Identification of host cellular targets of the Zika NS2B-NS3 protease
Location
Medical Education Building, LSUHSC-NO
Presentation Date
10-10-2019 10:00 AM
End Date
10-10-2019 12:00 PM
Description
Zika virus infection disrupts neuroblast proliferation and can result in severe syndromes for infants such as microcephaly and retinopathies. Recently, the Wojcik lab has shown that the Zika-encoded protease NS2B-NS3, hypothesized to be a key player in disease etiology, can cleave and inactivate HsEg5 in vitro.1,2,3 HsEg5 functions as a Kinesin-5 family microtubule motor protein required for mitosis and its loss of function is a known dominant monogenic cause of hereditary microcephaly and blindness.4 Given this connection, we propose to test whether the NS2B-NS3 protease can target HsEg5 in proliferating cells. To confirm a direct in vitro interaction between the protease and HsEg5, we probed for cellular targets that interact with the protease by proximity ligation experiments. In our proximity ligation assay, we created a stable NS2B-NS3 enzyme that incorporates a C-terminal biotin ligase moiety, known as MiniTurbo (MT), to promiscuously biotinylate all proteins that have NS2B-NS3 affinity. A preliminary NS2B-NS3-MT biotinylation assay has been executed and led to successful identification of biotinylated proteins via mass spectroscopy. However, further repetitions of the procedure are needed to confirm our preliminary results that potentially identify Kinesin-5 as a key target of the Zika protease in the causation of infant microcephaly.
Recommended Citation
Doiron, Jake E., "Identification of host cellular targets of the Zika NS2B-NS3 protease" (2019). Medical Student Research Poster Symposium. 12.
https://digitalscholar.lsuhsc.edu/sommrd/2019/posters/12
Identification of host cellular targets of the Zika NS2B-NS3 protease
Medical Education Building, LSUHSC-NO
Zika virus infection disrupts neuroblast proliferation and can result in severe syndromes for infants such as microcephaly and retinopathies. Recently, the Wojcik lab has shown that the Zika-encoded protease NS2B-NS3, hypothesized to be a key player in disease etiology, can cleave and inactivate HsEg5 in vitro.1,2,3 HsEg5 functions as a Kinesin-5 family microtubule motor protein required for mitosis and its loss of function is a known dominant monogenic cause of hereditary microcephaly and blindness.4 Given this connection, we propose to test whether the NS2B-NS3 protease can target HsEg5 in proliferating cells. To confirm a direct in vitro interaction between the protease and HsEg5, we probed for cellular targets that interact with the protease by proximity ligation experiments. In our proximity ligation assay, we created a stable NS2B-NS3 enzyme that incorporates a C-terminal biotin ligase moiety, known as MiniTurbo (MT), to promiscuously biotinylate all proteins that have NS2B-NS3 affinity. A preliminary NS2B-NS3-MT biotinylation assay has been executed and led to successful identification of biotinylated proteins via mass spectroscopy. However, further repetitions of the procedure are needed to confirm our preliminary results that potentially identify Kinesin-5 as a key target of the Zika protease in the causation of infant microcephaly.
Comments
Mentor: Dr. Edward Wojcik (Department of Biochemistry and Molecular Biology)