Maturation-Induced Cloaking of Neutralization Epitopes on HIV-1 Particles

To become infectious, HIV-1 particles undergo a maturation process involving proteolytic cleavage of the Gag and Gag-Pol polyproteins. Immature particles contain a highly stable spherical Gag lattice and are impaired for fusion with target cells. The fusion impairment is relieved by truncation of the gp41 cytoplasmic tail (CT), indicating that an interaction between the immature viral core and gp41 within the particle represses HIV-1 fusion by an unknown mechanism. We hypothesized that the conformation of Env on the viral surface is regulated allosterically by interactions with the HIV-1 core during particle maturation. To test this, we quantified the binding of a panel of monoclonal antibodies to mature and immature HIV-1 particles by immunofluorescence imaging. Surprisingly, immature particles exhibited markedly enhanced binding of several gp41-specific antibodies, including two that recognize the membrane proximal external region (MPER) and neutralize diverse HIV-1 strains. Several of the differences in epitope exposure on mature and immature particles were abolished by truncation of the gp41 CT, thus linking the immature HIV-1 fusion defect with altered Env conformation. Our results suggest that perturbation of fusion-dependent Env conformational changes contributes to the impaired fusion of immature particles. Masking of neutralization-sensitive epitopes during particle maturation may contribute to HIV-1 immune evasion and has practical implications for vaccine strategies targeting the gp41 MPER

Mutations in DCHS1 Cause Mitral Valve Prolapse

SUMMARY Mitral valve prolapse (MVP) is a common cardiac valve disease that affects nearly 1 in 40 individuals1–3. It can manifest as mitral regurgitation and is the leading indication for mitral valve surgery4,5. Despite a clear heritable component, the genetic etiology leading to non-syndromic MVP has remained elusive. Four affected individuals from a large multigenerational family segregating non-syndromic MVP underwent capture sequencing of the linked interval on chromosome 11. We report a missense mutation in the DCHS1 gene, the human homologue of the Drosophila cell polarity gene dachsous (ds) that segregates with MVP in the family. Morpholino knockdown of the zebrafish homolog dachsous1b resulted in a cardiac atrioventricular canal defect that could be rescued by wild-type human DCHS1, but not by DCHS1 mRNA with the familial mutation. Further genetic studies identified two additional families in which a second deleterious DCHS1 mutation segregates with MVP. Both DCHS1 mutations reduce protein stability as demonstrated in zebrafish, cultured cells, and, notably, in mitral valve interstitial cells (MVICs) obtained during mitral valve repair surgery of a proband. Dchs1+/− mice had prolapse of thickened mitral leaflets, which could be traced back to developmental errors in valve morphogenesis. DCHS1 deficiency in MVP patient MVICs as well as in Dchs1+/− mouse MVICs result in altered migration and cellular patterning, supporting these processes as etiological underpinnings for the disease. Understanding the role of DCHS1 in mitral valve development and MVP pathogenesis holds potential for therapeutic insights for this very common disease

Interruption of CXCL13-CXCR5 Axis Increases Upper Genital Tract Pathology and Activation of NKT Cells following Chlamydial Genital Infection

Peer reviewe

Immunization with MOMP-vaults reduces local bacterial burden following genital infection.

<p>The bacterial burden of chlamydiae following a challenge infection was determined from vaginal swabs to be statistically reduced in the MOMP-vault immunized group and the positive control group immunized i.n. with live <i>C. muridarum</i> (Live CM) compared to the control GL-vault immunized group (two-way RM ANOVA, *p<0.005). Dunn's post-hoc test showed no difference between the Live CM and the MOMP-vault immunized groups.</p

Design of recombinant vault nanoparticles containing immunogenic proteins.

<p>(a) ELISA assay configured using vaults with the z peptide (cp-MVP-z) and without (cp-MVP) reacted with either mouse IgG or IgA. Data points represent duplicates, SD = 0.004–0.034 nm. (b) Schematic diagram of constructs used to prepare baculovirus recombinant vaults containing MOMP indicating their approximate locations within a vault. (c) Western blot of high speed pellet extracts of MOMP-vaults (5 µg/lane). Molecular weight markers (lane 1). The gel was probed with a monoclonal antibody against the VD1 region of MOMP (MoPn-40)(lane 2) or mouse 1023C monoclonal antibody against MVP (lane 3). The size of MOMP fused to mINT is shown in the box and is approximately 58 kDa. (d) Negative stain EM of cp-MVP and cp-MVP-z/MOMP-mINT recombinant vaults. Bar, 100 nm.</p

MOMP-vault immunization enhances the number and induces the redistribution of Th1 cells in the ILN.

<p>(a) Representative histogram showing CD3+CD4+ cells and IFNγ vs. IL-4 intracellular cytokine staining of ILN cells. The number of (b) Th1 or (c) Th2 cells in the ILNs of individual mice on days 7 and 15 after challenge infection were compared by Students' t-test (*p<0.02). Representative experiment with 4–5 mice per group where at least 50,000 cells were analyzed per mouse. The level of (d) IL-1α, (e) IFNγ, and (f) CXCL10 was measured in triplicate in OD tissue homogenates at two time-points after challenge infection in mice immunized with MOMP-vaults or given a lung infection with <i>C. muridarum</i> using Luminex assays (Milipore Corp). Cytokine levels were compared by Students' t-test (*p<0.05). One representative experiment of 3–5 mice per group.</p

Dendritic cells are efficient at incorporating vaults.

<p>(a) BMDC (1×10⁶) were incubated with media, GL-vaults (500 µg) or FITC-dextran (250 µg) at 37°C for the indicated times. Cells were stained for a marker on mouse BMDC (CD11c-PE) and analyzed using a flow cytometer. (b) BMDC (red) incubated as above for 30 min were viewed on a fluorescent microscope (Carl Zeiss, LSM5 Pascal). The fluorescent particles (GL-vaults or FITC-dextran) appear green and mouse BMDC appear red due to staining with CD11c-PE. Results shown are representative of three experiments.</p

SLC6A1 variant pathogenicity, molecular function, and phenotype: a genetic and clinical analysis

Genetic variants in the SLC6A1 gene can cause a broad phenotypic disease spectrum by altering the protein function. Thus, systematically curated clinically relevant genotype-phenotype associations are needed to understand the disease mechanism and improve therapeutic decision-making. We aggregated genetic and clinical data from 172 individuals with likely pathogenic/pathogenic (lp/p) SLC6A1 variants and functional data for 184 variants (14.1% lp/p). Clinical and functional data were available for a subset of 126 individuals. We explored the potential associations of variant positions on the GAT1 3D structure with variant pathogenicity, altered molecular function, and phenotype severity using bioinformatic approaches. The GAT1 transmembrane domains 1, 6, and extracellular loop 4 (EL4) were enriched for patient over population variants. Across functionally tested missense variants (n = 156), the spatial proximity from the ligand was associated with loss-of-function in the GAT1 transporter activity. For variants with complete loss of in vitro GABA uptake, we found a 4.6-fold enrichment in patients having severe disease vs. non-severe disease (P = 2.9e-3, 95% CI: 1.5 - 15.3). In summary, we delineated associations between the 3D structure and variant pathogenicity, variant function, and phenotype in SLC6A1-related disorders. This knowledge supports biology-informed variant interpretation and research on GAT1 function. All our data can be interactively explored in the SLC6A1 Portal (https://slc6a1-portal.broadinstitute.org/)