Understanding kidney disease relies upon defining the complexity of cell types and states, their associated molecular profiles, and interactions within tissue neighborhoods. We have applied multiple single-cell or -nucleus assays (>400,000 nuclei/cells) and spatial imaging technologies to a broad spectrum of healthy reference (n = 42) and disease (n = 42) kidneys. This has provided a high resolution cellular atlas of 100 cell types that include rare and novel cell populations. The multi-omic approach provides detailed transcriptomic profiles, epigenomic regulatory factors, and spatial localizations for major cell types spanning the entire kidney. We further identify and define cellular states altered in kidney injury, encompassing cycling, adaptive or maladaptive repair, transitioning and degenerative states affecting several segments. Molecular signatures of these states permitted their localization within injury neighborhoods using spatial transcriptomics, and large-scale 3D imaging analysis of ~1.2 million neighborhoods provided linkages to active immune responses. These analyses further defined biological pathways relevant to injury niches, including signatures underlying the transition from reference to predicted maladaptive states that were associated with a decline in kidney function during chronic kidney disease. This human kidney cell atlas, including injury cell states and neighborhoods, will be a valuable resource for future studies.

Marburg virus (MARV) is a lipid-enveloped virus from the Filoviridae family containing a negative sense RNA genome. One of the seven MARV genes encodes the matrix protein VP40, which forms a matrix layer beneath the plasma membrane inner leaflet to facilitate budding from the host cell. MARV VP40 (mVP40) has been shown to be a dimeric peripheral protein with a broad and flat basic surface that can associate with anionic phospholipids such as phosphatidylserine. Although a number of mVP40 cationic residues have been shown to facilitate binding to membranes containing anionic lipids, much less is known on how mVP40 assembles to form the matrix layer following membrane binding. Here we have used hydrogen/deuterium exchange (HDX) mass spectrometry to determine the solvent accessibility of mVP40 residues in the absence and presence of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate. HDX analysis demonstrates that two basic loops in the mVP40 C-terminal domain make important contributions to anionic membrane binding and also reveals a potential oligomerization interface in the C-terminal domain as well as a conserved oligomerization interface in the mVP40 N-terminal domain. Lipid binding assays confirm the role of the two basic patches elucidated with HD/X measurements, whereas molecular dynamics simulations and membrane insertion measurements complement these studies to demonstrate that mVP40 does not appreciably insert into the hydrocarbon region of anionic membranes in contrast to the matrix protein from Ebola virus. Taken together, we propose a model by which association of the mVP40 dimer with the anionic plasma membrane facilitates assembly of mVP40 oligomers.

Descriptions of materials, metrological methods, computational methods, and supplementary results. Figures of HDX-MS publications and citations versus publication year, histogram of peptide sequence lengths, sequence coverage maps, performance of instrumentsoftware configurations, repeatability plots, %E corrected peptide t HDX versus log 10 (t HDX ) for eight peptides. Tables of instrumentation,software, peptide search methodology, and operating conditions of proteolytic, chromatographic components, and effects of peptide charge on deuterium uptake (PDF)

Marburg virus (MARV) is a highly pathogenic filovirus that is classified in a genus distinct from that of Ebola virus (EBOV) (genera Marburgvirus and Ebolavirus, respectively). Both viruses produce a multifunctional protein termed VP35, which acts as a polymerase cofactor, a viral protein chaperone, and an antagonist of the innate immune response. VP35 contains a central oligomerization domain with a predicted coiled-coil motif. This domain has been shown to be essential for RNA polymerase function. Here we present crystal structures of the MARV VP35 oligomerization domain. These structures and accompanying biophysical characterization suggest that MARV VP35 is a trimer. In contrast, EBOV VP35 is likely a tetramer in solution. Differences in the oligomeric state of this protein may explain mechanistic differences in replication and immune evasion observed for MARV and EBOV.IMPORTANCE Marburg virus can cause severe disease, with up to 90% human lethality. Its genome is concise, only producing seven proteins. One of the proteins, VP35, is essential for replication of the viral genome and for evasion of host immune responses. VP35 oligomerizes (self-assembles) in order to function, yet the structure by which it assembles has not been visualized. Here we present two crystal structures of this oligomerization domain. In both structures, three copies of VP35 twist about each other to form a coiled coil. This trimeric assembly is in contrast to tetrameric predictions for VP35 of Ebola virus and to known structures of homologous proteins in the measles, mumps, and Nipah viruses. Distinct oligomeric states of the Marburg and Ebola virus VP35 proteins may explain differences between them in polymerase function and immune evasion. These findings may provide a more accurate understanding of the mechanisms governing VP35's functions and inform the design of therapeutics. KEYWORDS VP35, filovirus, RNA-dependent RNA polymerase, phosphoprotein, coiled coil, oligomerization, Marburg virus, Ebola virus, X-ray crystallography M arburg virus (MARV) can cause severe hemorrhagic fever in humans with high case fatality rates. Though less well known than its relative Ebola virus (EBOV), MARV was the first filovirus identified. MARV has caused sporadic outbreaks since its identification in 1968, including the 1998 to 2000 outbreak in the Democratic Republic of the Congo, which occurred with 83% lethality, and the 2004 to 2005 outbreak in Angola, which occurred with 90% lethality (1). Currently, there are no approved vaccines or therapeutics available to treat individuals infected with MARV. Despite sharing ϳ50% nucleotide sequence identity, MARV and EBOV have several striking functional differences. MARV does not produce sGP or ssGP protein and does not require VP30 for transcription. MARV and EBOV also exhibit differences in their immune evasion strategies (2).

Understanding kidney disease relies on defining the complexity of cell types and states, their associated molecular profiles and interactions within tissue neighbourhoods1. Here we applied multiple single-cell and single-nucleus assays (>400,000 nuclei or cells) and spatial imaging technologies to a broad spectrum of healthy reference kidneys (45 donors) and diseased kidneys (48 patients). This has provided a high-resolution cellular atlas of 51 main cell types, which include rare and previously undescribed cell populations. The multi-omic approach provides detailed transcriptomic profiles, regulatory factors and spatial localizations spanning the entire kidney. We also define 28 cellular states across nephron segments and interstitium that were altered in kidney injury, encompassing cycling, adaptive (successful or maladaptive repair), transitioning and degenerative states. Molecular signatures permitted the localization of these states within injury neighbourhoods using spatial transcriptomics, while large-scale 3D imaging analysis (around 1.2 million neighbourhoods) provided corresponding linkages to active immune responses. These analyses defined biological pathways that are relevant to injury time-course and niches, including signatures underlying epithelial repair that predicted maladaptive states associated with a decline in kidney function. This integrated multimodal spatial cell atlas of healthy and diseased human kidneys represents a comprehensive benchmark of cellular states, neighbourhoods, outcome-associated signatures and publicly available interactive visualizations.

PIP3-dependent Rac exchanger 1 (P-Rex1) is activated downstream of G protein–coupled receptors to promote neutrophil migration and metastasis. The structure of more than half of the enzyme and its regulatory G protein binding site are unknown. Our 3.2 Å cryo-EM structure of the P-Rex1–Gβγ complex reveals that the carboxyl-terminal half of P-Rex1 adopts a complex fold most similar to those of Legionella phosphoinositide phosphatases. Although catalytically inert, the domain coalesces with a DEP domain and two PDZ domains to form an extensive docking site for Gβγ. Hydrogen-deuterium exchange mass spectrometry suggests that Gβγ binding induces allosteric changes in P-Rex1, but functional assays indicate that membrane localization is also required for full activation. Thus, a multidomain assembly is key to the regulation of P-Rex1 by Gβγ and the formation of a membrane-localized scaffold optimized for recruitment of other signaling proteins such as PKA and PTEN.

18Ebola virus (EBOV) causes sever hemorrhagic fever in humans, can cause death 19 in a large percentage of those infected, and still lacks FDA approved treatment 20 options. In this study, we investigated how the essential EBOV protein, VP40, 21 forms stable oligomers to mediate budding and assembly from the host cell 22 plasma membrane. An array of in vitro and cellular assays identified and 23 characterized two lysine rich regions that bind to PI(4,5)P 2 and serve distinct 24 VP40 is the matrix protein of EBOV and required for viral assembly, budding 48 and viral spread [11][12][13][14][15][16] . As the most abundantly expressed EBOV protein, VP40 49 achieves several distinct and essential tasks to ensure viral success. VP40 is a 50 transformer protein with several structures known, including a monomer 17 , dimer, 51 hexamer and octamer 18 . Initially, VP40 enters the nucleus of the host cell post 52 viral entry 19 , likely binds RNA as an octamer and regulates viral genome 53 replication 18,20 . Subsequently, VP40 dimers interact with host lipids such as 54 phosphatidylserine (PS) 21,22 to localize to the plasma membrane (PM) 18,22 and 55 form stable hexamers 18,21,23-25 . Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) 56 at the host PM inner leaflet is required for large VP40 oligomers to form 26 from 57 PS induced VP40 hexamers 18,[21][22][23][24][25] . 58Despite an important role for PS and PI(4,5)P 2 in the EBOV life cycle and 59 budding from the host cell PM, the molecular basis and structural consequences 60 of VP40-lipid interactions are mostly unknown. Notably, the VP40 dimer is 61 necessary for PM localization whereas the monomer is not sufficient for 62 trafficking to the PM 18,22 . Dimer interactions with PM lipids are purported to 63 induce structural changes to form VP40 hexamers 18,21,[23][24][25]27 and larger 64 oligomers forming the viral matrix layer 23,26 . Notably, the VP40 octamer has been 65 shown to play an important role in the regulation of viral transcription 18 and has 66 not been detected at the PM nor in virions or VLPs. Additionally, the VP40 67 octamer was found to have a significant reduction in PS affinity compared to the 68 VP40 dimer 22 . 69

Seasonal influenza virus infections can cause significant morbidity and mortality, but the threat from the emergence of a new pandemic influenza strain might have potentially even more devastating consequences. As such, there is intense interest in isolating and characterizing potent neutralizing antibodies that target the hemagglutinin (HA) viral surface glycoprotein. Here, we use cryo-electron microscopy (cryoEM) to decipher the mechanism of action of a potent HA head-directed monoclonal antibody (mAb) bound to an influenza H7 HA. The epitope of the antibody is not solvent accessible in the compact, prefusion conformation that typifies all HA structures to date. Instead, the antibody binds between HA head protomers to an epitope that must be partly or transiently exposed in the prefusion conformation. The “breathing” of the HA protomers is implied by the exposure of this epitope, which is consistent with metastability of class I fusion proteins. This structure likely therefore represents an early structural intermediate in the viral fusion process. Understanding the extent of transient exposure of conserved neutralizing epitopes also may lead to new opportunities to combat influenza that have not been appreciated previously.