Why do deoxy-didehydro-nucleotide triphosphates (ddhNTPs) accumulate in cells in response to viral infection?

Supervised by: Dr Liz Morris, Department of Biosciences, Durham University 

 The COVID-19 pandemic put the risk of viral infection on national and global health at the forefront of the general public's mind. The effects of COVID-19 on public health, economics, education, and social dynamics continue to affect many. However, as the immediate threat subsides, the focus has shifted to enhancing preparation for future health crises.

The COVID-19 vaccine was made over three years faster than any previous vaccine. A significant reason for this speed is years of prior research on related viruses². Research on families of viruses increases adaptability to unseen viral species such as Covid-19³. This demonstrates the importance of previous research and the need for future leaders in science to think ahead when planning their research.

My research focuses on understanding a cellular defence mechanism which inhibits viral replication for a family of viruses called retroviruses. Understanding this mechanism may be crucial for addressing future pandemics and epidemics and developing new therapies for existing challenges like HIV.

Viruses are microscopic agents that require a host, such as you or I, to replicate. They must integrate their genome into host genomes to hijack its cellular mechanisms. Retroviruses are a family of RNA viruses that must turn their RNA genome into DNA before integration into the host genome by reverse transcription. Deoxy-nucleotides are integral to DNA structure and are, therefore, required for reverse transcription. The enzyme SAMHD1 degrades these deoxy-nucleotides, preventing viral genome integration and subsequent replication⁴. Hosts use SAMHD1 as a defence mechanism against viral replication. 

DdhNTPs are synthesised by the enzyme Viperin from nucleotide triphosphates (NTPs) in response to viral infection and accumulate in cells⁵. The fact that they can accumulate in cells suggests that ddhNTPs can resist hydrolysis by SAMHD1. Deoxy-didehydro-cytosine triphosphates (ddhCTPs) have been shown to terminate RNA-dependent RNA polymerases, preventing viral replication in both Zika and SARS-CoV-2⁵. This antiviral effect may explain why ddhNTPs can resist hydrolysis. Harnessing natural defence mechanisms like this may encourage the development of new antiviral therapies and increase public health preparedness; therefore, understanding this mechanism is pertinent. 

SAMHD1 and Viperin are widely conserved enzymes with broad antiviral activity, making them important to study. ddhNTPs have low cytotoxicity despite having a similar structure to highly toxic nucleotide analogues. My research could help understand the toxicity of nucleotide analogues, which is valuable for developing safer antiviral and anticancer therapeutics.

Objectives:

• To understand the evolution of Viperin and SAMHD1 as antiviral factors by studying their activities in isolation and then in combination in vitro.

• To determine binding constants and thermodynamic parameters, providing measurable insights into the specificity and strength of interactions between ddhNTPs and SAMHD1, contributing to understanding how ddhNTPs resist hydrolysis by SAMHD1.
• To generate data highlighting distinctions between ddhNTPs and standard nucleotides with SAMHD1 contributing to understanding SAMHD1's selectivity. 

Hypothesis:

While SAMHD1 hydrolyses other nucleotides and antiviral nucleotide analogues, ddhNTPs resist hydrolysis by SAMHD1 and accumulate in cells, restricting viral replication. 

Methodology: 

Optimise conditions for SAMHD1 protein expression in E. coli and purify the protein. Conduct experiments that measure the breakdown rate of ddhNTPs and standard nucleotides with SAMHD1. Use techniques to study binding between SAMHD1 and ddhNTPs. Analyse the data collected to understand the mechanism of ddhNTP resistance and generate a report to summarise key findings and outline future directions. 

Timeline:

Week 1-2: I plan to express and purify SAMHD1 proteins in E. coli in the first two weeks using Ni-NTA, Mono S CE and Hydrophobic Interaction Chromatography⁶,⁷. I will confirm the enzymatic activity of purified SAMHD1 using standard nucleotides as substrates.

Week 3: Week 3 involves conducting SAMHD1 hydrolysis assays with ddhNTPs and standard nucleotides, quantifying hydrolysis rates via High-Performance Liquid Chromatography (HPLC) HPLC, and comparing hydrolysis rates between ddhNTPs and standard nucleotides.

Week 4: I'll use surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to study interactions between SAMHD1 and ddhNTPs. This will help me determine binding constants and thermodynamic parameters to understand the specificity of the interaction.

Week 5: I will analyse enzymatic kinetics and binding affinities to decipher the mechanisms of ddhNTP resistance. 

Week 6: I will write a comprehensive report summarising key findings and proposing future cell-based experiments in the final week.

1 Ball, P. (2020). The lightning-fast quest for COVID vaccines — And what it means for other diseases. Nature, 589(7841), 16-18. https://doi.org/10.1038/d41586-020-03626-1

2 List of Blueprint priority diseases. World Health Organization [Internet]. 2018 Jul 20 [cited 2024 Jan 11]; Available from: https://web.archive.org/web/20200301083134/http://origin.who.int/blueprint/priority-diseases/en/

3 Deutschmann, J., & Gramberg, T. (2021). SAMHD1 … and Viral Ways around It. Viruses, 13(3), 395. https://doi.org/10.3390/v13030395

4 Gizzi, A. S., Grove, T. L., Arnold, J. J., Jose, J., Jangra, R. K., Garforth, S. J., Du, Q., Cahill, S. M., Dulyaninova, N. G., Love, J. D., Chandran, K., Bresnick, A. R., Cameron, C. E., & Almo, S. C. (2018). A naturally occurring antiviral ribonucleotide encoded by the human genome. Nature, 558(7711), 610-614. https://doi.org/10.1038/s41586-018-0238-4

5 Seamon, K. J., Sun, Z., Shlyakhtenko, L. S., Lyubchenko, Y. L., & Stivers, J. T. (2015). SAMHD1 is a single-stranded nucleic acid binding protein with no active site-associated nuclease activity. Nucleic Acids Research, 43(13), 6486-6499. https://doi.org/10.1093/nar/gkv633

6 Morris, E. R., Caswell, S. J., Kunzelmann, S., Arnold, L. H., Purkiss, A. G., Kelly, G., & Taylor, I. A. (2020). Crystal structures of SAMHD1 inhibitor complexes reveal the mechanism of water-mediated dNTP hydrolysis. Nature Communications, 11(1), 1-14. https://doi.org/10.1038/s41467-020-16983-2