ANRS MIE - SFV 2024 thesis prizes awarded to Garima Joshi and Yang Yang

They answered our questions

What is your academic background? Why did you turn to research?

Garima Joshi : I did my Bachelor’s in Microbiology followed by a Master’s in Biomedical Science at the University of Delhi, India. I remember that I wanted to be a medical doctor as a child, but during my undergraduate studies, I realized that research is the backbone of medicine. I became fascinated by the cellular processes in health and disease, and it was clear that pursuing research offered a deeper understanding of these processes. Research, for me, is not just about solving scientific questions, but also requires creative thinking. The creative aspect of research, especially in terms of visualization and experimental design, has always attracted me.

I had learnt some French at school in India, but I had not imagined moving to France until I received the Charpak scholarship, which supported my research Master’s in Life Science and Chemistry at the Paris Sciences et Lettres university. The course I took up in France was application-based and involved keeping up with all the latest developments in the field. This balanced approach in practical and theoretical aspects of research that I developed in France and India, respectively, have collectively helped me solve scientific problems.

Yang Yang : After completing my master’s degree in China, the COVID-19 pandemic began to unfold. During this time, I gained a deep appreciation for the critical role of structural biology in understanding viruses and developing treatments. Driven by my passion for research, I applied to join the protein solid-state NMR group at the IBCP. With the support of a scholarship from the China Scholarship Council, I came to France to pursue my PhD. I was very fortunate to conduct my doctoral research under the joint supervision of Dr. Anja Böckmann (discover her portrait here) and Dr. Lauriane Lecoq, which has been an incredibly meaningful and rewarding experience

What is the subject of your thesis?

Garima Joshi : Hepatitis E virus (HEV) has been classified among the top 10 zoonotic viruses presenting the greatest risk of transmission to humans. We studied HEV gt3 that manifests an adverse clinical outcome in subjects with pre-existing medical conditions or suppressed immunity. Therefore, more information about the innate immune responses that control HEV infection would be instrumental in solving this burgeoning health crisis. Type I and III interferons protect the host against viral infections. Previous studies showed that only type III IFN response is triggered in hepatic cells upon hepatitis E virus (HEV) infection but not type I IFN. Type III IFN response alone is unable to stop viral spread. Therefore, we investigated the anti-HEV function of IFN secreted by plasmacytoid dendritic cells (pDCs), the specialized producers of type I IFNs. We showed that pDCs co-cultured with HEV-replicating cells secreted IFN in a cell-contact-dependent manner, much like other viruses. This pDC response depends on the endosomal nucleic-acid sensor TLR7 and adhesion molecules.

We also compared different HEV-replicating cell types for their immune response in terms of ISG and cytokine upregulation. One key finding of our study was the reduction in HEV spread mediated by the IFNs secreted by pDCs. The second part of our study focused on ORF2, the capsid protein of HEV, that can be produced in various forms by the infected cells. During infection, a fraction of ORF2 localizes into the nucleus, while another ORF2 fraction packages viral genomes in infectious virions. We showed that glycosylated ORF2 potentiates the recognition of infected cells by pDCs via regulation of cell contacts. We also explored the effect of ORF2 nuclear translocation on activation of cell immune response. Together, our results suggest that pDCs may be essential to control HEV replication.

Yang Yang : My doctoral research focused on understanding the structure and dynamics of the different forms of HDAg produced by cell-free synthesis (CFS) or in bacteria to study the HDV ribonucleoprotein (RNP), providing detailed structural information at atomic resolution.

What do you consider to be the most significant result of your thesis work?

Garima Joshi : The most striking result was the diminution of pDC IFN response to PLC3 cells replicating the STOP mutant and its complete abolition in case of 5R/5A mutant. The 5R/5A and the STOP mutant are deficient in the production of glycosylated (ORF2g/c) and nuclear ORF2, respectively. These observations suggested that ORF2 nuclear translocation and secretion of glycosylated ORF2g/c forms might modulate pDC response. We then hypothesized that this differential IFN response was probably regulated by the cell-contact formation propensity of the mutant replicating cells.

To put our hypothesis to test, we compared their pDC contact-forming propensity by confocal imaging. Only cells harboring the STOP mutant differed from wild-type HEV cells in their ability to form contacts with pDCs. These results support the hypothesis of a reduced attraction of pDCs towards STOP-expressing PLC3 cells, as compared to wild-type HEV replicating cells, due to the absence of ORF2g/c secretion. This suggests that ORF2g/c might be allowing the virus to disseminate by enhancing cell-cell contact formation, while concurrently enhancing its detection by immune cells. However, the underlying processes need to be studied further.

Yang Yang : One of the main results of my project was the successful determination of the 3D high-resolution solution NMR structure of S-HDAg^Δ60(residues 61-195), which has been deposited in the Protein Data Bank (PDB: 9FLG). In addition, my studies have characterised the N-terminal assembly structural domain (residues 1-60) of S-HDAg, a key component of the HDV RNP complex. Using CFPS and ¹H-detected fast magic angle spinning (MAS) solid-state NMR, we achieved de novo sequential assignment with less than 1mg of fully protonated protein.

Our results show that the N-terminal structural domain is the only rigid region of S-HDAg that remains fully conserved in the full-length protein, while the rest of the protein remains highly dynamic. These studies lay a crucial foundation for future investigations into the HDV RNP complex.

What would you suggest to continue your work? What do you see as the major challenges in this field?

Garima Joshi : The most pressing question would be how the ORF2g/c and nuclear ORF2 impact pDC viral sensing of HEV and must be investigated further. Additionally, we noted that absence of nuclear ORF2 results in complete abolition of pDC response to 5R/5A cells. This suggests that the nuclear ORF2 probably triggers certain immune pathways that might enhance sensing by pDCs. Therefore, investigating these nucleus-specific pathways triggered by viral nuclear proteins will also be quite useful in the context of other viruses. It is possible that some adhesion molecules are also interferon-stimulated genes, and consolidating these genes could provide valuable insights for the field of immunology. Therefore, a broader question to explore would be the impact of a robust immune response on immune cell recruitment and contact formation, as there is limited literature on this topic.

Our results also show that direct contacts mediated by ICAM-I and, at least in part, with α_Lβ₂-integrin, enable pDCs to sense and respond to HEV-replicating cells. In the field of pDC biology, a deeper understanding of other interactions occurring at the iterferogenic synapse could be highly beneficial. Moreover, elucidating the effects of IRF3 activation on pDC recruitment and contact formation could reveal new mechanisms of cross-talk between pDCs and infected cells.

Yang Yang : Structural research on the hepatitis D virus remains limited. Since S-HDAg is a very challenging protein to produce in vitro, the next step for future research on this project will focus on the NMR analysis of the HDAg^1-160 (residues 1-160) RNP complex. Resonance assignments of the additional peaks observed on RNP formation will allow to characterize the complex in more detail and reveal whether these signals belong to the assembly domain or the C-terminal S-HDAg^Δ60, thus further refining our understanding of the HDV RNP complex.

What are your career plans after your thesis?

Garima Joshi : Completing my thesis has significantly boosted my confidence, and I now feel that the sky is the limit. Since finishing my thesis, I have continued to pursue research, with a strong focus on expanding and diversifying my expertise. My goal is to bridge basic research with innovative therapies, translating scientific discoveries into practical solutions for patient care. France offers an outstanding ecosystem that fosters such innovation, providing the ideal environment for researchers to thrive. Over the course of my journey, I’ve honed my networking skills and developed a strong sense of interdisciplinary collaboration. I’m eager to build meaningful partnerships and forge lasting friendships to drive breakthroughs in the treatment of infections and diseases.

Yang Yang : I am currently exploring job opportunities in structural biology and biopharmaceutical research, both in academia and industry. I am particularly interested in roles where I can apply my expertise in protein characterization and drug development.