BTP2 restricts Tulane virus and human norovirus replication independent of store-operated calcium entry

Joseph Hyser, Baylor College of Medicine

Francesca J. Scribano1,2, J. Thomas Gebert1,2, Kristen A. Engevik1,2, Nicole M. Hayes1,2, Son Pham1,3, Soni Kaundal1,3, Janam J. Dave1,2, B V Venkataram Prasad1,3, Mary K. Estes1,2, Sasirekha Ramani1,2, Joseph M. Hyser1,2 1. Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA 2. Alkek Center for Metagenomics & Microbiome Research, Baylor College of Medicine, Houston, TX, USA 3. Verna and Marrs McLean Department of Biochemistry and Molecular Pharmacology, Baylor College of Medicine, Houston, TX, US

Prevention & Control (antivirals)

Human norovirus is the leading cause of viral gastroenteritis across all age groups. While there is a need for human norovirus antivirals, therapeutic development has been hindered by a lack of cell culture systems and animal models of infection. Surrogate viruses, such as Tulane virus (TV), have provided tractable systems to screen potential antiviral compounds. Our previous work demonstrated that TV encodes a viral ion channel, which dysregulates cytosolic calcium signaling. We set out to investigate whether host pathways triggered by viral ion channel activity, including store-operated calcium entry (SOCE), play a role in virus replication. Using pharmacologic inhibitors and genetically engineered cell lines, we establish that the SOCE inhibitor, BTP2, reduces TV replication in an SOCE-independent manner. We observed a significant reduction in TV replication, protein expression, and RNA synthesis in cells with both pre- and post-infection BTP2 treatment. By serial passage and plaque isolation, we demonstrate that TV quasi-species have mixed susceptibility and resistance to BTP2. Sequence comparison of the quasi-species revealed that amino acid changes in the structural proteins were associated with drug resistance. We utilized reverse genetics to generate TV with the resistance-associated VP1 and VP2 amino acid changes and found that a single amino acid change in VP1 (I380M) conferred BTP2 resistance. Further, expression of resistant VP2 alone was sufficient to partially rescue the replication of susceptible virus. Together, this supports that TV structural proteins are the targets of BTP2. Finally, using human intestinal organoids, we demonstrate that BTP2 significantly reduces human norovirus replication.