In this project, we aim to further understand influenza A virus (IAV) membrane fusion and cell entry. IAV utilizes the Hemagglutinin (HA) trimer, a type I viral fusion protein, to drive the formation of a fusion pore between the viral and endosomal membrane at low pH. The fusion pore is necessary for viral genome release into the host cell. However, neither the HA intermediates at low pH nor the IAV entry intermediates have yet been structurally characterized. The two major objectives of this project are as follows:

The first objective is to use cryo-electron microscopy (cEM) to provide further information on the structure of HA fusion intermediates at low pH. To this end, we will benefit from our previous work, where we found that liposomes with lipid compositions suboptimal for membrane fusion are prone to membrane rupture and thus allow trapping of shortlived HA intermediates present on the surface of IAV virus-like particles (VLP). Unpublished data show that low pH HA intermediates are thin with a diameter of 2 nm, suggesting that the HA trimer may transiently dissociate into monomers at low pH. We will perform cryo-electron tomography and subtomogram averaging (STA) to obtain a structure of the HA intermediates at a resolution <2 nm. To study the low pH HA intermediates at high resolution (<1 nm), we will use cryo-electron microscopy (cEM) and single-particle analysis (SPA) to analyze the structure of the HA ectodomains at low pH. HA ectodomains, which form rosette-like assemblies composed of thin HA spikes at low pH, will be purified in collaboration with Brügger/Kräusslich. We will compare the low pH structure of the HA ectodomains with the low pH HA intermediate structures trapped in the liposomes. Collectively this will allow to determine whether the HA intermediates at low pH are monomers.

The aim of the second objective is to study IAV entry comprising membrane fusion, disassembly and release of the viral genome inside the cells, by using correlative light and electron microscopy (CLEM) in collaboration with projects Brügger/Kräusslich and Funaya/Schwab. The structure of HA fusion intermediates as well as the structural changes accompanying virus uncoating during entry have not yet been studied in native conditions in situ, and, thus will likely provide novel opportunities to generate small chemicals to abrogate IAV entry. Two mechanistically different fusion pathways determined by cholesterol concentrations have recently been described in vitro, however, it has not been elucidated which of these predominates in physiological conditions in cells. We will use fluorescently labeled VLP whose entry will be followed and localized inside the cell by fluorescence microscopy, and the VLP entry intermediates will be structurally characterized by cET after cell thinning by cryo-focused ion beam (cFIB). The level of cellular cholesterol will be modulated by statins, overexpression of IFITM3 and by S1P knock-out, which disrupts cholesterol synthesis. The cET structural data corroborated with mass spectrometry quantitative lipid analysis of cellular cholesterol (collaboration Brügger) will clarify the role of cholesterol in HA-mediated membrane fusion in physiological settings. We will continue interacting with Briggs (associated member of SFB1129 in the next period), to seek advice on STA protocol optimization. Furthermore, we will use cET to structurally characterize Dengue virus (DENV) replication membrane vesicles and provide insight in the overall RNA distribution inside the vesicle in collaboration with Bartenschlager/Höfer.

Strukturelle Analyse der Einwirkung von Influenza Protein-Membranen während des Eintritts
Influenza A Virus (IAV) ist ein bedeutender menschlicher Erreger für den es keine universelle Impfung gibt. Wir werden Cryo-elektron mikroskopische Techniken anwenden um strukturelle Details von Hämagglutinin gesteuerten Membranfusionen in vitro und in situ, zu liefern. Wir werden unsere Forschung auf (i) Konformationsänderungen von Hämagglutinin bei niedrigem pH fokussieren und ebenso auf (ii) die Rolle von Cholesterin und Faktoren, die das Membrancholesterin des Wirtes während des IAV Eintritts und der Membranfusion modulieren. Wir wollen einen Arbeitsablauf in der Cryo-korrelativen Licht- und Elektronen Mikroskopie etablieren, der es uns zum ersten Mal erlaubt, den IAV Eintritt und die Membranfusion bei hoher Auflösung innerhalb der Zellen und unter nativen Verhältnisse, zu studieren. Dieses Projekt wird ein besseres Verständnis der Wirkprinzipien von IAV Eintritts-Hemmstoffen, wie von cholesterinsenkenden Medikamenten und Hämagglutinin-fusionshemmenden Peptiden, liefern.

Project Staff

Steffen Klein, PhD student