Mechanisms of membrane rupture and repair: viral assembly strategies. – membrane dynamics
membrane dynamics
understand the role of both lipids and proteins in the unconventional membrane assembly of large DNA viruses
study proteins and lipids
The objective of the proposal is to analyse the role of both lipids and viral proteins in the unconventional membrane assembly of the poxvirus vaccinia virus (VACV). During membrane assembly small vesicular carriers are recruited to the assembly site, these are opened via membrane rupture and contribute to the assembly of the viral inner membrane by fusion. The proposed working plan was based on two pieces of evidence; first the observation made in 2013 that the viral membrane is enriched in several lipid species among which phosphatic acid (PA) and phospho-inositides (PI). Second was the observation, based on characterization of a conditionally expressing recombinant VACV, that in the absence of a single VACV protein, the gene product of A11, membrane rupture does not occur and hence viral membrane assembly is blocked (Suarez et al., 2017). Thus, we proposed to study the role of lipids (PA and PI) and of A11 further both in vivo as well as in vitro. In vitro we planned to express A11 in liposomes of defined lipid composition and analyse effects on membranes (eg. Rupture) biochemically and by high-resolution cryo-EM.
The objective of the proposal is to analyse the role of both lipids and viral proteins in the unconventional membrane assembly of the poxvirus vaccinia virus (VACV). During membrane assembly small vesicular carriers are recruited to the assembly site, these are opened via membrane rupture and contribute to the assembly of the viral inner membrane by fusion. The proposed working plan was based on two pieces of evidence; first the observation made in 2013 that the viral membrane is enriched in several lipid species among which phosphatic acid (PA) and phospho-inositides (PI). Second was the observation, based on characterization of a conditionally expressing recombinant VACV, that in the absence of a single VACV protein, the gene product of A11, membrane rupture does not occur and hence viral membrane assembly is blocked (Suarez et al., 2017). Thus, we proposed to study the role of lipids (PA and PI) and of A11 further both in vivo as well as in vitro. In vitro we planned to express A11 in liposomes of defined lipid composition and analyse effects on membranes (eg. Rupture) biochemically and by high-resolution cryo-EM.
We carried out a study showing an important role the protein A11 in membrane biogenesis of VACV (Suarez et al., 2017). When A11 is not expressed VACV morphogenesis is blocked; by electron tomography we could show that this inhibition is due to the fact that cellular membranes are not ruptured providing an important piece of evidence for the fact that membrane rupture is a prerequisite for viral assembly. Recent publications on VACV proteins involved in membrane assembly have identified a complex of five minor membrane associated proteins with important roles in membrane biogenesis. Among these are the gene products of A11, A6, H7, L2 and A30,5. Together with the group of Fasseli Coulibaly, a collaborator on this proposal, we have started to purify these proteins individually and expressed them in liposomes with defined lipid composition. This has shown that at least two of these VMAPs require specific lipid composition to associate with membranes and both of these two VMAP considerably modify membranes. These preliminary results will now be used to solve their structure by cryo-EM. The other three proteins are either not cloned yet or present complications when expressed in E. coli. The characterization of these three proteins is thus work for the future.
In order to study the function of these five proteins in infected cells we wish to establish complementation assays.
To test for a role of lipids in infected cells we have carried out drug experiments with little results so far. Hence, we have turned towards the localization of lipids with respect to the virally modified membranes by light microscopy. We put a specific emphasis on PA and PI using lipid binding probes bound to GFP. This study is currently underway with some preliminary encouraging results.
continue the work as described under main results
no patents
several peer reviewed publications, one submitted for publication
Abstract
Within eukaryotic cells membrane compartments are closed entities that communicate by vesicular transport, controlled by specific protein machineries. Being obligatory intracellular parasites, viruses acquire their membrane from the host, in a process mimicking the formation of vesicles. Nucleo-cytoplasmic large DNA viruses (NCLDVs), a family of large DNA viruses, are an exception to this rule and follow an unconventional pathway of membrane acquisition. During infection they form open membrane intermediates, likely the result of membrane rupture, from which they build an open membrane sphere. The latter is shaped by the viral scaffold protein, a protein conserved among most NCLDVs. The membrane sphere remains open until the viral genome has been taken up, upon which the membrane closes and the particle matures into an infectious virion. We proposed that this unusual membrane assembly is controlled by a machinery, common to NCLDVs, which includes both (viral) proteins and lipids. Together they mediate membrane rupture, stabilization of open membrane ends, the formation of the open sphere and its subsequent closure after DNA-uptake.
Our recent lipid mass spectrometry (MS) results of purified vaccinia virus (VACV, member of the NCLDVs) showed that its envelope is enriched in lipid species that have been implicated in membrane rupture/destabilization. Moreover, preliminary data obtained by 3D-electron tomography (ET) identified a VACV protein, required for membrane rupture in infected cells. Within the frame of this proposal we will search for interacting partners of this VACV protein during infection. The VACV-protein will be expressed and purified with or without its interacting partners and its structure analyzed by X-ray crystallography as well as by cryo-EM. For the former we will rely on the collaboration with Dr. Coulibaly (Monash, Melbourne), an expert on X-ray crystallography of viral proteins, in particular of VACV proteins. In parallel we will continue lipid-analyses and identify minor lipid species and specific lipid classes of purified VACV. We propose to complement these data by lipid-MS of open VACV membranes, isolated and affinity-purified from infected cells. The role of specific lipids in membrane rupture will be analyzed in vitro as well as in vivo. In vitro the VACV protein required for rupture will be reconstituted in artificial membranes (liposomes) containing lipids identify by MS to be enriched in the viral membrane. Its effect on liposomes will be analyzed by content release and cryo-ET. In vivo we aim at inhibiting the synthesis of specific lipids in infected cells and assess effects on VACV assembly by ET.
Our study sheds light on mechanisms of membrane rupture and repair that may find applications in targeted delivery of compounds in cells. Evolutionary, it may shed light on a mechanism that is either rare in modern eukaryotes or was present in an early eukaryote/archaea/bacteria but was lost during evolution.
Project coordination
Guillaume Dumenil (INSTITUT PASTEUR (BP))
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.
Partnership
Monash university Structural Virology Laboratory
Heidelberg University Biochemie Zentrum Heidelberg
INSTITUT PASTEUR (BP)
Help of the ANR 259,382 euros
Beginning and duration of the scientific project:
October 2016
- 36 Months
