A role for the HSV1 UL13 protein kinase in regulating the interferon response
Posted on February 14, 2022 by Microbiology Society
Each year, the Microbiology Society awards a number of grants that enable undergraduates to work on microbiological research projects during the summer vacation and provide early and mid-career scientists the opportunity to gain supervision experience. Over the next few months, we’ll be posting a series of blogs from members who were awarded Harry Smith Vacation Studentships in 2021. This week is Research Fellow Dr Emma Wise, University of Surrey and undergraduate student Rowan Casey, University of York.
From the student: Rowan Casey
My project investigated the effect of the Herpes Simplex Virus 1 (HSV1) UL13 protein on the cGAS-STING signalling pathway and consequently its effect on the expression of Type 1 interferon and Interferon Stimulated Genes. Key to this project was a UL13 knockout HSV1 mutant.
By infecting human cells with wildtype and UL13 knockout viruses, we extracted samples for Reverse Transcription quantitative Polymerase Chain Reaction (RTqPCR) and Western Blotting. We carried out RTqPCR of the transcripts for Interferon Stimulated Genes IFIT1 and IFIT2 and Western Blotted for unphosphorylated and phosphorylated components of the cGAS-STING signalling pathway, namely IRF3, Phospho-IRF3, STING and Phospho-STING. We then used a luciferase reporter assay of the Interferon-β promoter in cells transfected with cGAS and STING coding plasmids to measure the effect on Interferon-β expression of introducing different concentrations of a UL13-expressing plasmid. I was thrilled to present these results at the weekly laboratory meetings.
During my studentship, I also had the opportunity to develop practical skills outside the scope of the project by carrying out an immunofluorescence staining experiment of IRF3 and ICP4. This investigated the nuclear localisation of these proteins in cells infected with different strains and mutants of HSV1. We used this to produce preliminary results on the differences in the extent of IRF3 translocation to the nucleus when infecting with different strains and mutants of HSV1.
We found that UL13 knockout HSV1 has significantly more expression of the Interferon Stimulated Genes IFIT1 and IFIT2 compared to wildtype viral infections. UL13 knockout and wildtype virus-infected cells have no significant difference in the phosphorylation of STING and IRF3. In a luciferase reporter assay, UL13 significantly decreased the expression of the IFN-β reporter. Therefore, we hypothesize that UL13 reduces the expression of Interferon Stimulated Genes in vitro via reduced IFN-β expression. This suggests UL13 is involved in downregulating the innate immune response against HSV1, via a currently unknown mechanism.
With the current SARS-Cov-2 pandemic, I have had less than 12 hours to learn virology laboratory techniques at university. Thus, it is quite understandable I started this studentship with next to no confidence in my practical abilities. However, by the end of the project, I felt on the path to becoming an independent scientist, being able to be given objectives and carry them out in the lab (in some cases alone), a testament to just how quickly you can gain confidence with the right support. The laboratory techniques taught by Dr Wise will allow me to seek new opportunities in my development as a research scientist.
In my time at the University of Surrey, I was tasked with writing a short literature review, a new skill for me, which was then subjected to rigorous scrutiny. It was highlighted to me the importance of precision in my wording, something I will take forward to my dissertation and to papers I will (hopefully) write in the future. Keeping my first professional lab book has given me an understanding of how to ensure my experiments can be easily replicated.
From the supervisor: Emma Wise
As an early career researcher aspiring to become an independent researcher, gaining a track record in funding acquisition is essential for me to progress in my career. This award, combined with the experience of grant writing, has helped me develop in this manner. I also hoped to develop my leadership and supervisory experience via the studentship. I was able to do this by acting as the student’s primary supervisor; I gained experience in setting and managing workload, training in laboratory techniques, giving feedback on both written and practical work, and troubleshooting results. I found it a rewarding experience to see the student develop from day one through the final weeks of the project, and to hopefully contribute positively to their career development.
Applications for our 2022 scheme are now open and will close on 20 February 2022. Find out more and apply on our website . For any questions, please contact [email protected].