Crystal structure determination of the M1 matrix protein at different pHs and structural analyses of the mechanism of M1 polymerization on membranes.
The budding of influenza virus is independent of the ESCRT system and our hypothesis is that the matrix protein M1 exerts some of the ESCRT-III-like functions. Our first objective is to determine the structural basis of M1 assembly. The disassembly of M1 is induced by low pH in the endosome and we thus want to determine the structure of M1 at low pH in order to study the structural transitions induced during the viral life cycle.
Our aim is to crystallize M1 alone from influenza virus. In parallel we established protocols to purify and crystallize M1 in complex with antibody Fab fragments that may facilitate crystallization. Once we have obtained crystals we will use them to solve the structure by X-ray crystallography. Secondly, we study the polymerization of M1 on membranes by employing the GUV system as well as liposomes to reconstitute the M1 capsid that can be further analyzed by electron microscopy.
The purification protocols of M1 proteins from different influenza viruses as well as complex formation with antibody Fab fragments have been established. The different M1 proteins have been analyzed by small angle X-ray scattering and low resolution envelopes of M1 have been constructed. Incubation of fluorescently labeled M1 with giant unilamellar vesicles (GUVs) demonstrated the formation of membrane tubes at neutral pH.
We will continue our efforts to obtain structural information on the complete M1 protein at high resolution. The crystallization effort will continue and in case we don’t obtain crystals we will switch to NMR analyses of M1. We will also continue to study the effect of M1 on membranes by the GUV system and confocal microscopy. Furthermore, we study M1 polymerization on liposomes by electron microscopy.
No manuscript is yet in preparation. Publication of results is foreseen for the second period of the project.
Influenza viruses are negative stranded, enveloped, segmented RNA viruses belonging to the Orthomyxoviridae family. Their genome codes for three major sub-viral components. The viral membrane envelope, which contains hemagglutinin (HA), neuraminidase (NA) and the ion channel M2, a submembrane protein coat formed by the matrix protein M1 and the viral helical ribonucleocapsid composed of the nucleoprotein in complex with RNA and the viral polymerase complex. Influenza enters cells via endocytosis and acidification in the endosome leads to fusion of viral and cellular membranes catalysed by HA. Concomitantly, acidification of the virion interior by M2 induces uncoating of the M1 -RNP complexes. M1 then plays a second major role during assembly and budding from the plasma membrane that seems to occur independent of the ESCRT (Endosomal Sorting Complexes Required for Transport) machinery. ESCRTs are otherwise recruited by some enveloped viruses to facilitate budding. The structural basis of the role of M1 during low pH-induced uncoating as well as during assembly and budding are still poorly understood.
The project concentrates on the structural biology of the complete viral matrix protein M1. Although the crystal structure of the N-terminal domain is known, no structural information of the C-terminal domain is available. We hypothesize that the crystal structure of a complete M1 protein will provide important insight into its function during uncoating and during assembly and budding. The project will thus have the following objectives: (i) Determine the crystal structure of M1 at neutral pH and at the endosomal low pH and (ii) characterize the structural determinants of M1 polymerisation and coat formation. This work will thus give important insight into the conformational differences of M1 that accompany the uncoating process in the endosome during entry. Structural details of M1 at neutral and low pH can be further employed to develop anti-viral agents that target the entry process. Secondly our results will provide important insight into the hypothesis that M1 might have similar structural properties than ESCRT-III. Our results will provide a low-resolution image of M1 polymer and protein coat formation and these structural analyses might explain why influenza virus can bud independent of the ESCRT machinery. These structural details of M1 at neutral pH will help to develop anti-viral agents that target assembly.
Monsieur Winfried Weissenhorn (CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE RHONE-ALPES SECTEUR ALPES) – email@example.com
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
CNRS-EMBL CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE RHONE-ALPES SECTEUR ALPES
Help of the ANR 200,000 euros
Beginning and duration of the scientific project: - 30 Months