The Rift Valley Fever Virus (RVFV) is an RNA virus identified as a category A pathogen by the National Institute of Allergy and Infectious Diseases (NIAID) because of its devastating effects on both livestock and human populations. In ruminants, RVFV causes "abortion storms," in which up to 100% of infected pregnant animals abort their pregnancies. Within Africa, the Rift Valley Fever Virus has had a high economic impact of up to $470 million in losses. Globally, RVFV is listed by the World Health Organization as a virus that is likely to cause the next global pandemic.
The right open reading frame kinase (RIOK) family consists of three proteins: RIOK1, RIOK2, and RIOK3. Both RIOK1 and RIOK2 are conserved from archaea to humans, but RIOK3 is exclusively found in multi-cellular eukaryotes, including humans. During infection with the Rift Valley Fever Virus, the protein RIOK3 is required for creation of the antiviral interferon (IFN) response within the human body. RIOK3 is an atypical serine/threonine kinase. However, the biological function of RIOK3 is largely unknown, making it a ‘dark’ kinase.
RIOK3 is hypothesised to bind ubiquitin via a domain in the N-terminus of the protein; however, any further detail is unknown. Structural studies employing native and intact mass spectrometry, cryo-EM, small-angle x-ray scattering and circular dichroism spectroscopy reveal new insights into RIOK3's conformation in solution. Interaction studies using analytical ultracentrifugation, crosslinking mass spectrometry, kinase activity assays and surface plasmon resonance identify potential ubiquitin binding sites and the specific ubiquitin chains preferred by RIOK3. Through this research, we aim to elucidate the structure and function of RIOK3, providing new knowledge on its ubiquitination mechanisms.