The primary objective of this research project is to comprehensively characterize the protein composition of the intracellular replication complex (RC) associated with the yellow fever virus, a representative member of the Flavivirus genus. This study aims to compare the interactomes of replication complexes across different species and identify cellular proteins crucial for viral replication. The significance of this project is underscored by the recently ended pandemic, emphasizing the critical need for a deeper understanding of viral replication processes to accelerate drug development. Currently, the intricate protein composition of the RC, the intracellular structure responsible for replicating the viral genome, remains understudied for many mammalian and human viruses. Our proposed methodology involves engineering recombinant yellow fever viruses, wherein specific non-structural proteins, namely NS1 or NS2A, are fused with the green fluorescent protein (GFP). These labeled proteins facilitate the infection of different cell lines, enabling the isolation and purification of RCs. Subsequent steps include the separation of intracellular fractions enriched in viral RCs using density gradient fractionation and immunoprecipitation of GFP-labeled proteins. The goal is to purity viral RCs through immunoaffinity chromatography conditions on anti-GFP sorbents. Furthermore, mass spectrometry-based identification of RC proteins will be conducted using liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). This approach, demonstrated using the yellow fever virus, presents an adaptable method that can be applied to various viruses. It opens avenues for the identification of intracellular targets crucial for the development of antiviral drugs. The distinctiveness of our proposed approach lies in its focus on the interactions between viral and cellular proteins within the RC. In contrast to existing literature that primarily emphasizes viral protein-protein interactions or relies on genetic approaches, our project suggests a mass spectrometry analysis of viral replication complexes from different virus/cell line combinations. This innovative approach will facilitate the identification of cellular proteins integral to the formation of the RC and essential for the viral replication process. Additionally, hydrogen exchange mass spectrometry will be employed in this project to provide dynamic insights into the structural stability of the viral replication complex proteins, offering a complementary approach that enhances our understanding of conformational changes critical for viral replication and potential drug targeting.
This study marks a step forward in advancing our understanding of virus-host interactions and may contribute significantly to the development of targeted antiviral interventions. From a socio-economic perspective, the outcomes of this research could contribute to the development of a knowledge-based economy in Kazakhstan. The knowledge and expertise gained from this project can catalyze the growth of a skilled workforce in the fields of virology, molecular biology, and advanced analytical techniques, fostering a culture of innovation and research within the country since these directions are not well-developed yet.
The primary objective of this project is to comprehensively characterize the protein composition of the intracellular replication complex (RC) associated with the yellow fever virus, compare interactomes in replication complexes functioning in different species, and identify cellular proteins essential for replication.
As a part of the project, the following steps will be carried out:
Pavel Tarlykov – Ph.D., Associate Professor, Head of the Laboratory of Proteomics and Mass Spectrometry of LLP “National Center for Biotechnology”.
Specialist in the field of genomics, proteomics and mass spectrometry, the author of more than 100 scientific papers, Hirsch index – 11 (Scopus). Studied in the master’s program “Bolashak” (USA, Montana State University, 2007-2009). Scholarship holder of the World Federation of Scientists (Switzerland, 2010). COmpleted internships in France (Institute Gustave-Roussy, 2011-2012), Russia (“Medical and Genetic center of N.P. Bochkov”, 2012). Hirsch index – 11 (Scopus), ResearcherID C-2587-2012, ORCID 0000-0003-2075-307, Scopus Author ID 35076539700.
Alexander Shustov – PhD in Biological Sciences, Head of the Genetic Engineering Laboratory at LLP “National Center for Biotechnology.” He has held research positions at the Department of Microbiology, University of Alabama (USA), and the Department of Microbiology and Immunology, University of Texas Medical Branch (USA), as well as at the State Research Center of Virology and Biotechnology “Vector” (Russia). Dr. Shustov is a specialist in the fields of genetic engineering of microorganisms, virology, bioengineering of expression systems in mammalian and bacterial cells, and recombinant protein production. Over the past five years, he has led projects focused on the development of virus-like particles and expression systems for antigens in the form of virus-like particles. Hirsch index is 12 (Scopus), ORCID ID: 0000-0001-9880-9382, and Scopus ID: 57211989685.
Sabina Atavlieva, researcher at the Laboratory of proteomics and mass spectrometry of the NCB, Master of Science in Natural Sciences. Hirsch index – 5 (Scopus), ORCID 0000-0002-7565-9454, Scopus Author ID 57204157988. Specialist in proteomics and mass spectrometry.
Dana Auganova – Junior Researcher at the Laboratory of Proteomics and Mass Spectrometry of the NCB, Master of Science in Natural Sciences. Specialist in molecular biology with expertise in proteomics, proteolysis, and HDX-MS. Hirsch index – 3 (Scopus), Scopus Author ID: 57367752100.