High-throughput Screening to Discover Host Factors Involved in Filoviral Entry.

摘要

Emerging infectious agents are responsible for death and disease worldwide and in all life forms. Humans strive to combat them by developing antibiotics, antivirals and a variety of drug treatments which unfortunately become obsolete as the targeted infectious agent rapidly evolves. Emerging diseases are often associated with high morbidity and mortality, as the human body is naive and susceptible to the new infection, and hospitals are not prepared with prophylactic and treatment options. In order to keep abreast with the challenges presented by emerging diseases, the focus of this dissertation is (1), to design and carry out a HTS that can efficiently and thoroughly identify host factors involved in viral infection, and (2), to develop our knowledge of the entry process of some of the world's most deadly pathogens, filoviruses. Implementing key factors into a HTS format including viral pseudotyping, RNAi, and a parallel screening technique allowed us to efficiently identify host factors that are utilized by the surface proteins of two highly pathogenic viruses, Avian Influenza virus and Marburg virus, on a genome-wide scale. This protocol is beneficial in that it alleviates the safety concerns of highly pathogenic viruses and also allows for a large-scale search for human factors utilized by the viruses during their infection process. The roles of the identified factors were investigated further for their mechanism of action, in hopes of finding new targets for antiviral development. EXT1, a primary player in the process of heparan sulfate biosynthesis, was identified as a Marburg-specific host factor involved in infection. Development of this finding led us to the discovery that heparan sulfate binds Marburg and Ebola surface glycoproteins during infection. Also, other GAGs, primarily heparin, can also interact with filoviruses and effectively block their infection in cell culture. This opens the possibility of heparin being utilized as a readily available option to combat filovirus infection.