From Acid Activation Mechanisms of Proton Conduction to Design of Inhibitors of the M2 Proton Channel of Influenza A Virus

With an estimated 1 billion people affected across the globe, influenza is one of the most serious health concerns worldwide. Therapeutic treatments have encompassed a number of key functional viral proteins, mainly focused on the M2 proton channel and neuraminidase. This review highlights the efforts spent in targeting the M2 proton channel, which mediates the proton transport toward the interior of the viral particle as a preliminary step leading to the release of the fusion peptide in hemagglutinin and the fusion of the viral and endosomal membranes. Besides the structural and mechanistic aspects of the M2 proton channel, attention is paid to the challenges posed by the development of efficient small molecule inhibitors and the evolution toward novel ligands and scaffolds motivated by the emergence of resistant strains.

SARS-CoV-2 treatment effects induced by ACE2-expressing microparticles are explained by the oxidized cholesterol-increased endosomal pH of alveolar macrophages

Exploring the cross-talk between the immune system and advanced biomaterials to treat SARS-CoV-2 infection is a promising strategy. Here, we show that ACE2-overexpressing A549 cell-derived microparticles (AO-MPs) are a potential therapeutic agent against SARS-CoV-2 infection. Intranasally administered AO-MPs dexterously navigate the anatomical and biological features of the lungs to enter the alveoli and are taken up by alveolar macrophages (AMs). Then, AO-MPs increase the endosomal pH but decrease the lysosomal pH in AMs, thus escorting bound SARS-CoV-2 from phago-endosomes to lysosomes for degradation. This pH regulation is attributable to oxidized cholesterol, which is enriched in AO-MPs and translocated to endosomal membranes, thus interfering with proton pumps and impairing endosomal acidification. In addition to promoting viral degradation, AO-MPs also inhibit the proinflammatory phenotype of AMs, leading to increased treatment efficacy in a SARS-CoV-2-infected mouse model without side effects. These findings highlight the potential use of AO-MPs to treat SARS-CoV-2-infected patients and showcase the feasibility of MP therapies for combatting emerging respiratory viruses in the future.

FgRab5 and FgRab7 are essential for endosomes biogenesis and non-redundantly recruit the retromer complex to the endosomes in Fusarium graminearum

The retromer complex, composed of the cargo-selective complex (CSC) Vps35-Vps29-Vps26 in complex with the sorting nexin dimer Vps5-Vps17, mediates the sorting and retrograde transport of cargo proteins from the endosomes to the trans-Golgi network in eukaryotic cells. Rab proteins belong to the Ras superfamily of small GTPases and regulate many trafficking events including vesicle formation, budding, transport, tethering, docking and fusion with target membranes. Herein, we investigated the potential functional relationship between the retromer complex and the 11 Rab proteins that exist in Fusarium graminearum using genetic and high-resolution laser confocal microscopic approaches. We found that only FgRab5 (FgRab5A and FgRab5B) and FgRab7 associate with the retromer complex. Both FgVps35-GFP and FgVps17-GFP are mis-localized and appear diffused in the cytoplasm of ΔFgrab5A, ΔFgrab5B and ΔFgrab7 mutants as compared to their punctate localization within the endosomes of the wild-type. FgRab7 and FgRab5B were found to co-localize with the retromer on endosomal membranes. Most strikingly, we found that these three Rab GTPases are indispensable for endosome biogenesis as both early and late endosomes could not be detected in the cells of the mutants after FM4-64 staining of the cells, while they were very clearly seen in the wild-type PH-1. Furthermore, FgRab7 was found to recruit FgVps35 but not FgVps17 to the endosomal membranes, whereas FgRab5B recruits both FgVps35 and FgVps17 to the membranes. Thus, we conclude that the Rab proteins FgRab5A, FgRab5B and FgRab7 play critical roles in the biogenesis of endosomes and in regulating retromer-mediated trafficking in F. graminearum.

Mechanisms of Extracellular Vesicle Biogenesis, Cargo Loading, and Release

Extracellular vesicles (EVs) are carriers of various biomolecules including bioactive enzymes, lipids, proteins, nucleic acids, and metabolites. EVs are classified into three main types based on their size, biogenesis, and cargo. Exosomes originate from endosomal membranes and are the smallest type of EV. Microvesicles (MVs) or microparticles are larger in size, and like apoptotic bodies which represent the largest type of EVs, both of these vesicles originate from outward budding of the plasma membrane. As discussed in this chapter, cargo loading of EVs and their release into the extracellular space where they can be taken up by neighboring or distant cells plays an important role in physiology and pathophysiology. This chapter will outline specific mechanisms involved in the loading and enrichment of miRNAs, proteins, and lipids within EVs. As explained here, various external and biological stimuli play a role in EV release. Finally, recent studies have shown that the biogenesis, cargo loading, and release of EVs are governed by circadian rhythms. Although EVs were once thought to serve as garbage disposals of cells, the numerous roles they serve in physiology and pathophysiology are now being appreciated.

Tensin Phosphatase-Like System of Hantavirus Facilitates Membrane Fusion to Disrupt Vascular permeability

Increased vascular permeability is a characteristic of Hantavirus illness, for which there is now no treatment. We employed the domain search method to investigate the Hantavirus protein in this present work. The results indicated that the membrane glycoprotein E protein (containing Gn-Gc) of Hantavirus had lipid phosphatase and C2-like domains. The E protein was a tensin phosphatase-like (PTEN) enzyme that could shuttle in the cytoplasm and cell membrane. In an acidic endosomal environment, Gn dissociates, exposing Gc's autophosphorylation region to complete autophosphorylation and activating the C2 domain. The C2 domain facilitates Gc's conformational transition, which is followed by Gc binding to the endosomal membrane. After being inserted into the endosomal membrane, the phosphatase domain of Gc phosphorylates PI(3,4,5)P3 on the endosomal membrane. Then converted PI(3,4,5)P3 to PI(4,5)P2 . PI(4,5)P2 bound to the N-terminal of Gc, completely anchoring the tetramer-shaped Gc to the endosomal membrane and forming a fusion hole. Then analogous to PTEN, phosphorylation of PI(3,4,5)P3 directly induced the disintegration of Gc tetramer. The enlargement of the fusion pore speeded up the fusion of the viral and endosomal membranes. Through the fusion hole, the virus's intracellular material was swiftly discharged into the cytoplasm. The C2 domain promoted the PKC signaling route during Hantavirus membrane fusion, whereas the phosphatase inhibited the PI3K signaling pathway. E protein's PTEN-like action impaired lipid metabolism and endothelial cell remodeling, increasing blood vessel permeability and resulting in renal and cardiac syndromes. Additionally, E protein inhibited the immune system and Akt-mediated eNOS activation, resulting in a cascade of consequences.

Evaluating Large Spontaneous Deletions in a Bovine Cell Line Selected for Bovine Viral Diarrhea Virus Resistance

Bovine viral diarrhea virus’s (BVDV) entry into bovine cells involves attachment of virions to cellular receptors, internalization, and pH-dependent fusion with endosomal membranes. The primary host receptor for BVDV is CD46; however, the complete set of host factors required for virus entry is unknown. The Madin-Darby bovine kidney (MDBK) cell line is susceptible to BVDV infection, while a derivative cell line (CRIB) is resistant at the level of virus entry. We performed complete genome sequencing of each to identify genomic variation underlying the resistant phenotype with the aim of identifying host factors essential for BVDV entry. Three large compound deletions in the BVDV-resistant CRIB cell line were identified and predicted to disrupt the function or expression of the genes PTPN12, GRID2, and RABGAP1L. However, CRISPR/Cas9 mediated knockout of these genes, individually or in combination, in the parental MDBK cell line did not impact virus entry or replication. Therefore, resistance to BVDV in the CRIB cell line is not due to the apparent spontaneous loss of PTPN12, GRID2, or RABGAP1L gene function. Identifying the functional cause of BVDV resistance in the CRIB cell line may require more detailed comparisons of the genomes and epigenomes.

Flash nanoprecipitation for the production of endosomolytic polymersomes for cytosolic drug delivery

The delivery of biomacromolecular drugs to cytosolic targets has been a long-standing engineering challenge due to the presence of multiple biological barriers including cellular and endosomal membranes. Although many promising carriers designed to facilitate endosomal escape have been developed, the clinical translation of these carriers is often limited by complex production processes that are not amenable to scaled-up manufacturing. In this study, we employed flash nanoprecipitation (FNP) for the rapid, scalable, and reproducible assembly of nanocarriers composed of the pH-responsive, endosomolytic diblock copolymer poly[(ethylene glycol)x-block-[((2-diethylamino) ethyl methacrylate)0.6-co-(butyl methacrylate)0.4]y (PEG-b-DEAEMA-co-BMA). We found that varying the second block molecular weight, while holding the first block molecular weight constant, significantly influenced nanoparticle self-assembly and hence nanocarrier properties and function – including drug encapsulation, endosomolytic capacity, cytotoxicity, and in vitro activity of a cytosolically-active drug cargo, a cyclic dinucleotide (CDN) stimulator of interferon genes (STING) agonist. We found that while increasing second block molecular weight enhanced the capacity of nanocarriers to induce endosomal destabilization, larger second block molecular weights also lead to increased cytotoxicity, increased particle size and heterogeneity, increased the encapsulation efficiency of small (<0.5 kDa) hydrophilic drugs, decreased the encapsulation efficiency of large (10 kDa) hydrophilic biomacromolecules, and decreased long-term particle stability. Collectively, these results demonstrate the utility of FNP for the rapid and scalable production of uniform PEG-b-DEAEMA-co-BMA nanocarriers and implicate an optimal hydrophilic mass fraction for balancing desirable nanoparticle properties with cytosolic cargo delivery efficiency.

PI4P and BLOC-1 remodel endosomal membranes into tubules

Intracellular trafficking is mediated by transport carriers that originate by membrane remodeling from donor organelles. Tubular carriers play major roles in the flux of membrane lipids and proteins to acceptor organelles. However, how lipids and proteins impose a tubular geometry on the carriers is incompletely understood. By exploiting imaging approaches at different scales on cells and in vitro membrane systems, we show that phosphatidylinositol-4-phosphate (PI4P) and biogenesis of lysosome-related organelles complex 1 (BLOC-1) govern the formation, stability and functions of recycling endosomal tubules. Endosomal PI4P production by type II PI4-kinases is needed to form nascent curved tubules through binding of BLOC-1 that stabilize and elongate them. Membrane remodeling by the PI4P/ BLOC-1 module functions not only in the recycling of endosomal cargoes, but also in the lifecycles of intracellular pathogens such as Chlamydia bacteria and influenza virus. This study demonstrates how a phospholipid and a protein complex coordinate as a minimal machinery to remodel cellular membranes into functional tubes.

PDZD-8 and TEX-2 regulate endosomal PI(4,5)P2 homeostasis via lipid transport to promote embryogenesis in C. elegans

Different types of cellular membranes have unique lipid compositions that are important for their functional identity. PI(4,5)P2 is enriched in the plasma membrane where it contributes to local activation of key cellular events, including actomyosin contraction and cytokinesis. However, how cells prevent PI(4,5)P2 from accumulating in intracellular membrane compartments, despite constant intermixing and exchange of lipid membranes, is poorly understood. Using the C. elegans early embryo as our model system, we show that the evolutionarily conserved lipid transfer proteins, PDZD-8 and TEX-2, act together with the PI(4,5)P2 phosphatases, OCRL-1 and UNC-26/synaptojanin, to prevent the build-up of PI(4,5)P2 on endosomal membranes. In the absence of these four proteins, large amounts of PI(4,5)P2 accumulate on endosomes, leading to embryonic lethality due to ectopic recruitment of proteins involved in actomyosin contractility. PDZD-8 localizes to the endoplasmic reticulum and regulates endosomal PI(4,5)P2 levels via its lipid harboring SMP domain. Accumulation of PI(4,5)P2 on endosomes is accompanied by impairment of their degradative capacity. Thus, cells use multiple redundant systems to maintain endosomal PI(4,5)P2 homeostasis.

The Manifold Roles of Sphingolipids in Viral Infections

Sphingolipids are essential components of eukaryotic cells. In this review, we want to exemplarily illustrate what is known about the interactions of sphingolipids with various viruses at different steps of their replication cycles. This includes structural interactions during entry at the plasma membrane or endosomal membranes, early interactions leading to sphingolipid-mediated signal transduction, interactions with internal membranes and lipids during replication, and interactions during virus assembly and budding. Targeted interventions in sphingolipid metabolism – as far as they can be tolerated by cells and organisms – may open novel possibilities to support antiviral therapies. Human immunodeficiency virus type 1 (HIV-1) infections have intensively been studied, but for other viral infections, such as influenza A virus (IAV), measles virus (MV), hepatitis C virus (HCV), dengue virus, Ebola virus, and severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), investigations are still in their beginnings. As many inhibitors of sphingolipid metabolism are already in clinical use against other diseases, repurposing studies for applications in some viral infections appear to be a promising approach.