RESUMEN
Type II topoisomerases are ubiquitous enzymes that play a pivotal role in modulating the topological configuration of double-stranded DNA. These topoisomerases are required for DNA metabolism and have been extensively studied in both prokaryotic and eukaryotic organisms. However, our understanding of virus-encoded type II topoisomerases remains limited. One intriguing example is the African swine fever virus, which stands as the sole mammalian-infecting virus encoding a type II topoisomerase. In this work, we use several approaches including cryo-EM, X-ray crystallography, and biochemical assays to investigate the structure and function of the African swine fever virus type II topoisomerase, pP1192R. We determine the structures of pP1192R in different conformational states and confirm its enzymatic activity in vitro. Collectively, our results illustrate the basic mechanisms of viral type II topoisomerases, increasing our understanding of these enzymes and presenting a potential avenue for intervention strategies to mitigate the impact of the African swine fever virus.
Asunto(s)
Virus de la Fiebre Porcina Africana , Microscopía por Crioelectrón , ADN-Topoisomerasas de Tipo II , Virus de la Fiebre Porcina Africana/enzimología , Virus de la Fiebre Porcina Africana/genética , ADN-Topoisomerasas de Tipo II/metabolismo , ADN-Topoisomerasas de Tipo II/química , Animales , Cristalografía por Rayos X , Porcinos , Proteínas Virales/metabolismo , Proteínas Virales/química , Proteínas Virales/genética , Modelos Moleculares , Conformación Proteica , Fiebre Porcina Africana/virologíaRESUMEN
Junin virus (JUNV) causes Argentine hemorrhagic fever, a debilitating human disease of high mortality rates and a great risk to public health worldwide. Studying the L protein that replicates and transcribes the genome of JUNV, and its regulator Z protein should provide critical clues to identify therapeutic targets for disrupting the life cycle of JUNV. Here we report the 3.54 Å cryo-EM structure of the JUNV L protein complexed with regulator Z protein. JUNV L structure reveals a conserved architecture containing signature motifs found in other L proteins. Structural analysis shows that L protein is regulated by binding of Z protein at the RNA product exit site. Based on these findings, we propose a model for the role of Z protein as a switch to turn on/off the viral RNA synthesis via its interaction with L protein. Our work unveils the mechanism of JUNV transcription, replication and regulation, which provides a framework for the rational design of antivirals for combating viral infections.