A bioinformatics approach to identifying novel genes involved in ebolavirus entry

Ebolavirus (EBOV) is a negative sense, single stranded RNA virus that causes Ebola hemorrhagic fever. This disease causes substantial morbidity and mortality in humans, with death occurring in 50-90% of cases. Despite years of intensive research, much of the molecular mechanism underlying the entry of EBOV remains unknown. We performed a bioinformatics screen to identify novel entry cofactors by correlating mRNA expression in a panel of human cancer cell lines with permissivity to the EBOV entry glycoprotein. This assay identified several known EBOV entry cofactors such as actin and the tyrosine kinase Axl. In addition, several genes involved in macropinocytosis and endosomal maturation were also correlated with EBOV permissivity. Subsequent evaluation of plasma membrane proteins correlated by this screen showed T-cell immunoglobulin and mucin domain-1 (TIM-1) mRNA expression correlated extremely well with EBOV pseudovirion transduction. Depletion of TIM-1 from highly-permissive cells inhibits EBOV pseudovirion transduction. Conversely, expression of TIM-1 in poorly-permissive cells significantly and specifically enhances EBOV pseudovirion transduction and infection. TIM-1 binds to EBOV GP and this binding is important in the initial interaction between the virus and the host cell. ARD5, a TIM-1 mAb, significantly inhibits EBOV GP-mediated entry into several cell lines and primary human airway epithelia in a dose and time-dependent manner. Therefore, TIM-1 is the first receptor identified for EBOV. 2 Additionally, AMP-activated protein kinase (AMPK) mRNA correlated strongly with EBOV pseudovirion transduction. Compound C, a specific AMPK inhibitor, inhibited EBOV pseudovirion transduction and infection in a time and dose-dependent manner into several cell lines and primary human monocyte derived macrophages. Mouse embryonic fibroblasts (MEFs) lacking functional AMPK were significantly less permissive to EBOV GP-mediated infection that WT MEFs. Visualization of virus entry into these cells revealed that EBOV causes actin polymerization independently of AMPK, but AMPK-/cells do not form lamellipodia in the presence of EBOV and, consequently, cannot internalize virus into cells by macropinocytosis. Abstract Approved: _______________________________ Thesis Supervisor _______________________________ Title and Department _______________________________ DateApproved: _______________________________ Thesis Supervisor _______________________________ Title and Department _______________________________ Date 1 A BIOINFORMATICS APPROACH TO IDENTIFYING NOVEL GENES INVOLVED IN EBOLAVIRUS ENTRY by Andrew Steven Kondratowicz A thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree in Microbiology in the Graduate College of The University of Iowa December 2011 Thesis Supervisor: Associate Professor Wendy J. Maury Graduate College The University of Iowa Iowa City, Iowa CERTIFICATE OF APPROVAL _______________________ PH.D. THESIS _______________ This is to certify that the Ph. D. thesis of Andrew Steven Kondratowicz has been approved by the Examining Committee for the thesis requirement for the Doctor of Philosophy degree in Microbiology at the December 2011 graduation. Thesis Committee: ______________________________ Wendy Maury, Thesis Supervisor _______________________________ Richard Roller _______________________________ Paul B. McCray Jr. _______________________________ Aloysius Klingelhutz _______________________________ Joseph Zabner