IFITM3 restricts influenza A virus entry by blocking the formation of fusion pores following virus-endosome hemifusion

Interferon-induced transmembrane proteins (IFITMs) inhibit infection of diverse enveloped viruses, including the influenza A virus (IAV) which is thought to enter from late endosomes. Recent evidence suggests that IFITMs block virus hemifusion (lipid mixing in the absence of viral content release) by altering the properties of cell membranes. Consistent with this mechanism, excess cholesterol in late endosomes of IFITM-expressing cells has been reported to inhibit IAV entry. Here, we examined IAV restriction by IFITM3 protein using direct virus-cell fusion assay and single virus imaging in live cells. IFITM3 over-expression did not inhibit lipid mixing, but abrogated the release of viral content into the cytoplasm. Although late endosomes of IFITM3-expressing cells accumulated cholesterol, other interventions leading to aberrantly high levels of this lipid did not inhibit virus fusion. These results imply that excess cholesterol in late endosomes is not the mechanism by which IFITM3 inhibits the transition from hemifusion to full fusion. The IFITM3\u27s ability to block fusion pore formation at a post-hemifusion stage shows that this protein stabilizes the cytoplasmic leaflet of endosomal membranes without adversely affecting the lumenal leaflet. We propose that IFITM3 interferes with pore formation either directly, through partitioning into the cytoplasmic leaflet of a hemifusion intermediate, or indirectly, by modulating the lipid/protein composition of this leaflet. Alternatively, IFITM3 may redirect IAV fusion to a non-productive pathway, perhaps by promoting fusion with intralumenal vesicles within multivesicular bodies/late endosomes

The Effect of Electrostatics on the Marginal Cooperativity of an Ultrafast Folding Protein*

Proteins fold up by coordinating the different segments of their polypeptide chain through a network of weak cooperative interactions. Such cooperativity results in unfolding curves that are typically sigmoidal. However, we still do not know what factors modulate folding cooperativity or the minimal amount that ensures folding into specific three-dimensional structures. Here, we address these issues on BBL, a small helical protein that folds in microseconds via a marginally cooperative downhill process (Li, P., Oliva, F. Y., Naganathan, A. N., and Muñoz, V. (2009) Proc. Natl. Acad. Sci. USA. 106, 103–108). Particularly, we explore the effects of salt-induced screening of the electrostatic interactions in BBL at neutral pH and in acid-denatured BBL. Our results show that electrostatic screening stabilizes the native state of the neutral and protonated forms, inducing complete refolding of acid-denatured BBL. Furthermore, without net electrostatic interactions, the unfolding process becomes much less cooperative, as judged by the broadness of the equilibrium unfolding curve and the relaxation rate. Our experiments show that the marginally cooperative unfolding of BBL can still be made twice as broad while the protein retains its ability to fold into the native three-dimensional structure in microseconds. This result demonstrates experimentally that efficient folding does not require cooperativity, confirming predictions from theory and computer simulations and challenging the conventional biochemical paradigm. Furthermore, we conclude that electrostatic interactions are an important factor in determining folding cooperativity. Thus, electrostatic modulation by pH-salt and/or mutagenesis of charged residues emerges as an attractive tool for tuning folding cooperativity

Molecular Details of Olfactomedin Domains Provide Pathway to Structure-Function Studies.

Olfactomedin (OLF) domains are found within extracellular, multidomain proteins in numerous tissues of multicellular organisms. Even though these proteins have been implicated in human disorders ranging from cancers to attention deficit disorder to glaucoma, little is known about their structure(s) and function(s). Here we biophysically, biochemically, and structurally characterize OLF domains from H. sapiens olfactomedin-1 (npoh-OLF, also called noelin, pancortin, OLFM1, and hOlfA), and M. musculus gliomedin (glio-OLF, also called collomin, collmin, and CRG-L2), and compare them with available structures of myocilin (myoc-OLF) recently reported by us and R. norvegicus glio-OLF and M. musculus latrophilin-3 (lat3-OLF) by others. Although the five-bladed β-propeller architecture remains unchanged, numerous physicochemical characteristics differ among these OLF domains. First, npoh-OLF and glio-OLF exhibit prominent, yet distinct, positive surface charges and copurify with polynucleotides. Second, whereas npoh-OLF and myoc-OLF exhibit thermal stabilities typical of human proteins near 55°C, and most myoc-OLF variants are destabilized and highly prone to aggregation, glio-OLF is nearly 20°C more stable and significantly more resistant to chemical denaturation. Phylogenetically, glio-OLF is most similar to primitive OLFs, and structurally, glio-OLF is missing distinguishing features seen in OLFs such as the disulfide bond formed by N- and C- terminal cysteines, the sequestered Ca2+ ion within the propeller central hydrophilic cavity, and a key loop-stabilizing cation-π interaction on the top face of npoh-OLF and myoc-OLF. While deciphering the explicit biological functions, ligands, and binding partners for OLF domains will likely continue to be a challenging long-term experimental pursuit, we used structural insights gained here to generate a new antibody selective for myoc-OLF over npoh-OLF and glio-OLF as a first step in overcoming the impasse in detailed functional characterization of these biomedically important protein domains

Analysis of Thermal Stabilization of OLF domains.

<p>^aT_m measured in 10 mM Hepes, 200 mM NaCl pH 7.5 (Buffer A) with or without 0.75 mg/mL of GAG. For myoc-OLF, T_m is ~53°C in Buffer A [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130888#pone.0130888.ref061" target="_blank">61</a>].</p><p>Analysis of Thermal Stabilization of OLF domains.</p

Electrostatic surface representations and biochemical analysis of nucleotide and heparin binding for npoh-OLF.

<p>(a) Electrostatic surfaces of npoh-OLF and glio-OLF at top and bottom faces. (b) Electrostatic surfaces of myoc-OLF (PDB code 4WXU) and lat3-OLF (PDB code 5AFB). Surface potentials are colored negative (red, -5 kT/e^-) to positive (blue, + 5 kT/e^-). (c) Extraction analysis reveals small nucleotide stretches bound to npoh-OLF. (d) Low affinity binding of npoh-OLF to heparin column; no binding occurs with buffers at physiological ionic strength. (e) Commercial antibodies, anti-npoh-OLF and anti-myocilin (H130), lack specificity and detect npoh-OLF, myoc-OLF, and glio-OLF compared to custom myocilin antibody prepared in this study.</p

Structural features of npoh-OLF (yellow) and glio-OLF (purple).

<p>(a) Overlay of npoh-OLF and glio-OLF in two orientations with strands and blades labeled (r.m.s.d. over Cα atoms is 1.456 Å). (b) Disulfide bond in npoh-OLF (left) and corresponding cation-π interaction in glio-OLF (right). (c) Overview of molecular clasp region highlighting Pro, Tyr residues discussed in text; polar contacts < 3.5 Å are depicted as black dashes (left). Crystal contact of npoh-OLF involving residues from the molecular clasp and an outer strand of blade A from an adjacent symmetry-related molecule (right).</p