STRUCTURAL REARRANGEMENTS IN THE PARAMYXOVIRUS FUSION COMPLEX IN THE PRESENCE OF A NOVEL ANTIVIRAL.

Human parainfluenza virus type 3 (HPIV3), an enveloped virus representative of human paramyxoviruses, enters cells by fusing directly with the cell membrane. The viral receptor-binding glycoprotein hemagglutinin-neuraminidase (HN) binds to cellular receptors and activates the viral fusion protein (F) to its fusion-ready state; the transition of F to its final post-fusion state enables the merger of viral and cell membranes. HN is comprised of a transmembrane domain, a stalk that interacts with F, and a globular head, and is present on the infected cell as a dimer of dimers. Crystal structures show HN either with the heads flipped down partially covering the stalk, or with heads up exposing the stalk. Our previous analysis of HPIV3 particles using negative-stain electron tomography showed that prior to receptor engagement the surface of viral particles bear a continuous coat of spikes with two layers of alternating density reflecting the configuration of complexed HN and F with the globular heads of HN oriented upwards. HN and F are complexed prior to receptor binding, and the transition to a heads-up conformation does not drive the F-activation that occurs upon HN’s receptor engagement. We aim to discover how receptor engagement activates the structural changes in HN and F that lead to membrane merger and infection. To capture intermediate states in this process, we used a small molecule receptor-mimic (CM9) that we have shown to interact with HN and thereby induce F-triggering in the absence of receptor engagement, thus inactivating the virus. We hypothesize that in the presence of this small molecule HN and F are in contact and F is converted to its post-fusion state. To this end, purified HPIV3 preparations were incubated with CM9 and the structure and arrangement of HN/F are being analyzed by negative stain electron microscopy and cryo-electron tomography. Parallel biological and biochemical experiments demonstrate that CM9 inactivates virus and inhibits viral infectivity in cell culture, ex vivo, and in animals, and that interaction of CM9 with HN results in conversion of F to protease sensitivity as an indicator of conformational transition. We expect to observe the intermediate between pre- and post-fusion F in the presence of a lipid-conjugated peptide that binds the transitional state of F, and in future studies we aim to develop this new antiviral target while identifying the key interactions between native HN and F that result in fusion activation