Elucidation of the Oropouche virus structure based on its protein interactions

Ketleen Lucas do Carmo1,2, Laís Durço Coimbra2, Alice Nagai2, Amanda Bellini Silva1,2, Ana Julia de Melo Couto1,2, Rafael Elias Marques 2, Talita Diniz Melo Hanchuk2

1. UNICAMP, Universidade Estadual de Campinas; Cidade Universitária \"Zeferino Vaz\" Barão Geraldo, Rua Roxo Moreira, N° 1402 Campinas-SP CEP: 13083-970
2. CNPEM, Centro Nacional de Pesquisa em Energias e Materiais; Rua Giuseppe Máximo Scolfaro, 10.000 – Polo II de Alta Tecnologia de Campinas, Campinas (SP).

The Oropouche virus (OROV), an arbovirus belonging to the Orthobunyavirus genus, has emerged as a growing public health threat in Brazil, with over 24,000 reported cases between 2024 and 2025, including fatalities and instances of vertical transmission. Despite its epidemiological relevance, the structural characterization of the OROV viral particle remains limited, hindering progress in the development of vaccines and antiviral strategies. The main obstacle to structural elucidation is its pleomorphic morphology, which prevents the effective use of conventional techniques such as X-ray crystallography and cryo-electron microscopy (Cryo-EM), commonly applied to icosahedral viruses like Dengue and Zika. This project aims to understand how protein-protein interactions drive the organization and assembly of the OROV viral particle, using crosslinking mass spectrometry (XL-MS) as the main experimental approach. The strategy includes viral purification, preliminary characterization by electron microscopy, application of chemical crosslinkers, and structural data analysis through computational modeling. Key interactions identified will be functionally validated by generating infectious OROV clones with site-directed mutations. As preliminary results, we have initiated the standardization of OROV purification using sucrose cushion ultracentrifugation, aiming to obtain suitable samples for crosslinking and XL-MS analysis. In conclusion, this doctoral project proposes to generate unprecedented data on the internal architecture and molecular interaction dynamics of OROV, overcoming the limitations imposed by pleomorphism and contributing to the structural virology of neglected arboviruses.
 

Agradecimentos: Agradeço à Universidade Estadual de Campinas e ao Instituto de Biologia (UNICAMP/IB) ao Centro Nacional de Pesquisa em Energia e Materiais e ao Laboratório Nacional de Biociências (CNPEM/LNBio). Agradeço ao Programa de Pós-Graduação em Genética e Biologia Molecular (PPG-GBM) e à Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). Ao Instituto Serrapilheira e à Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).