Periodic Reporting for period 1 - INTERACT (SCRUTINIZING VIRUS-CELL INTERACTIONS: FROM ENSEMBLE TO SINGLE-MOLECULE STUDIES)
Período documentado: 2017-03-01 hasta 2019-02-28
Viral capsids are nanometer-sized containers made of proteins, whose main function is to encapsidate the viral genome, to transport it and to subsequently release it inside a new host cell. During infection, viruses are reproduced inside the infected host cell. These self-assembled capsids are kinetically in a metastable state, meaning that, on one hand they show a high degree of stability outside of the host cell to effectively protect the packaged genome. On the other hand, they become unstable after docking onto the receptors in the host cell, and this induced instability is an essential precursor to genome release. During the binding process the proteins in the viral capsid go through conformational changes which trigger a stability switch of the capsids. Quantitative information about these steps are currently lacking. In the proposed research (INTERACT 751404), I sought to investigate these steps using real time imaging at the nanoscale by High Speed-Atomic Force Microscopy (HS-AFM).
During the period of my MSCA Individual fellowship I had the opportunity to work in the Roos lab (Molecular Biophysics department) at the University of Groningen, the Netherlands. During that time, I extended my knowledge and expertise in the field of biophysics and nanotechnology, and became more experienced in team work and collaborative research. I was involved in several national and international collaborations, where I have learned a variety of biological and technological skills, boosting my own expertise in the field. Specifically, I studied virus mechanics, stability and dynamics. Sample preparation protocols for HS-AFM were optimized to achieve the needed high spatio-temporal resolution for the dynamic studies. Conformational changes in the capsid proteins of one type of virus upon binding of host proteins were captured at a video rate of 2 frame/second. The stability, disassembly and structural properties of another virus were also studied using HS-AFM.
The virus sample preparations were carried out through international collaborations. In the beginning of the project, I learned to work with the HS-AFM in the Roos lab and got experienced in this technique yielding already many new results and insights into the different biological systems. I also improved the HS-AFM performance to make even better use of the technique. After I had gained all this HS-AFM expertise, hands-on knowledge and insights in the host lab, we decided to go for a secondment in order to even further deepen my knowledge. For this, a short visit (one week) in the lab of Prof. Toshio Ando at the Kanazawa University, Japan was planned. After this successful visit, another visit (one month) in the same lab was carried out to focus on specific aspects of the HS-AFM instrumentation.
The scientific results were presented in different national and international conferences, namely, AFM Bio-med conference, 2017, Krakow, Poland (talk); Dutch Biophysics conference 2018 (poster); Dutch SPM day 2018, Utrecht University, The Netherlands (talk); Linz winter workshop 2017 (poster), 2018 (talk), Austria, and BPS annual meeting 2019, USA (poster). I received the Journal of molecular Recognition Young Investigator award, 2017 in the venue of AFM-BioMed conference, 2017, Poland.
In addition to experimental reporting, I was involved in other activities, for example, participation as guest lecturer and practical trainer to BSc. and MSc. students at the University of Groningen; supervising MSc. and PhD. students; engagement in academic and non-academic public interactions through lab visit, open-day, and public seminars and conferences.
In the quest to optimise the HS-AFM performance, I got an opportunity to work on a project in collaboration with Prof. Winfried Weissenhorn of Grenoble University, France, where I have studied the effect of VPS4 and the Endosomal Sorting Complex Required for Transport (ESCRT) machinery during the vesicle budding. In particular, the ESCRT III system and the AAA-ATPase VPS4 are recruited during membrane scission, but their exact functions were unknown. During HIV budding, specific ESCRT III subunits called CHMP 2A and 3 form helical filament in the budded neck. Previous studies from the Weissenhorn lab (Grenoble) showed that VPS4 complexes are capable of disassembling the filament. It remained however unclear how the ~60 nm diameter budded neck is able to constrict, finally leading to membrane scission. In this study, we have captured the action of VPS4 to constrict the ESCRT tube that can lead to the membrane scission. The work was published in Science Advances in 2019 (Apr 10;5(4):eaau7198).
The virus sample preparations were carried out through international collaborations. In the beginning of the project, I learned to work with the HS-AFM in the Roos lab and got experienced in this technique yielding already many new results and insights into the different biological systems. I also improved the HS-AFM performance to make even better use of the technique. After I had gained all this HS-AFM expertise, hands-on knowledge and insights in the host lab, we decided to go for a secondment in order to even further deepen my knowledge. For this, a short visit (one week) in the lab of Prof. Toshio Ando at the Kanazawa University, Japan was planned. After this successful visit, another visit (one month) in the same lab was carried out to focus on specific aspects of the HS-AFM instrumentation.
The scientific results were presented in different national and international conferences, namely, AFM Bio-med conference, 2017, Krakow, Poland (talk); Dutch Biophysics conference 2018 (poster); Dutch SPM day 2018, Utrecht University, The Netherlands (talk); Linz winter workshop 2017 (poster), 2018 (talk), Austria, and BPS annual meeting 2019, USA (poster). I received the Journal of molecular Recognition Young Investigator award, 2017 in the venue of AFM-BioMed conference, 2017, Poland.
In addition to experimental reporting, I was involved in other activities, for example, participation as guest lecturer and practical trainer to BSc. and MSc. students at the University of Groningen; supervising MSc. and PhD. students; engagement in academic and non-academic public interactions through lab visit, open-day, and public seminars and conferences.
In the quest to optimise the HS-AFM performance, I got an opportunity to work on a project in collaboration with Prof. Winfried Weissenhorn of Grenoble University, France, where I have studied the effect of VPS4 and the Endosomal Sorting Complex Required for Transport (ESCRT) machinery during the vesicle budding. In particular, the ESCRT III system and the AAA-ATPase VPS4 are recruited during membrane scission, but their exact functions were unknown. During HIV budding, specific ESCRT III subunits called CHMP 2A and 3 form helical filament in the budded neck. Previous studies from the Weissenhorn lab (Grenoble) showed that VPS4 complexes are capable of disassembling the filament. It remained however unclear how the ~60 nm diameter budded neck is able to constrict, finally leading to membrane scission. In this study, we have captured the action of VPS4 to constrict the ESCRT tube that can lead to the membrane scission. The work was published in Science Advances in 2019 (Apr 10;5(4):eaau7198).
A complete understanding of a viral life cycle is an essential task in virology. Different phases involved in the virus infectivity are often dynamic processes, which lead to the challenges in studying them. The proposed research was a step forward to make a unique platform for studying and capturing those dynamic stages using the advanced technique of HS-AFM. The results obtained during the period of INTERACT have/will have high impact in the scientific and non-scientific community. We are expecting to summarize the different results for publication in high impact journals (one result related to HIV budding is already published in a high impact journal).