Elucidating the molecular determinants of novel anti-henipavirus antiviral compounds - Project Summary/Abstract Henipaviruses are recently emerged zoonotic viruses in the family Paramyxoviridae that can spill over from their pteropid bat reservoir into humans to cause severe respiratory disease with high mortality. There are no approved antivirals to treat infection by Hendra virus (HeV) and Nipah virus (NiV), the two henipaviruses that cause disease in humans, and identifying antivirals is challenging due to their classification as biosafety level 4 (BSL4) pathogens. We have assembled a library of 44,000 small molecules containing compounds with diverse structures, including 1,000 nucleoside analogs, and screened for antiviral activity against a recombinant, non-pathogenic henipavirus, Cedar virus, that expresses GFP (CedV-GFP). We screened in Calu-3 lung epithelial cells, which are permissive to respiratory virus infection and resemble primary cells in their morphology and signaling pathways. We identified 64 compounds that inhibit infection, including two lead compounds: CV-002 and UPGNUC255. Their antiviral activity was validated in diverse cellular models. CV-002 is a non-nucleoside compound with no previously described antiviral activity, so to start to determine the mechanism of action, we generated 5 CV-002-resistant strains of CedV-GFP (CV002Rs) by selection through serial passaging with increasing doses of CV-002. By sequencing the RNA genomes of the CV002Rs, we identified mutations in the L gene that encodes the RNA-dependent RNA polymerase (RdRp) of CedV-GFP. Modeling the locations of the residues changed in the CV002Rs showed that they are in close proximity to the active site of the RdRp (GDN). Thus, I hypothesize that CV-002 blocks viral replication at the viral RNA level by directly targeting the RdRp activity of Cedar virus. In contrast, UPGNUC255 is a nucleoside analog, a class of antivirals that typically targets the viral RdRp and acts through mutagenesis or (delayed) chain termination during RNA replication. However, the antiviral mechanism of action for UPGNUC255 is unknown and will be explored. Furthermore, the Cherry lab has shown that nucleoside analog antivirals can synergize with nucleoside biosynthesis inhibitors to inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and rubella infections. Thus, I hypothesize that UPGNUC255 targets the Cedar virus RdRp for its antiviral activity and its activity can be improved through co-administration with at least one class of nucleoside biosynthesis inhibitors. In Aim 1, I will confirm the amino acid residues in Cedar virus L protein that are essential for CV-002 antiviral activity and use structure activity relationships (SAR) to optimize this activity. In Aim 2, I will generate UPGNUC255-resistant mutant strains of CedV-GFP and sequence their RNA genomes to determine UPGNUC255’s viral protein target. I will also co-administer UPGNUC255 with nucleoside biosynthesis inhibitors to determine if there is synergistic antiviral activity with these combinations. The proposed experiments will provide insight into the mechanisms of action of these newly identified antiviral compounds and may facilitate future development of antivirals against Hendra virus and Nipah virus.
