"Investigation of Membrane Fusion Interactions of Enveloped Viruses using Magnetically-Labeled Liposomes"

Program Director/Principal Investigator: Santra, Santimukul PROJECT SUMMARY/ABSTRACT This application proposes to develop liposome-coated iron oxide nanosensors (LIONs) mimicking as magnetically-labeled host membranes to detect fusion interactions of enveloped viruses. Spin-spin T2 relaxation technique will be established as a generic bioanalytical method to detect dynamic conformational changes in viral glycoproteins that play vital roles in fusion interactions. Within the first aim, influenza fusion protein interactions with LIONs membrane will be studied, considering diverse fusion triggering environmental factors including low pH, protease activation and receptor binding. Further studies to investigate the sensitivity of this LIONs technology in quantifying similar fusion interactions with more native configurations of HA is demonstrated using virus-like particle (VLPs) and shorter domains derived from HA. Similarly, fusion experiments will be carried out using ganglioside-conjugated LIONs. The promising outcome of this research will be screening potential antiviral candidates including small molecules and neutralizing antibodies. The second aim establishes the broad adaptability of this sensitive LIONs technology for evaluation of fusion interactions of enveloped viruses where presence of membrane receptor is an important trigger, for example, SARS-CoV-2. Receptor-conjugated LIONs (R-LIONs) will be developed for the elucidation of this vital process using spike proteins and reporter virus particles (RVPs) of SARS-CoV-2. R-LIONs offers a novel approach of exploring the role of variety of entry receptors that may play an important role in SARS-CoV-2 membrane fusion. The third aim of this proposal determines further advantage of nanosensor technology for the real-time monitoring of virus-mediated cell fusion. SARS-CoV-2 virus entry into ACE2 receptor overexpressing mammalian cells (HEK-293T) will be examined by developing magnetically labeled reporter virus particles (M-RVPs). Parallel experiments will be carried out in the presence of fusion inhibitors and neutralizing antibodies. The important application of this experiment will be demonstrated by screening potential new fusion inhibitors and antiviral candidates. The proposed AREA proposal, if successful, will develop novel nanosensor technology for the rapid detection of fusion interactions of enveloped viruses including SARS-CoV-2 and influenza. This adaptable technology will allow for rapid screening of potential drug candidates and fusion inhibitors of many other enveloped viruses, in a timely fashion. Most importantly, this AREA proposal will initiate the proposed PURE program for undergraduate education, and increase the hands-on biomedical research opportunity and learning for students in our undergraduate-focused institution. OMB No. 0925-0001 and 0925-0002 (Rev. 03/20 Approved Through 02/28/2023) Page Project Summary