HSPA5 Promotes Attachment and Internalization of Porcine Epidemic Diarrhea Virus through Interaction with the Spike Protein and the Endo-/Lysosomal Pathway.

Porcine epidemic diarrhea virus (PEDV) has caused huge economic losses to the global pig industry. The swine enteric coronavirus spike (S) protein recognizes various cell surface molecules to regulate viral infection. In this study, we identified 211 host membrane proteins related to the S1 protein by pulldown combined with liquid-chromatography tandem mass spectrometry (LC-MS/MS) analysis. Among these, heat shock protein family A member 5 (HSPA5) was identified through screening as having a specific interaction with the PEDV S protein, and positive regulation of PEDV infection was validated by knockdown and overexpression tests. Further studies verified the role of HSPA5 in viral attachment and internalization. In addition, we found that HSPA5 interacts with S proteins through its nucleotide-binding structural domain (NBD) and that polyclonal antibodies can block viral infection. In detail, HSPA5 was found to be involved in viral trafficking via the endo-/lysosomal pathway. Inhibition of HSPA5 activity during internalization would reduce the subcellular colocalization of PEDV with lysosomes in the endo-/lysosomal pathway. Together, these findings show that HSPA5 is a novel PEDV potential target for the creation of therapeutic drugs. IMPORTANCE PEDV infection causes severe piglet mortality and threatens the global pig industry. However, the complex invasion mechanism of PEDV makes its prevention and control difficult. Here, we determined that HSPA5 is a novel target for PEDV which interacts with its S protein and is involved in viral attachment and internalization, influencing its transport via the endo-/lysosomal pathway. Our work extends knowledge about the relationship between the PEDV S and host proteins and provides a new therapeutic target against PEDV infection.

Lysosomal nanotoxicity: Impact of nanomedicines on lysosomal function.

Although several nanomedicines got clinical approval over the past two decades, the clinical translation rate is relatively small so far. There are many post-surveillance withdrawals of nanomedicines caused by various safety issues. For successful clinical advancement of nanotechnology, it is of unmet need to realize cellular and molecular foundation of nanotoxicity. Current data suggest that lysosomal dysfunction caused by nanoparticles is emerging as the most common intracellular trigger of nanotoxicity. This review analyzes prospect mechanisms of lysosomal dysfunction-mediated toxicity induced by nanoparticles. We summarized and critically assessed adverse drug reactions of current clinically approved nanomedicines. Importantly, we show that physicochemical properties have great impact on nanoparticles interaction with cells, excretion route and kinetics, and subsequently on toxicity. We analyzed literature on adverse reactions of current nanomedicines and hypothesized that adverse reactions might be linked with lysosomal dysfunction caused by nanomedicines. Finally, from our analysis it becomes clear that it is unjustifiable to generalize safety and toxicity of nanoparticles, since different particles possess distinct toxicological properties. We propose that the biological mechanism of the disease progression and treatment should be central in the optimization of nanoparticle design.

SARS-CoV-2 ORF3a expression in brain disrupts the autophagy-lysosomal pathway, impairs sphingolipid homeostasis, and drives neuropathogenesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes injury to multiple organ systems, including the brain. SARS-CoV-2's neuropathological mechanisms may include systemic inflammation and hypoxia, as well as direct cell damage resulting from viral infections of neurons and glia. How the virus directly causes injury to brain cells, acutely and over the long term, is not well understood. In order to gain insight into this process, we studied the neuropathological effects of open reading frame 3a (ORF3a), a SARS-CoV-2 accessory protein that is a key pathological factor of the virus. Forced ORF3a brain expression in mice caused the rapid onset of neurological impairment, neurodegeneration, and neuroinflammation-key neuropathological features found in coronavirus disease (COVID-19, which is caused by SARS-CoV-2 infection). Furthermore, ORF3a expression blocked autophagy progression in the brain and caused the neuronal accumulation of α-synuclein and glycosphingolipids, all of which are linked to neurodegenerative disease. Studies with ORF3-expressing HeLa cells confirmed that ORF3a disrupted the autophagy-lysosomal pathway and blocked glycosphingolipid degradation, resulting in their accumulation. These findings indicate that, in the event of neuroinvasion by SARS-CoV-2, ORF3a expression in brain cells may drive neuropathogenesis and be an important mediator of both short- and long-term neurological manifestations of COVID-19.

SARS-CoV-2 infection alkalinizes the ERGIC and lysosomes through the viroporin activity of the viral envelope protein.

The coronavirus SARS-CoV-2, the agent of the deadly COVID-19 pandemic, is an enveloped virus propagating within the endocytic and secretory organelles of host mammalian cells. Enveloped viruses modify the ionic homeostasis of organelles to render their intra-luminal milieu permissive for viral entry, replication and egress. Here, we show that infection of Vero E6 cells with the delta variant of the SARS-CoV-2 alkalinizes the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) as well as lysosomes, mimicking the effect of inhibitors of vacuolar proton ATPases. We further show the envelope protein of SARS-CoV-2 accumulates in the ERGIC when expressed in mammalian cells and selectively dissipates the ERGIC pH. This viroporin action is prevented by mutations of Val25 but not Asn15 within the channel pore of the envelope (E) protein. We conclude that the envelope protein acts as a proton channel in the ERGIC to mitigate the acidity of this intermediate compartment. The altered pH homeostasis of the ERGIC likely contributes to the virus fitness and pathogenicity, making the E channel an attractive drug target for the treatment of COVID-19.

Autophagy in Inflammatory Response against SARS-CoV-2.

The coronavirus disease pandemic, which profoundly reshaped the world in 2019 (COVID-19), and is currently ongoing, has affected over 200 countries, caused over 500 million cumulative cases, and claimed the lives of over 6.4 million people worldwide as of August 2022. The causative agent is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Depicting this virus' life cycle and pathogenic mechanisms, as well as the cellular host factors and pathways involved during infection, has great relevance for the development of therapeutic strategies. Autophagy is a catabolic process that sequesters damaged cell organelles, proteins, and external invading microbes, and delivers them to the lysosomes for degradation. Autophagy would be involved in the entry, endo, and release, as well as the transcription and translation, of the viral particles in the host cell. Secretory autophagy would also be involved in developing the thrombotic immune-inflammatory syndrome seen in a significant number of COVID-19 patients that can lead to severe illness and even death. This review aims to review the main aspects that characterize the complex and not yet fully elucidated relationship between SARS-CoV-2 infection and autophagy. It briefly describes the key concepts regarding autophagy and mentions its pro- and antiviral roles, while also noting the reciprocal effect of viral infection in autophagic pathways and their clinical aspects.

Coronaviruses, Lysosomes, and Secondary Bacterial Infections: Coronaviruses Outsmart the Host.

Lysosomes are key organelles that contribute to homeostatic functions such as autophagy-mediated recycling of cellular components and innate immune response through phagocytosis-mediated pathogen killing during infections. Viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has developed unique adaptation to not only avoid lysosome-mediated destruction but also actively utilize lysosomal machinery to both enter and exit cells. To survive the highly hostile lysosomal environment, coronaviruses deacidify the lysosomes, potentially by manipulating H+ ion exchange across the lysosomal lumen, ensuring coronavirus survival. At the same time, this deacidification not only impairs cellular homeostatic functions such as autophagy but also renders the host susceptible to secondary bacterial infections. Furthermore, lysosomal enzymes promote extensive cell death and tissue damage during secondary bacterial infections. Thus, targeting lysosomal pathways provide a great opportunity to limit both viral replication and subsequent negative impact on host immunity against secondary bacterial infections.

SARS-CoV-2 down-regulates ACE2 through lysosomal degradation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes its Spike (S) glycoprotein to bind to the angiotensin-converting enzyme 2 (ACE2) receptor for cellular entry. ACE2 is a critical negative regulator of the renin-angiotensin system and plays a protective role in preventing tissue injury. Expression of ACE2 has been shown to decrease upon infection by SARS-CoV. However, whether SARS-CoV-2 down-regulates ACE2 and the underlying mechanism and biological impact of this down-regulation have not been well defined. Here we show that the SARS-CoV-2 infection down-regulates ACE2 in vivo in an animal model, and in cultured cells in vitro, by inducing clathrin- and AP2-dependent endocytosis, leading to its degradation in the lysosome. SARS-CoV-2 S-treated cells and ACE2 knockdown cells exhibit similar alterations in downstream gene expression, with a pattern indicative of activated cytokine signaling that is associated with respiratory distress and inflammatory diseases often observed in COVID-19 patients. Finally, we have identified a soluble ACE2 fragment with a stronger binding to SARS-CoV-2 S that can efficiently block ACE2 down-regulation and viral infection. Thus, our study suggests that ACE2 down-regulation represents an important mechanism underlying SARS-CoV-2-associated pathology, and blocking this process could be a promising therapeutic strategy.

MERS-CoV nsp1 regulates autophagic flux via mTOR signalling and dysfunctional lysosomes.

Autophagy, a cellular surveillance mechanism, plays an important role in combating invading pathogens. However, viruses have evolved various strategies to disrupt autophagy and even hijack it for replication and release. Here, we demonstrated that Middle East respiratory syndrome coronavirus (MERS-CoV) non-structural protein 1(nsp1) induces autophagy but inhibits autophagic activity. MERS-CoV nsp1 expression increased ROS and reduced ATP levels in cells, which activated AMPK and inhibited the mTOR signalling pathway, resulting in autophagy induction. Meanwhile, as an endonuclease, MERS-CoV nsp1 downregulated the mRNA of lysosome-related genes that were enriched in nsp1-located granules, which diminished lysosomal biogenesis and acidification, and inhibited autophagic flux. Importantly, MERS-CoV nsp1-induced autophagy can lead to cell death in vitro and in vivo. These findings clarify the mechanism by which MERS-CoV nsp1-mediated autophagy regulation, providing new insights for the prevention and treatment of the coronavirus.

Lysosomal enzyme trafficking factor LYSET enables nutritional usage of extracellular proteins.

Mammalian cells can generate amino acids through macropinocytosis and lysosomal breakdown of extracellular proteins, which is exploited by cancer cells to grow in nutrient-poor tumors. Through genetic screens in defined nutrient conditions, we characterized LYSET, a transmembrane protein (TMEM251) selectively required when cells consume extracellular proteins. LYSET was found to associate in the Golgi with GlcNAc-1-phosphotransferase, which targets catabolic enzymes to lysosomes through mannose-6-phosphate modification. Without LYSET, GlcNAc-1-phosphotransferase was unstable because of a hydrophilic transmembrane domain. Consequently, LYSET-deficient cells were depleted of lysosomal enzymes and impaired in turnover of macropinocytic and autophagic cargoes. Thus, LYSET represents a core component of the lysosomal enzyme trafficking pathway, underlies the pathomechanism for hereditary lysosomal storage disorders, and may represent a target to suppress metabolic adaptations in cancer.

Endolysosomal cholesterol export: More than just NPC1.

NPC1 plays a central role in cholesterol egress from endolysosomes, a critical step for maintaining intracellular cholesterol homeostasis. Despite recent advances in the field, the full repertoire of molecules and pathways involved in this process remains unknown. Emerging evidence suggests the existence of NPC1-independent, alternative routes. These may involve vesicular and non-vesicular mechanisms, as well as release of extracellular vesicles. Understanding the underlying molecular mechanisms that bypass NPC1 function could have important implications for the development of therapies for lysosomal storage disorders. Here we discuss how cholesterol may be exported from lysosomes in which NPC1 function is impaired.

Coronavirus Lung Infection Impairs Host Immunity against Secondary Bacterial Infection by Promoting Lysosomal Dysfunction.

Postviral bacterial infections are a major health care challenge in coronavirus infections, including COVID-19; however, the coronavirus-specific mechanisms of increased host susceptibility to secondary infections remain unknown. In humans, coronaviruses, including SARS-CoV-2, infect lung immune cells, including alveolar macrophages, a phenotype poorly replicated in mouse models of SARS-CoV-2. To overcome this, we used a mouse model of native murine ß-coronavirus that infects both immune and structural cells to investigate coronavirus-enhanced susceptibility to bacterial infections. Our data show that coronavirus infection impairs the host ability to clear invading bacterial pathogens and potentiates lung tissue damage in mice. Mechanistically, coronavirus limits the bacterial killing ability of macrophages by impairing lysosomal acidification and fusion with engulfed bacteria. In addition, coronavirus-induced lysosomal dysfunction promotes pyroptotic cell death and the release of IL-1ß. Inhibition of cathepsin B decreased cell death and IL-1ß release and promoted bacterial clearance in mice with postcoronavirus bacterial infection.

Stress granules and mTOR are regulated by membrane atg8ylation during lysosomal damage.

We report that lysosomal damage is a hitherto unknown inducer of stress granule (SG) formation and that the process termed membrane atg8ylation coordinates SG formation with mTOR inactivation during lysosomal stress. SGs were induced by lysosome-damaging agents including SARS-CoV-2ORF3a, Mycobacterium tuberculosis, and proteopathic tau. During damage, mammalian ATG8s directly interacted with the core SG proteins NUFIP2 and G3BP1. Atg8ylation was needed for their recruitment to damaged lysosomes independently of SG condensates whereupon NUFIP2 contributed to mTOR inactivation via the Ragulator-RagA/B complex. Thus, cells employ membrane atg8ylation to control and coordinate SG and mTOR responses to lysosomal damage.

A phosphoinositide signalling pathway mediates rapid lysosomal repair.

Lysosomal dysfunction has been increasingly linked to disease and normal ageing1,2. Lysosomal membrane permeabilization (LMP), a hallmark of lysosome-related diseases, can be triggered by diverse cellular stressors3. Given the damaging contents of lysosomes, LMP must be rapidly resolved, although the underlying mechanisms are poorly understood. Here, using an unbiased proteomic approach, we show that LMP stimulates a phosphoinositide-initiated membrane tethering and lipid transport (PITT) pathway for rapid lysosomal repair. Upon LMP, phosphatidylinositol-4 kinase type 2α (PI4K2A) accumulates rapidly on damaged lysosomes, generating high levels of the lipid messenger phosphatidylinositol-4-phosphate. Lysosomal phosphatidylinositol-4-phosphate in turn recruits multiple oxysterol-binding protein (OSBP)-related protein (ORP) family members, including ORP9, ORP10, ORP11 and OSBP, to orchestrate extensive new membrane contact sites between damaged lysosomes and the endoplasmic reticulum. The ORPs subsequently catalyse robust endoplasmic reticulum-to-lysosome transfer of phosphatidylserine and cholesterol to support rapid lysosomal repair. Finally, the lipid transfer protein ATG2 is also recruited to damaged lysosomes where its activity is potently stimulated by phosphatidylserine. Independent of macroautophagy, ATG2 mediates rapid membrane repair through direct lysosomal lipid transfer. Together, our findings identify that the PITT pathway maintains lysosomal membrane integrity, with important implications for numerous age-related diseases characterized by impaired lysosomal function.

The Key Role of Lysosomal Protease Cathepsins in Viral Infections.

Cathepsins encompass a family of lysosomal proteases that mediate protein degradation and turnover. Although mainly localized in the endolysosomal compartment, cathepsins are also found in the cytoplasm, nucleus, and extracellular space, where they are involved in cell signaling, extracellular matrix assembly/disassembly, and protein processing and trafficking through the plasma and nuclear membrane and between intracellular organelles. Ubiquitously expressed in the body, cathepsins play regulatory roles in a wide range of physiological processes including coagulation, hormone secretion, immune responses, and others. A dysregulation of cathepsin expression and/or activity has been associated with many human diseases, including cancer, diabetes, obesity, cardiovascular and inflammatory diseases, kidney dysfunctions, and neurodegenerative disorders, as well as infectious diseases. In viral infections, cathepsins may promote (1) activation of the viral attachment glycoproteins and entry of the virus into target cells; (2) antigen processing and presentation, enabling the virus to replicate in infected cells; (3) up-regulation and processing of heparanase that facilitates the release of viral progeny and the spread of infection; and (4) activation of cell death that may either favor viral clearance or assist viral propagation. In this review, we report the most relevant findings on the molecular mechanisms underlying cathepsin involvement in viral infection physiopathology, and we discuss the potential of cathepsin inhibitors for therapeutical applications in viral infectious diseases.

The human disease gene LYSET is essential for lysosomal enzyme transport and viral infection.

Lysosomes are key degradative compartments of the cell. Transport to lysosomes relies on GlcNAc-1-phosphotransferase-mediated tagging of soluble enzymes with mannose 6-phosphate (M6P). GlcNAc-1-phosphotransferase deficiency leads to the severe lysosomal storage disorder mucolipidosis II (MLII). Several viruses require lysosomal cathepsins to cleave structural proteins and thus depend on functional GlcNAc-1-phosphotransferase. We used genome-scale CRISPR screens to identify lysosomal enzyme trafficking factor (LYSET, also named TMEM251) as essential for infection by cathepsin-dependent viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). LYSET deficiency resulted in global loss of M6P tagging and mislocalization of GlcNAc-1-phosphotransferase from the Golgi complex to lysosomes. Lyset knockout mice exhibited MLII-like phenotypes, and human pathogenic LYSET alleles failed to restore lysosomal sorting defects. Thus, LYSET is required for correct functioning of the M6P trafficking machinery and mutations in LYSET can explain the phenotype of the associated disorder.

Application of meso-CF3-Fluorophore BODIPY with Phenyl and Pyrazolyl Substituents for Lifetime Visualization of Lysosomes.

A bright far-red emitting unsymmetrical meso-CF3-BODIPY fluorescent dye with phenyl and pyrazolyl substituents was synthesized by condensation of trifluoropyrrolylethanol with pyrazolyl-pyrrole, with subsequent oxidation and complexation of the formed dipyrromethane. This BODIPY dye exhibits optical absorption at λab ≈ 610-620 nm and emission at λem ≈ 640-650 nm. The BODIPY was studied on Ehrlich carcinoma cells as a lysosome-specific fluorescent dye that allows intravital staining of cell structures with subsequent real-time monitoring of changes occurring in the cells. It was also shown that the rate of uptake by cells, the rate of intracellular transport into lysosomes, and the rate of saturation of cells with the dye depend on its concentration in the culture medium. A concentration of 5 µM was chosen as the most suitable BODIPY concentration for fluorescent staining of living cell lysosomes, while a concentration of 100 µM was found to be toxic to Ehrlich carcinoma cells.

mRNA-carrying lipid nanoparticles that induce lysosomal rupture activate NLRP3 inflammasome and reduce mRNA transfection efficiency.

In the last several years, countless developments have been made to engineer more efficient and potent mRNA lipid nanoparticle vaccines, culminating in the rapid development of effective mRNA vaccines against COVID-19. However, despite these advancements and materials approaches, there is still a lack of understanding of the resultant immunogenicity of mRNA lipid nanoparticles. Therefore, a more mechanistic, design-driven approach needs to be taken to determine which biophysical characteristics, especially related to changes in lipid compositions, drive nanoparticle immunogenicity. Here, we synthesized a panel of six mRNA lipid nanoparticle formulations, varying the concentrations of different lipid components and systematically studied their effect on NLRP3 inflammasome activation; a key intracellular protein complex that controls various inflammatory responses. Initial experiments aimed to determine differences in nanoparticle activation of NLRP3 inflammasomes by IL-1ß ELISA, which unveiled that nanoparticles with high concentrations of ionizable lipid DLin-MC3-DMA in tandem with high cationic lipid DPTAP and low cholesterol concentration induced the greatest activation of the NLRP3 inflammasome. These results were further corroborated by the measurement of ASC specks indicative of NLRP3 complex assembly, as well as cleaved gasdermin-D and caspase-1 expression indicating complex activation. We also uncovered these activation profiles to be mechanistically correlated primarily with lysosomal rupturing caused by the delayed membrane disruption capabilities of ionizable lipids until the lysosomal stage, as well as by mitochondrial reactive oxygen species (ROS) production and calcium influx for some of the particles. Therefore, we report that the specific, combined effects of each lipid type, most notably ionizable, cationic lipids, and cholesterol, is a crucial mRNA lipid nanoparticle characteristic that varies the endo/lysosomal rupture capabilities of the formulation and activate NLRP3 inflammasomes in a lysosomal rupture dependent manner. These results provide a more concrete understanding of mRNA lipid Nanoparticle-Associated Molecular Patterns for the activation of molecular-level immune responses and provide new lipid composition design considerations for future mRNA-delivery approaches.

Autophagy: A Key Regulator of Homeostasis and Disease: An Overview of Molecular Mechanisms and Modulators.

Autophagy is a highly conserved lysosomal degradation pathway active at basal levels in all cells. However, under stress conditions, such as a lack of nutrients or trophic factors, it works as a survival mechanism that allows the generation of metabolic precursors for the proper functioning of the cells until the nutrients are available. Neurons, as post-mitotic cells, depend largely on autophagy to maintain cell homeostasis to get rid of damaged and/or old organelles and misfolded or aggregated proteins. Therefore, the dysfunction of this process contributes to the pathologies of many human diseases. Furthermore, autophagy is highly active during differentiation and development. In this review, we describe the current knowledge of the different pathways, molecular mechanisms, factors that induce it, and the regulation of mammalian autophagy. We also discuss its relevant role in development and disease. Finally, here we summarize several investigations demonstrating that autophagic abnormalities have been considered the underlying reasons for many human diseases, including liver disease, cardiovascular, cerebrovascular diseases, neurodegenerative diseases, neoplastic diseases, cancers, and, more recently, infectious diseases, such as SARS-CoV-2 caused COVID-19 disease.

Friend or Foe? Implication of the autophagy-lysosome pathway in SARS-CoV-2 infection and COVID-19.

There is increasing amount of evidence indicating the close interplays between the replication cycle of SARS-CoV-2 and the autophagy-lysosome pathway in the host cells. While autophagy machinery is known to either assist or inhibit the viral replication process, the reciprocal effects of the SARS-CoV-2 on the autophagy-lysosome pathway have also been increasingly appreciated. More importantly, despite the disappointing results from the clinical trials of chloroquine and hydroxychloroquine in treatment of COVID-19, there is still ongoing effort in discovering new therapeutics targeting the autophagy-lysosome pathway. In this review, we provide an update-to-date summary of the interplays between the autophagy-lysosome pathway in the host cells and the pathogen SARS-CoV-2 at the molecular level, to highlight the prognostic value of autophagy markers in COVID-19 patients and to discuss the potential of developing novel therapeutic strategies for COVID-19 by targeting the autophagy-lysosome pathway. Thus, understanding the nature of such interactions between SARS-CoV-2 and the autophagy-lysosome pathway in the host cells is expected to provide novel strategies in battling against this global pandemic.