction.

5. Lista, M.J., Ficarelli, M., Wilson, H., Kmiec,

D., Youle, R.L., Wanford, J., . . Swanson, C.M. (2023)

A Nuclear Export Signal in KHNYN Required for Its Antiviral Activity Evolved as ZAP Emerged in Tetrapods.