Advanced cell-based biosensors for detection of label-free viral pathogens

• Autor(a) principal: Silva, Ana Catarina Martins Mandeiro da
• Data de Publicação: 2019
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/89378
• topic: Switch on sensor protease activated; activated; virus detection and quantification; Cre recombinase; adenovirus; chikungunya virus; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

The last decades have been marked by a rising number of diseases attributed to newly discovered viruses, as well as an increasing incidence of diseases caused by viral pathogens outside of their endemic areas. Simultaneously, interest on virus-based biopharmaceuticals (VBBs), for therapeutic or prophylactic ends, has increased dramatically. However, fundamental and applied research in virology depend on fast, reliable, and high-throughput approaches for detection and quantification of infectious viruses, which the most commonly used methods fail to deliver. Herein, we developed a novel cell-based sensing system using a switch-on Cre recombinase strategy. This modular strategy consists on a protease-sensing Cre recombinase (ProCre) and a reporter module. The latter is activated by Cre-mediated DNA recombination events, resulting in a signal output. Cre always-on activity was successfully controlled by structural distortion of a truncated Cre, harbouring a viral protease specific cleavable linker. Transient transfection results showed that Cre activity was effectively constrained, delivering a “vigilant state”. As proof-of-concept, sensor activation with tobacco etch virus protease indicated that Cre activity could be efficiently recovered upon specific proteolysis at the cleavable linker. ProCre capacity to detect proteolytic activity of clinically relevant human viruses was demonstrated with the human rhinovirus protease. Moreover, ProCre was also adapted to detect adenovirus, chikungunya and Zika viruses proteolytic activity. Although no substantial increase in GFP expression was observed, sensor activation upon proteolysis was confirmed. Further optimizations are currently ongoing to allow robust detection of these viruses. Overall, we developed a novel strategy to control the always-on nature of Cre recombinase, enabling its use as a protease-dependent sensor. The versatility of this system further allows its adaptation to detect other proteases, either viral or cellular, providing a permanent and robust output signal of interest.