IFITM3 blocks influenza virus entry by sorting lipids and stabilizing hemifusion.

Interferon-induced transmembrane protein 3 (IFITM3) inhibits the entry of numerous viruses through undefined molecular mechanisms. IFITM3 localizes in the endosomal-lysosomal system and specifically affects virus fusion with target cell membranes. We found that IFITM3 induces local lipid sorting, resulting in an increased concentration of lipids disfavoring viral fusion at the hemifusion site. This increases the energy barrier for fusion pore formation and the hemifusion dwell time, promoting viral degradation in lysosomes. In situ cryo-electron tomography captured IFITM3-mediated arrest of influenza A virus membrane fusion. Observation of hemifusion diaphragms between viral particles and late endosomal membranes confirmed hemifusion stabilization as a molecular mechanism of IFITM3. The presence of the influenza fusion protein hemagglutinin in post-fusion conformation close to hemifusion sites further indicated that IFITM3 does not interfere with the viral fusion machinery. Collectively, these findings show that IFITM3 induces lipid sorting to stabilize hemifusion and prevent virus entry into target cells.

Scission of COPI and COPII vesicles is independent of GTP hydrolysis.

Intracellular transport and maintenance of the endomembrane system in eukaryotes depends on formation and fusion of vesicular carriers. A seeming discrepancy exists in the literature about the basic mechanism in the scission of transport vesicles that depend on GTP-binding proteins. Some reports describe that the scission of COP-coated vesicles is dependent on GTP hydrolysis, whereas others found that GTP hydrolysis is not required. In order to investigate this pivotal mechanism in vesicle formation, we analyzed formation of COPI- and COPII-coated vesicles utilizing semi-intact cells. The small GTPases Sar1 and Arf1 together with their corresponding coat proteins, the Sec23/24 and Sec13/31 complexes for COPII and coatomer for COPI vesicles were required and sufficient to drive vesicle formation. Both types of vesicles were efficiently generated when GTP hydrolysis was blocked either by utilizing the poorly hydrolyzable GTP analogs GTPγS and GMP-PNP, or with constitutively active mutants of the small GTPases. Thus, GTP hydrolysis is not required for the formation and release of COP vesicles.

Differential transport of Influenza A neuraminidase signal anchor peptides to the plasma membrane.

Influenza A Neuraminidase is essential for virus release from the cell surface of host cells. Given differential structures of the N-terminal sequences including the transmembrane domains of neuraminidase subtypes, we investigated their contribution to transport and localization of subtypes N1, N2 and N8 to the plasma membrane. We generated consensus sequences from all protein entries available for these subtypes. We found that 40N-terminal the forty N-terminal amino acids are sufficient to confer plasma membrane localization of fusion proteins, albeit with different efficiencies. Strikingly, subtle differences in the primary structure of the part of the transmembrane domain that resides in the exoplasmic leaflet of the membrane have a major impact on transport efficiency, providing a potential target for the inhibition of virus release.

Distinct role of subcomplexes of the COPI coat in the regulation of ArfGAP2 activity.

COPI vesicles serve for transport of proteins and membrane lipids in the early secretory pathway. Their coat protein (coatomer) is a heptameric complex that is recruited to the Golgi by the small GTPase Arf1. Although recruited en bloc, coatomer can be viewed as a stable assembly of an adaptin-like tetrameric subcomplex (CM4) and a trimeric 'cage' subcomplex (CM3). Following recruitment, coatomer stimulates ArfGAP-dependent GTP hydrolysis on Arf1. Here, we employed recombinant coatomer subcomplexes to study the role of coatomer components in the regulation of ArfGAP2, an ArfGAP whose activity is strictly coatomer-dependent. Within CM4, we define a novel hydrophobic pocket for ArfGAP2 interaction on the appendage domain of γ1-COP. The CM4 subcomplex (but not CM3) is recruited to membranes through Arf1 and can subsequently recruit ArfGAP2. Neither CM3 nor CM4 in itself is effective in stimulating ArfGAP2 activity, but stimulation is regained when both subcomplexes are present. Our findings point to a distinct role of each of the two coatomer subcomplexes in the regulation of ArfGAP2-dependent GTP hydrolysis on Arf1, where the CM4 subcomplex functions in GAP recruitment, while, similarly to the COPII system, the cage-like CM3 subcomplex stimulates the catalytic reaction.

Interaction between DLX2 and EGFR regulates proliferation and neurogenesis of SVZ precursors.

In the postnatal subventricular zone (SVZ) neural stem cells (NSCs) give rise to transit-amplifying precursors (TAPs) expressing high levels of epidermal growth factor receptor (EGFR) that in turn generate neuroblasts. Both TAPs and neuroblasts express distal-less (DLX)2 homeobox transcription factor but the latter proliferate less. Modulation of its expression in vivo has revealed that DLX2 affects both neurogenesis and proliferation in the postnatal SVZ. However, the mechanisms underlying these effects are not clear. To investigate this issue we have here forced the expression of DLX2 in SVZ isolated NSCs growing in defined in vitro conditions. This analysis revealed that DLX2 affects the proliferation of SVZ precursors by regulating two distinct steps of neural lineage progression. Firstly, it promotes the lineage transition from NSCs to TAPs. Secondly it enhances the proliferative response of neuronal progenitors to EGF. Thus DLX2 and EGFR signalling interact at multiple levels to coordinate proliferation in the postnatal SVZ.

Improvement of gemcitabine-based therapy of pancreatic carcinoma by means of oncolytic parvovirus H-1PV.

UNLABELLED: Pancreatic carcinoma is a gastrointestinal malignancy with poor prognosis. Treatment with gemcitabine, the most potent chemotherapeutic against this cancer up to date, is not curative, and resistance may appear. Complementary treatment with an oncolytic virus, such as the rat parvovirus H-1PV, which is infectious but nonpathogenic in humans, emerges as an innovative option. PURPOSE: To prove that combining gemcitabine and H-1PV in a model of pancreatic carcinoma may reduce the dosage of the toxic drug and/or improve the overall anticancer effect. EXPERIMENTAL DESIGN: Pancreatic tumors were implanted orthotopically in Lewis rats or subcutaneously in nude mice and treated with gemcitabine, H-1PV, or both according to different regimens. Tumor size was monitored by micro-computed tomography, whereas bone marrow, liver, and kidney functions were monitored by measuring clinically relevant markers. Human pancreatic cell lines and gemcitabine-resistant derivatives were tested in vitro for sensitivity to H-1PV infection with or without gemcitabine. RESULTS: In vitro studies proved that combining gemcitabine with H-1PV resulted in synergistic cytotoxic effects and achieved an up to 15-fold reduction in the 50% effective concentration of the drug, with drug-resistant cells remaining sensitive to virus killing. Toxicologic screening showed that H-1PV had an excellent safety profile when applied alone or in combination with gemcitabine. The benefits of applying H-1PV as a second-line treatment after gemcitabine included reduction of tumor growth, prolonged survival of the animals, and absence of metastases on CT-scans. CONCLUSION: In addition to their potential use as monotherapy for pancreatic cancer, parvoviruses can be best combined with gemcitabine in a two-step protocol.