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A chimeric vesicular stomatitis virus with the glycoprotein of the lymphocytic choriomeningitis virus, VSV-GP, is a potent viral vaccine vector that overcomes several of the limitations of wild-type VSV. Here, we evaluated the potential of VSV-GP as an HIV vaccine vector. We introduced genes for different variants of the HIV-1 envelope protein Env, i.e., secreted or membrane-anchored, intact or mutated furin cleavage site or different C-termini, into the genome of VSV-GP. We found that the addition of the Env antigen did not attenuate VSV-GP replication. All HIV-1 Env variants were expressed in VSV-GP infected cells and some were incorporated very efficiently into VSV-GP particles. Crucial epitopes for binding of broadly neutralizing antibodies against HIV-1 such as MPER (membrane-proximal external region), CD4 binding site, V1V2 and V3 loop were present on the surface of VSV-GP-Env particles. Binding of quaternary antibodies indicated a trimeric structure of VSV-GP incorporated Env. We detected high HIV-1 antibody titers in mice and showed that vectors expressing membrane-anchored Env elicited higher antibody titers than vectors that secreted Envs. In rabbits, Tier 1A HIV-1 neutralizing antibodies were detectable after prime immunization and titers further increased after boosting with a second immunization. Taken together, VSV-GP-Env is a promising vector vaccine against HIV-1 infection since this vector permits incorporation of native monomeric and/or trimeric HIV-1 Env into a viral membrane.
The multifunctional virion protein 35 (VP35) of ebolaviruses is a critical determinant of virulence and pathogenesis indispensable for viral replication and host innate immune evasion. Essential for VP35 function is homo-oligomerization via a coiled-coil motif. Here we report crystal structures of VP35 oligomerization domains from the prototypic Ebola virus (EBOV) and the non-pathogenic Reston virus (RESTV), together with a comparative biophysical characterization of the domains from all known species of the Ebolavirus genus. EBOV and RESTV VP35 oligomerization domains form bipartite parallel helix bundles with a canonical coiled coil in the N-terminal half and increased plasticity in the highly conserved C-terminal half. The domain assembles into trimers and tetramers in EBOV, whereas it exclusively forms tetramers in all other ebolavirus species. Substitution of coiled-coil leucine residues critical for immune antagonism leads to aberrant oligomerization. A conserved arginine involved in inter-chain salt bridges stabilizes the VP35 oligomerization domain and modulates between coiled-coil oligomeric states.
Endosomal sorting complexes required for transport (ESCRT) recognize ubiquitinated cargo and catalyze diverse budding processes including multivesicular body biogenesis, enveloped virus egress, and cytokinesis. We present the crystal structure of an N-terminal fragment of the deubiquitinating enzyme AMSH (AMSHΔC) in complex with the C-terminal region of ESCRT-III CHMP3 (CHMP3ΔN). AMSHΔC folds into an elongated 90 Å long helical assembly that includes an unusual MIT domain. CHMP3ΔN is unstructured in solution and helical in complex with AMSHΔC, revealing a novel MIT domain interacting motif (MIM) that does not overlap with the CHMP1-AMSH binding site. ITC and SPR measurements demonstrate an unusual high-affinity MIM-MIT interaction. Structural analysis suggests a regulatory role for the N-terminal helical segment of AMSHΔC and its destabilization leads to a loss of function during HIV-1 budding. Our results indicate a tight coupling of ESCRT-III CHMP3 and AMSH functions and provide insight into the regulation of ESCRT-III.
TRiC/CCT is a highly conserved and essential chaperonin that uses ATP cycling to facilitate folding of approximately 10% of the eukaryotic proteome. This 1 MDa hetero-oligomeric complex consists of two stacked rings of eight paralogous subunits each. Previously proposed TRiC models differ substantially in their subunit arrangements and ring register. Here, we integrate chemical crosslinking, mass spectrometry, and combinatorial modeling to reveal the definitive subunit arrangement of TRiC. In vivo disulfide mapping provided additional validation for the crosslinking-derived arrangement as the definitive TRiC topology. This subunit arrangement allowed the refinement of a structural model using existing X-ray diffraction data. The structure described here explains all available crosslink experiments, provides a rationale for previously unexplained structural features, and reveals a surprising asymmetry of charges within the chaperonin folding chamber.
Down-regulation of plasma membrane receptors via the endocytic pathway involves their monoubiquitylation, transport to endosomal membranes and eventual sorting into multi vesicular bodies (MVB) destined for lysosomal degradation. Successive assemblies of Endosomal Sorting Complexes Required for Transport (ESCRT-I, -II and III) largely mediate sorting of plasma membrane receptors at endosomal membranes, the formation of multivesicular bodies and their release into the endosomal lumen. In addition, the human ESCRT-II has been shown to form a complex with RNA polymerase II elongation factor ELL in order to exert transcriptional control activity.
Unprecedented by number of casualties and socio-economic burden occurring worldwide, the coronavirus disease 2019 (Covid-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the worst health crisis of this century. In order to develop adequate countermeasures against Covid-19, identification and structural characterization of suitable antiviral targets within the SARS-CoV-2 protein repertoire is urgently needed. The nucleocapsid phosphoprotein (N) is a multifunctional and highly immunogenic determinant of virulence and pathogenicity, whose main functions consist in oligomerizing and packaging the single-stranded RNA (ssRNA) viral genome. Here we report the structural and biophysical characterization of the SARS-CoV-2 N C-terminal domain (CTD), on which both N homo-oligomerization and ssRNA binding depend. Crystal structures solved at 1.44 Å and 1.36 Å resolution describe a rhombus-shape N CTD dimer, which stably exists in solution as validated by size-exclusion chromatography coupled to multi-angle light scattering and analytical ultracentrifugation. Differential scanning fluorimetry revealed moderate thermal stability and a tendency towards conformational change. Microscale thermophoresis demonstrated binding to a 7-bp SARS-CoV-2 genomic ssRNA fragment at micromolar affinity. Furthermore, a low-resolution preliminary model of the full-length SARS-CoV N in complex with ssRNA, obtained by cryo-electron microscopy, provides an initial understanding of self-associating and RNA binding functions exerted by the SARS-CoV-2 N.
Potent and broadly neutralizing antibodies (bnAbs) are the hallmark of HIV-1 protection by vaccination. The membrane-proximal external region (MPER) of the HIV-1 gp41 fusion protein is targeted by the most broadly reactive HIV-1 neutralizing antibodies. Here, we examine the structural and molecular mechansims of neutralization by anti-MPER bnAb, LN01, which was isolated from lymph-node-derived germinal center B cells of an elite controller and exhibits broad neutralization breadth. LN01 engages both MPER and the transmembrane (TM) region, which together form a continuous helix in complex with LN01. The tilted TM orientation allows LN01 to interact simultaneously with the peptidic component of the MPER epitope and membrane via two specific lipid binding sites of the antibody paratope. Although LN01 carries a high load of somatic mutations, most key residues interacting with the MPER epitope and lipids are germline encoded, lending support for the LN01 epitope as a candidate for lineage-based vaccine development.
Classical antagonistic antibodies (Abs) targeting PD-1, such as pembrolizumab and nivolumab, act through blockade of the PD-1-PDL-1 interaction. Here, we have identified novel antagonistic anti-PD-1 Abs not blocking the PD-1-PDL-1 interaction. The nonblocking Abs recognize epitopes on PD-1 located on the opposing face of the PDL-1 interaction and overlap with a newly identified evolutionarily conserved patch. These nonblocking Abs act predominantly through the CD28 coreceptor. Importantly, a combination of blocking and nonblocking Abs synergize in the functional recovery of antigen-specific exhausted CD8 T cells. Interestingly, nonblocking anti-PD-1 Abs have equivalent antitumor activity compared with blocker Abs in two mouse tumor models, and combination therapy using both classes of Abs enhanced tumor suppression in the mouse immunogenic tumor model. The identification of the novel nonblocker anti-PD-1 Abs and their synergy with classical blocker Abs may be instrumental in potentiating immunotherapy strategies and antitumor activity.
The GroEL/ES chaperonin system functions as a protein folding cage. Many obligate substrates of GroEL share the (βα)8 TIM-barrel fold, but how the chaperonin promotes folding of these proteins is not known. Here, we analyzed the folding of DapA at peptide resolution using hydrogen/deuterium exchange and mass spectrometry. During spontaneous folding, all elements of the DapA TIM barrel acquire structure simultaneously in a process associated with a long search time. In contrast, GroEL/ES accelerates folding more than 30-fold by catalyzing segmental structure formation in the TIM barrel. Segmental structure formation is also observed during the fast spontaneous folding of a structural homolog of DapA from a bacterium that lacks GroEL/ES. Thus, chaperonin independence correlates with folding properties otherwise enforced by protein confinement in the GroEL/ES cage. We suggest that folding catalysis by GroEL/ES is required by a set of proteins to reach native state at a biologically relevant timescale, avoiding aggregation or degradation.
Pseudomonas aeruginosa is the agent of opportunistic infections in immunocompromised individuals and chronic respiratory illnesses in cystic fibrosis patients. Pseudomonas aeruginosa utilizes a type III secretion system for injection of toxins into the host cell cytoplasm through a channel on the target membrane (the 'translocon'). Here, we have functionally and structurally characterized PopB and PopD, membrane proteins implicated in the formation of the P.aeruginosa translocon. PopB and PopD form soluble complexes with their common chaperone, PcrH, either as stable heterodimers or as metastable heterooligomers. Only oligomeric forms are able to bind to and disrupt cholesterol-rich membranes, which occurs within a pH range of 5-7 in the case of PopB/PcrH, and only at acidic pH for PcrH-free PopD. Electron microscopy reveals that upon membrane association PopB and PopD form 80 A wide rings which encircle 40 A wide cavities. Thus, formation of metastable oligomers precedes membrane association and ring generation in the formation of the Pseudomonas translocon, a mechanism which may be similar for other pathogens that employ type III secretion systems.
Gephyrin is a bi-functional modular protein involved in molybdenum cofactor biosynthesis and in postsynaptic clustering of inhibitory glycine receptors (GlyRs). Here, we show that full-length gephyrin is a trimer and that its proteolysis in vitro causes the spontaneous dimerization of its C-terminal region (gephyrin-E), which binds a GlyR beta-subunit-derived peptide with high and low affinity. The crystal structure of the tetra-domain gephyrin-E in complex with the beta-peptide bound to domain IV indicates how membrane-embedded GlyRs may interact with subsynaptic gephyrin. In vitro, trimeric full-length gephyrin forms a network upon lowering the pH, and this process can be reversed to produce stable full-length dimeric gephyrin. Our data suggest a mechanism by which induced conformational transitions of trimeric gephyrin may generate a reversible postsynaptic scaffold for GlyR recruitment, which allows for dynamic receptor movement in and out of postsynaptic GlyR clusters, and thus for synaptic plasticity.
ESCRT-III proteins catalyze membrane fission during multi vesicular body biogenesis, budding of some enveloped viruses and cell division. We suggest and analyze a novel mechanism of membrane fission by the mammalian ESCRT-III subunits CHMP2 and CHMP3. We propose that the CHMP2-CHMP3 complexes self-assemble into hemi-spherical dome-like structures within the necks of the initial membrane buds generated by CHMP4 filaments. The dome formation is accompanied by the membrane attachment to the dome surface, which drives narrowing of the membrane neck and accumulation of the elastic stresses leading, ultimately, to the neck fission. Based on the bending elastic model of lipid bilayers, we determine the degree of the membrane attachment to the dome enabling the neck fission and compute the required values of the protein-membrane binding energy. We estimate the feasible values of this energy and predict a high efficiency for the CHMP2-CHMP3 complexes in mediating membrane fission. We support the computational model by electron tomography imaging of CHMP2-CHMP3 assemblies in vitro. We predict a high efficiency for the CHMP2-CHMP3 complexes in mediating membrane fission.
The membrane-proximal external region (MPER) of gp41 is considered as a prime target for the induction of neutralizing antibodies, since it contains the epitopes for three broadly neutralizing antibodies (2F5, 4E10 and Z13). Here we present a novel gp41 construct (HA-gp41) comprising gp41 HR2 and MPER fused to two triple-stranded coiled-coil domains at both ends. HA-gp41 is trimeric, has a high helical content in solution and forms rod-like structures as revealed by negative staining electron microscopy. Immunization of rabbits with HA-gp41 induced antibodies directed against MPER, which failed to exert significant neutralization capacity against envelopes from primary isolates. Thus trimerisation of MPER regions does not suffice to induce a potent neutralizing antibody response specific for conserved regions within gp41.
One unique feature of the foamy virus (FV) capsid protein Gag is the absence of Cys-His motifs, which in orthoretroviruses are irreplaceable for multitude functions including viral RNA genome recognition and packaging. Instead, FV Gag contains glycine-arginine-rich (GR) sequences at its C-terminus. In case of prototype FV (PFV) these are historically grouped in three boxes, which have been shown to play essential functions in genome reverse transcription, virion infectivity and particle morphogenesis. Additional functions for RNA packaging and Pol encapsidation were suggested, but have not been conclusively addressed.
Plasmacytoid dendritic cells (pDCs) constitute a major source of type-I interferon (IFN-I) production during acute HIV infection. Their activation results primarily from TLR7-mediated sensing of HIV-infected cells. However, the interactions between HIV-infected T cells and pDCs that modulate this sensing process remain poorly understood. BST2/Tetherin is a restriction factor that inhibits HIV release by cross-linking virions onto infected cell surface. BST2 was also shown to engage the ILT7 pDC-specific inhibitory receptor and repress TLR7/9-mediated IFN-I production by activated pDCs. Here, we show that Vpu, the HIV-1 antagonist of BST2, suppresses TLR7-mediated IFN-I production by pDC through a mechanism that relies on the interaction of BST2 on HIV-producing cells with ILT7. Even though Vpu downregulates surface BST2 as a mean to counteract the restriction on HIV-1 release, we also find that the viral protein re-locates remaining BST2 molecules outside viral assembly sites where they are free to bind and activate ILT7 upon cell-to-cell contact. This study shows that through a targeted regulation of surface BST2, Vpu promotes HIV-1 release and limits pDC antiviral responses upon sensing of infected cells. This mechanism of innate immune evasion is likely to be important for an efficient early viral dissemination during acute infection.
The restriction factor BST-2/tetherin contains two membrane anchors employed to retain some enveloped viruses, including HIV-1 tethered to the plasma membrane in the absence of virus-encoded antagonists. The 2.77 A crystal structure of the BST-2/tetherin extracellular core presented here reveals a parallel 90 A long disulfide-linked coiled-coil domain, while the complete extracellular domain forms an extended 170 A long rod-like structure based on small-angle X-ray scattering data. Mutagenesis analyses indicate that both the coiled coil and the N-terminal region are required for retention of HIV-1, suggesting that the elongated structure can function as a molecular ruler to bridge long distances. The structure reveals substantial irregularities and instabilities throughout the coiled coil, which contribute to its low stability in the absence of disulfide bonds. We propose that the irregular coiled coil provides conformational flexibility, ensuring that BST-2/tetherin anchoring both in the plasma membrane and in the newly formed virus membrane is maintained during virus budding.
The vacuolar protein sorting machinery regulates multivesicular body biogenesis and is selectively recruited by enveloped viruses to support budding. Here we report the crystal structure of the human ESCRT-III protein CHMP3 at 2.8 A resolution. The core structure of CHMP3 folds into a flat helical arrangement that assembles into a lattice, mainly via two different dimerization modes, and unilaterally exposes a highly basic surface. The C terminus, the target for Vps4-induced ESCRT disassembly, extends from the opposite side of the membrane targeting region. Mutations within the basic and dimerization regions hinder bilayer interaction in vivo and reverse the dominant-negative effect of a truncated CHMP3 fusion protein on HIV-1 budding. Thus, the final steps in the budding process may include CHMP protein polymerization and lattice formation on membranes by employing different bilayer-recognizing surfaces, a function shared by all CHMP family members.
Broad and potent neutralizing llama single domain antibodies (VHH) against HIV-1 targeting the CD4 binding site (CD4bs) have previously been isolated upon llama immunization. Here we describe the epitopes of three additional VHH groups selected from phage libraries. The 2E7 group binds to a new linear epitope in the first heptad repeat of gp41 that is only exposed in the fusion-intermediate conformation. The 1B5 group competes with co-receptor binding and the 1F10 group interacts with the crown of the gp120 V3 loop, occluded in native Env. We present biophysical and structural details on the 2E7 interaction with gp41. In order to further increase breadth and potency, we constructed bi-specific VHH. The combination of CD4bs VHH (J3/3E3) with 2E7 group VHH enhanced strain-specific neutralization with potencies up to 1400-fold higher than the mixture of the individual VHHs. Thus, these new bivalent VHH are potent new tools to develop therapeutic approaches or microbicide intervention.
Rubisco, the key enzyme of CO2 fixation in photosynthesis, is prone to inactivation by inhibitory sugar phosphates. Inhibited Rubisco undergoes conformational repair by the hexameric AAA+ chaperone Rubisco activase (Rca) in a process that is not well understood. Here, we performed a structural and mechanistic analysis of cyanobacterial Rca, a close homolog of plant Rca. In the Rca:Rubisco complex, Rca is positioned over the Rubisco catalytic site under repair and pulls the N-terminal tail of the large Rubisco subunit (RbcL) into the hexamer pore. Simultaneous displacement of the C terminus of the adjacent RbcL opens the catalytic site for inhibitor release. An alternative interaction of Rca with Rubisco is mediated by C-terminal domains that resemble the small Rubisco subunit. These domains, together with the N-terminal AAA+ hexamer, ensure that Rca is packaged with Rubisco into carboxysomes. The cyanobacterial Rca is a dual-purpose protein with functions in Rubisco repair and carboxysome organization.
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