Halorubrum pleomorphic virus-6 Membrane Fusion Is Triggered by an S-Layer Component of Its Haloarchaeal Host

(1) Background: Haloarchaea comprise extremely halophilic organisms of the Archaea domain. They are single-cell organisms with distinctive membrane lipids and a protein-based cell wall or surface layer (S-layer) formed by a glycoprotein array. Pleolipoviruses, which infect haloarchaeal cells, have an envelope analogous to eukaryotic enveloped viruses. One such member, Halorubrum pleomorphic virus 6 (HRPV-6), has been shown to enter host cells through virus-cell membrane fusion. The HRPV-6 fusion activity was attributed to its VP4-like spike protein, but the physiological trigger required to induce membrane fusion remains yet unknown. (2) Methods: We used SDS-PAGE mass spectroscopy to characterize the S-layer extract, established a proteoliposome system, and used R18-fluorescence dequenching to measure membrane fusion. (3) Results: We show that the S-layer extraction by Mg2+ chelating from the HRPV-6 host, Halorubrum sp. SS7-4, abrogates HRPV-6 membrane fusion. When we in turn reconstituted the S-layer extract from Hrr. sp. SS7-4 onto liposomes in the presence of Mg2+, HRPV-6 membrane fusion with the proteoliposomes could be readily observed. This was not the case with liposomes alone or with proteoliposomes carrying the S-layer extract from other haloarchaea, such as Haloferax volcanii. (4) Conclusions: The S-layer extract from the host, Hrr. sp. SS7-4, corresponds to the physiological fusion trigger of HRPV-6

Halorubrum pleomorphic virus-6 Membrane Fusion Is Triggered by an S-Layer Component of Its Haloarchaeal Host

(1) Background: Haloarchaea comprise extremely halophilic organisms of the Archaea domain. They are single-cell organisms with distinctive membrane lipids and a protein-based cell wall or surface layer (S-layer) formed by a glycoprotein array. Pleolipoviruses, which infect haloarchaeal cells, have an envelope analogous to eukaryotic enveloped viruses. One such member, Halorubrum pleomorphic virus 6 (HRPV-6), has been shown to enter host cells through virus-cell membrane fusion. The HRPV-6 fusion activity was attributed to its VP4-like spike protein, but the physiological trigger required to induce membrane fusion remains yet unknown. (2) Methods: We used SDS-PAGE mass spectroscopy to characterize the S-layer extract, established a proteoliposome system, and used R18-fluorescence dequenching to measure membrane fusion. (3) Results: We show that the S-layer extraction by Mg2+ chelating from the HRPV-6 host, Halorubrum sp. SS7-4, abrogates HRPV-6 membrane fusion. When we in turn reconstituted the S-layer extract from Hrr. sp. SS7-4 onto liposomes in the presence of Mg2+, HRPV-6 membrane fusion with the proteoliposomes could be readily observed. This was not the case with liposomes alone or with proteoliposomes carrying the S-layer extract from other haloarchaea, such as Haloferax volcanii. (4) Conclusions: The S-layer extract from the host, Hrr. sp. SS7-4, corresponds to the physiological fusion trigger of HRPV-6

The structure of a prokaryotic viral envelope protein expands the landscape of membrane fusion proteins

Lipid membrane fusion is an essential function in many biological processes. Detailed mechanisms of membrane fusion and the protein structures involved have been mainly studied in eukaryotic systems, whereas very little is known about membrane fusion in prokaryotes. Haloarchaeal pleomorphic viruses (HRPVs) have a membrane envelope decorated with spikes that are presumed to be responsible for host attachment and membrane fusion. Here we determine atomic structures of the ectodomains of the 57-kDa spike protein VP5 from two related HRPVs revealing a previously unreported V-shaped fold. By Volta phase plate cryo-electron tomography we show that VP5 is monomeric on the viral surface, and we establish the orientation of the molecules with respect to the viral membrane. We also show that the viral membrane fuses with the host cytoplasmic membrane in a process mediated by VP5. This sheds light on protein structures involved in prokaryotic membrane fusion.Peer reviewe

Mutations involving the SRY-related gene SOX8 are associated with a spectrum of human reproductive anomalies.

© The Author(s) 2018. Published by Oxford University Press. All rights reserved. SOX8 is an HMG-box transcription factor closely related to SRY and SOX9. Deletion of the gene encoding Sox8 in mice causes reproductive dysfunction but the role of SOX8 in humans is unknown. Here, we show that SOX8 is expressed in the somatic cells of the early developing gonad in the human and influences human sex determination. We identified two individuals with 46, XY disorders/differences in sex development (DSD) and chromosomal rearrangements encompassing the SOX8 locus and a third individual with 46, XY DSD and a missense mutation in the HMG-box of SOX8. In vitro functional assays indicate that this mutation alters the biological activity of the protein. As an emerging body of evidence suggests that DSDs and infertility can have common etiologies, we also analysed SOX8 in a cohort of infertile men (n=274) and two independent cohorts of women with primary ovarian insufficiency (POI; n=153 and n=104). SOX8 mutations were found at increased frequency in oligozoospermic men (3.5%; P < 0.05) and POI (5.06%; P=4.5×10 -5 ) as compared with fertile/normospermic control populations (0.74%). The mutant proteins identified altered SOX8 biological activity as compared with the wild-type protein. These data demonstrate that SOX8 plays an important role in human reproduction and SOX8 mutations contribute to a spectrum of phenotypes including 46, XY DSD, male infertility and 46, XX POI.Link_to_subscribed_fulltex

Molecular organization and dynamics of the fusion protein Gc at the hantavirus surface

International audienceThe hantavirus envelope glycoproteins Gn and Gc mediate virion assembly and cell entry, with Gc driving fusion of viral and endosomal membranes. Although the X-ray structures and overall arrangement of Gn and Gc on the hantavirus spikes are known, their detailed interactions are not. Here we show that the lateral contacts between spikes are mediated by the same 2-fold contacts observed in Gc crystals at neutral pH, allowing the engineering of disulfide bonds to cross-link spikes. Disrupting the observed dimer interface affects particle assembly and overall spike stability. We further show that the spikes display a temperature-dependent dynamic behavior at neutral pH, alternating between ‘open’ and ‘closed’ forms. We show that the open form exposes the Gc fusion loops but is off-pathway for productive Gc-induced membrane fusion and cell entry. These data also provide crucial new insights for the design of optimized Gn/Gc immunogens to elicit protective immune responses

The Hantavirus Surface Glycoprotein Lattice and Its Fusion Control Mechanism

Hantaviruses are rodent-borne viruses causing serious zoonotic outbreaks worldwide for which no treatment is available. Hantavirus particles are pleomorphic and display a characteristic square surface lattice. The envelope glycoproteins Gn and Gc form heterodimers that further assemble into tetrameric spikes, the lattice building blocks. The glycoproteins, which are the sole targets of neutralizing antibodies, drive virus entry via receptor-mediated endocytosis and endosomal membrane fusion. Here we describe the high-resolution X-ray structures of the heterodimer of Gc and the Gn head and of the homotetrameric Gn base. Docking them into an 11.4-angstrom-resolution cryoelectron tomography map of the hantavirus surface accounted for the complete extramembrane portion of the viral glycoprotein shell and allowed a detailed description of the surface organization of these pleomorphic virions. Our results, which further revealed a built-in mechanism controlling Gc membrane insertion for fusion, pave the way for immunogen design to protect against pathogenic hantaviruses.Peer reviewe

Inter-protomer interactions unique to PUUV G_C.

<p>(A) Strand A₀ at the N-terminus of domain I extends the B₀-I₀-H₀-G₀ β-sheet of the adjacent protomer. The donor protomer (protomer 1) is indicated in the same color scheme as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005948#ppat.1005948.g001" target="_blank">Fig 1</a> while the neighboring protomer (protomer 2) is shown in faded colors. (B) Inter-trimer salt bridge at the membrane proximal part of domain II. Ionic pairs are in sticks representation. The boundaries of each protomer are highlighted. (C) The glycosylation on N937 mediates interactions between protomers. Right: view of the trimer from the membrane, down the crystallographic three-fold axis. Left: Close-up view on the glycosylation groove between the protomers. N937 and the glycans are in sticks representation. 2F_O-F_C electron density map at 1σ is shown in light blue mesh.</p

Structural and evolutionary relationships in class II fusion proteins.

<p>Cladograms representing the structural relationships between different class II fusion proteins. The coordinates of PUUV G_C were submitted to the DALI server. Atomic coordinates were obtained from the Protein Data Bank (PDB). Structure based alignment of the collected coordinates was performed with MUSTANG. A dendrogram was estimated based on a neighbor-joining analysis of the aligned sequences and guided by the BLOSUM62 substitution matrix. Abbreviations and their respective PDBs are as follows: dengue virus 2 (DENV-2, 1OK8-A), tick-borne encephalitis virus (TBEV, 1SVB-A), dengue virus 3 (DENV-3, 1UZG-A), Semliki forest virus (SFV, 1RER-A), West Nile virus (WNV, 2I69-A), Dengue virus 1 (DENV-1, 3G7T-A), Sindbis virus (SINV, 3MUU-A), Chikugunya virus (CHV, 3N41-F), Japanese encephalitis virus (JEV, 3P54-A), rubella virus (RV, 4ADG-A), St. Louis encephalitis virus (SLEV, 4FG0-A), Rift valley fever virus (RVFV, 4HJ1). <i>C</i>.<i>elegans</i> EFF1 (4OJC-A) was added as an out-group. Color scheme is per legend. Icosahedron icon represents icosahedral envelope, <b>P</b>- pleomorphic envelope. Genome type and transmission vectors are indicated by representative symbols.</p

Hinge motions in PUUV sG_C protomers.

<p>(A) Superposition of individual domains of sG_C^XF1 (color) and sG_C^XF2 (light grey). Root mean square deviation (RMSD, calculated in PyMol) for domain I, II and III are 0.339 Å, 0.483 Å, 0.227 Å respectively. (B) B-factor putty representation of the two crystal structures of PUUV sG_C. Cold colors (blue-green) represent lower B-factors whereas warm colors (yellow-red) represent high B-factors. In the inset is a ribbon representation of sG_C^XF1 (color) and sG_C^XF2 (light grey) in the same orientation as the putty representation. In red is the crystallographic 3-fold axis. (C) A quantifying B-factor analysis of the two PUUV sG_C crystal forms. Analysis was executed using bavarage module in CCP4 program suite (61). In black is sG_C^XF1 and red is sG_C^XF2. Linear domain organization is shown for orientation. Color scheme and domains borders are as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005948#ppat.1005948.g001" target="_blank">Fig 1</a>. (D) A view on the fusion loop down the three-fold axis of sG_C^XF1 (color) and sG_C^XF2 (light grey) superposition. Triangles represent the distances between the C_α^W773 of the protomers. The distance in sG_C^XF1 (pH 6.0) is 11.0 Å whereas in sG_C^XF2 (pH 8.0) it is 14.9 Å. Triangles area for pH 6.0 and pH 8.0 are 52.4 Ų and 114.1 Ų, respectively. (E) E770-R902 inter-protomer salt bridge at the two crystal forms. Color scheme is as in panel B.</p