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International audience; The Cdv proteins constitute the cell division system of the Crenarchaea, a machinery closely related to the ESCRT system of eukaryotes. Using a combination of TEM imaging and biochemical assays, we here present an in vitro study of Metallosphaera sedula CdvB1, the Cdv protein that is believed to play a major role in the constricting ring that drives cell division in the Crenarchaea. We show that CdvB1 self-assembles into filaments that are depolymerized by the Vps4-homolog ATPase CdvC. Furthermore, we find that CdvB1 binds to negatively charged lipid membranes and can be detached from the membrane by the action of CdvC. Our findings provide novel insight into one of the main components of the archaeal cell division machinery.

Stabilization of the HIV-1 Envelope glycoprotein trimer (Env) in its native pre-fusion closed conformation is regarded as one of several requirements for the induction of neutralizing antibody (nAb) responses, which, in turn, will most likely be a prerequisite for the development of an efficacious preventive vaccine. Here, we systematically analyzed how the stepwise stabilization of a clade C consensus (ConC) Env immunogen impacts biochemical and biophysical protein traits such as antigenicity, thermal stability, structural integrity, and particle size distribution. The increasing degree of conformational rigidification positively correlates with favorable protein characteristics, leading to optimized homogeneity of the protein preparations, increased thermal stability, and an overall favorable binding profile of structure-dependent broadly neutralizing antibodies (bnAbs) and non-neutralizing antibodies (non-nAbs). We confirmed that increasing the structural integrity and stability of the Env trimers positively correlates with the quality of induced antibody responses by the immunogens. These and other data contribute to the selection of ConCv5 KIKO as novel Env immunogens for use within the European Union’s H2020 Research Consortium EHVA (European HIV Alliance) for further preclinical analysis and phase 1 clinical development.

Additional file 4: Figure S4. Deformation of bare vesicles and vesicles covered with CHMP2A + CHMP3 or CHMP2B + CHMP3. HS-AFM images of a vesicle covered with CHMP2A and CHMP 3 (left column) and a vesicle covered with CHMP2B and CHMP3 proteins (right column). The deformability of the SUVs coated with corresponding proteins upon increased applied force are shown at intermediate force increments of 27% (second panels) and at higher force increment, 81% (third panels).

Additional file 2: Figure S2. Study of CHMP protein-membrane interaction. (A) Optimization of the buffer conditions to optimize the binding of CHMP2B-ΔC (noted here CHMP2B) at 500 nM. Pre-formed vesicles were incubated with CHMP2B-ΔC in buffers with different salt concentrations ranging from 0 mM to 100 mM NaCl (+Tris 25 mM at pH 7.5) and imaged with confocal microscope after 30 min incubation. Lipid signal is shown in magenta and protein signal in green. Scale bar: 5 μm. (B) Confocal image of MBP-CHMP2A-ΔC (noted here CHMP2A) without TEV (Top line) and in the presence of TEV to cleave the MBP tag (Bottom line). Saturated protein fluorescent signal is represented in yellow. Cleavage of MBP tag slightly increases the interaction but induces aggregation. Scale bar: 30 μm. (C) Spinning disk image of GUV incubated with MBP-CHMP2B-ΔC (noted here MBP-CHMP2B) at a concentration of 50 nM (left image, scale bar = 10 μm) and at 200 nm (right image; scale bar = 5 μm). (D) Confocal images of GUV incubated with MBP-CHMP2B-ΔC at a concentration of 1 μM (noted here MBP-CHMP2B), submitted to an osmotic pressure difference equal to 150% (Osmolarity inside and outside the GUV are respectively 120 mOsm.L− 1 and 315 mOsm.L− 1). Scale bar: 10 μm.

Additional file 7: Figures S6. Test of mutual interaction between CHMP2B and CHMP3 in solution. CHMP2B-ΔC (first gel) and CHMP3-FL (second gel) in Hepes Buffer have been deposited on a sucrose gradient. 100 μL of CHMP2B-ΔC at 10 μM has been incubated with 100 μl CHMP3FL at 10 μM and deposit on a sucrose gradient (third gel). No aggregation is observed on the bottom of the gradient. The presence of CHMP3 does not induce CHMP2B aggregation.

Additional file 6: Figure S5. Deformation of bare vesicles and vesicles covered with CHMP proteins. (A) Reduction of vesicle height under increasing force for bare vesicles. ‘Zero’ force increment represents the minimum imaging force (~ 150 pN). (B) Example of deformation for a ~ 60 nm vesicle over increasing force up to 80% of the initial imaging force. (C) Represents the transformation of vesicle height to relative height for each point for the curve in D. (D) represents the relative height vs force increment for all the curves from panel A for bare SUVs. (E) represents the relative height vs force increment for SUV covered with CHMP2B (left), CHMP2B + CHMP3 (middle) and CHMP2A + CHMP3 (right).

Additional file 1: Figure S1. Evolution of the ESCRT-III complex. (A) Table illustrating the ESCRT-III complex function, origin and homologs in S. cerevisiae and H. sapiens. (B) Distribution of Vps2 and Vps24 genes across Eukaryotes showing the presence of two Vps2 genes in high organisms. (C) Table illustrating the implication of ESCRT-III subunits in different subcellular locations in S. cerevisiae and H. sapiens. The names are the human homologs in case of S. cerevisiae.