Nicholas Lennemann, Ph.D., assistant professor in the Department of Microbiology, aimed to develop and characterize a fluorescent CoV infection reporter, and identify antivirals in relevant mouse models using the reporter, with his grant from the Urgent COVID-19 Clinical Research and Laboratory Research Fund.

Lennemann’s team has continued to develop an infection reporter that streamlines infection assays in the bio-safety level 3 lab (BSL3). Unlike enteroviruses, CoV replicates in association with the ER membrane, as do flaviviruses. Lennemann made modifications to an existing enterovirus reporter in parallel for CoV and flaviviruses to determine if reporters can be developed for viruses that replicate at the ER.

They found that substitution of the PVR TM domain in the initial construct for the TfR TM domain and a flavivirus protease cleavage site allowed for flavivirus cleavage; however, introduction of 10 different CoV protease recognition sites (PLpro or 3CLpro) did not yield a functional reporter.

Interestingly, the team included this site for both SARS-CoV2 and OC43 in the reporters previously and did not observe cleavage by OC43 or 3CLpro expression, suggesting protease recognition is more complex.

The future of his research includes modifying the split fluorescent GFP reporter to include Sec61B TM and several viral protease recognition sites to determine if they are recognized by SARS-CoV2 and/or OC43. These are useful tools for work in the BSL3, but also for understanding the molecular determinants of CoV protease recognition/cleavage.