Biogenesis of Influenza A Virus Hemagglutinin Cross-Protective Stem Epitopes

Antigenic variation in the globular domain of influenza A virus (IAV) hemagglutinin (HA) precludes effective immunity to this major human pathogen. Although the HA stem is highly conserved between influenza virus strains, HA stem-reactive antibodies (StRAbs) were long considered biologically inert. It is now clear, however, that StRAbs reduce viral replication in animal models and protect against pathogenicity and death, supporting the potential of HA stem-based immunogens as drift-resistant vaccines. Optimally designing StRAb-inducing immunogens and understanding StRAb effector functions require thorough comprehension of HA stem structure and antigenicity. Here, we study the biogenesis of HA stem epitopes recognized in cells infected with various drifted IAV H1N1 strains using mouse and human StRAbs. Using a novel immunofluorescence (IF)-based assay, we find that human StRAbs bind monomeric HA in the endoplasmic reticulum (ER) and trimerized HA in the Golgi complex (GC) with similar high avidity, potentially good news for producing effective monomeric HA stem immunogens. Though HA stem epitopes are nestled among several N-linked oligosaccharides, glycosylation is not required for full antigenicity. Rather, as N-linked glycans increase in size during intracellular transport of HA through the GC, StRAb binding becomes temperature-sensitive, binding poorly to HA at 4°C and well at 37°C. A de novo designed, 65-residue protein binds the mature HA stem independently of temperature, consistent with a lack of N-linked oligosaccharide steric hindrance due to its small size. Likewise, StRAbs bind recombinant HA carrying simple N-linked glycans in a temperature-independent manner. Chemical cross-linking experiments show that N-linked oligosaccharides likely influence StRAb binding by direct local effects rather than by globally modifying the conformational flexibility of HA. Our findings indicate that StRAb binding to HA is precarious, raising the possibility that sufficient immune pressure on the HA stem region could select for viral escape mutants with increased steric hindrance from N-linked glycans.</p

Multilayered Mechanism of CD4 Downregulation by HIV-1 Vpu Involving Distinct ER Retention and ERAD Targeting Steps

A key function of the Vpu protein of HIV-1 is the targeting of newly-synthesized CD4 for proteasomal degradation. This function has been proposed to occur by a mechanism that is fundamentally distinct from the cellular ER-associated degradation (ERAD) pathway. However, using a combination of genetic, biochemical and morphological methodologies, we find that CD4 degradation induced by Vpu is dependent on a key component of the ERAD machinery, the VCP-UFD1L-NPL4 complex, as well as on SCFβ-TrCP-dependent ubiquitination of the CD4 cytosolic tail on lysine and serine/threonine residues. When degradation of CD4 is blocked by either inactivation of the VCP-UFD1L-NPL4 complex or prevention of CD4 ubiquitination, Vpu still retains the bulk of CD4 in the ER mainly through transmembrane domain interactions. Addition of a strong ER export signal from the VSV-G protein overrides this retention. Thus, Vpu exerts two distinct activities in the process of downregulating CD4: ER retention followed by targeting to late stages of ERAD. The multiple levels at which Vpu engages these cellular quality control mechanisms underscore the importance of ensuring profound suppression of CD4 to the life cycle of HIV-1

Gut microbiota in a mouse model of fish allergy

9th World Congress onTargeting Microbiota, October 19-21, ParisN

Fish allergy sensitization and gut microbiome: a metagenomic approach in a mouse model

3rd Food Chemistry Conference, 10-12 October 2023, Dresden, GermanyFood allergy is considered one of the major concerns in food safety, being estimated to affect worldwide 6-8% of children and about 2-4% of adults. Fish is one of the most frequent causes of immunoglobulin E (IgE)-mediated food allergy (Type I hypersensitivity). Currently, there is no cure for fish allergy and sensitized individuals have to practice strict, life-long avoidance of fish products in their diets. Parvalbumins beta (β-PRVBs) are considered the major fish allergens. The relationship between the gut microbiome and food allergies is currently a major topic of discussion since food allergies involve the development of dysbiosis, a microbial imbalance caused by immune-related mechanisms. Recent studies provide evidence that individuals, suffering from food allergies, display an intestinal microbiota with a different microbial composition than that present in healthy subjects. Moreover, previous investigations suggest that species belonging to two major bacterial groups, Clostridiales and Bacteroidales, can inhibit the development of food allergies in mice via the induction of different mechanisms that suppress IgE, while enhancing IgA production. The present work describes a designed protocol to evaluate differences in the gut microbiome in a mouse model of fish allergy with/without previous sensitization to beta-Parvalbumin (beta-PRVB). The analysis to identify the microbiome composition is performed by metagenomic methodology in feces samples. 16S rRNA V3-V4 regions are amplified, taxonomic assignment is performed, and differential abundance of taxa is tested. Results show that oral allergen challenge in beta-PRVB sensitized animals did not affect both alpha-diversity (local diversity of the species in a community) and beta-diversity (difference in taxa between communities), but it was able to alter the relative abundance of specific taxa. These results should contribute to identifying novel connections between the development of fish allergy and microbiome modifications and dysbiosisN

Rab22a Regulates the Sorting of Transferrin to Recycling Endosomes

Rab22a is a member of the Rab family of small GTPases that localizes in the endocytic pathway. In CHO cells, expression of canine Rab22a (cRab22a) causes a dramatic enlargement of early endocytic compartments. We wondered whether transferrin recycling is altered in these cells. Expression of the wild-type protein and a GTP hydrolysis-deficient mutant led to the redistribution of transferrin receptor to large cRab22a-positive structures in the periphery of the cell and to a significant decrease in the plasma membrane receptor. Kinetic analysis of transferrin uptake indicates that internalization and early recycling were not affected by cRab22a expression. However, recycling from large cRab22a-positive compartments was strongly inhibited. A similar effect on transferrin transport was observed when human but not canine Rab22a was expressed in HeLa cells. After internalization for short periods of time (5 to 8 min) or at a reduced temperature (16°C), transferrin localized with endogenous Rab22a in small vesicles that did not tubulate with brefeldin A, suggesting that the endogenous protein is present in early/sorting endosomes. Rab22a depletion by small interfering RNA disorganized the perinuclear recycling center and strongly inhibited transferrin recycling. We speculate that Rab22a controls the transport of the transferrin receptor from sorting to recycling endosomes

Linkage analysis of high frequency variants that emerged during rSV12-HA passage.

<p><b>(A)</b> Graphical representation of genetic linkage between selected variants within the three parallel passage 3 rSV12-HA populations, based on PrimerID sequencing. Node size is proportional to relative frequency of the indicated allele across the three populations. Aqua nodes indicate parental amino acids and red nodes represent variant amino acids. Edges connect pairs of amino acids that were linked in greater than 0.02% of all primerID consensus reads examined. <b>(B)</b> Multi-step growth comparison of the indicated viruses in eggs.</p

PrimerID sequencing of HA genes from recombinant WT and SV12-HA following passage.

<p>Recombinant PR8 (WT) and rSV12-HA (SV12) viruses were rescued via reverse genetics. <b>(A)</b> Amino acid variant frequencies within WT (black) and SV12 HA (red) populations collected from rescue supernatants without amplification, as determined by primerID sequencing. (<b>B,C</b>) Amino acid variant frequencies within WT <b>(B)</b> and rSV12-HA <b>(C)</b> populations following three passages in eggs. For each population, all variants over 0.1% are colored to allow them to be visually distinguished. <b>(D)</b> Comparison of amino acid variant frequencies within rSV12-HA populations before and after 3 passages in eggs. Passage 3 data represents mean +/- SD of three independent passage lines. All amino acid numbering indicated on graphs <b>A-C</b> is from initiating methionine, not according to H3 system.</p

Bio-layer interferometry comparison of binding properties of WT, SV12 and SV12-N133T.

<p>To measure binding properties of WT PR8, SV12, and the compensatory glycan addition mutant N133T, we bound biotinylated 3’SLN to sensor at different concentrations, and measured the K_on and K_off of intact purified virions in the presence and absence of the NA inhibitor oseltamivir, respectively. We fit two-phase (association then dissociation) nonlinear regression curves based on the average of 2–3 experiments.</p