In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Guidelines for the use and interpretatoin of assays for monitoring autophagy

Viruses manipulate cellular metabolism and macromolecule recycling processes like autophagy. Dysregulated metabolism might lead to excessive inflammatory and autoimmune responses as observed in severe and long COVID-19 patients. Here we show that SARS-CoV-2 modulates cellular metabolism and reduces autophagy. Accordingly, compound-driven induction of autophagy limits SARS-CoV-2 propagation. In detail, SARS-CoV-2-infected cells show accumulation of key metabolites, activation of autophagy inhibitors (AKT1, SKP2) and reduction of proteins responsible for autophagy initiation (AMPK, TSC2, ULK1), membrane nucleation, and phagophore formation (BECN1, VPS34, ATG14), as well as autophagosome-lysosome fusion (BECN1, ATG14 oligomers). Consequently, phagophore-incorporated autophagy markers LC3B-II and P62 accumulate, which we confirm in a hamster model and lung samples of COVID-19 patients. Single-nucleus and single-cell sequencing of patient-derived lung and mucosal samples show differential transcriptional regulation of autophagy and immune genes depending on cell type, disease duration, and SARS-CoV-2 replication levels. Targeting of autophagic pathways by exogenous administration of the polyamines spermidine and spermine, the selective AKT1 inhibitor MK-2206, and the BECN1-stabilizing anthelmintic drug niclosamide inhibit SARS-CoV-2 propagation in vitro with IC50 values of 136.7, 7.67, 0.11, and 0.13 μM, respectively. Autophagy-inducing compounds reduce SARS-CoV-2 propagation in primary human lung cells and intestinal organoids emphasizing their potential as treatment options against COVID-19. Viruses manipulate host cell pathways to support infection. Here the authors show that SARS-CoV-2 infection modulates cellular metabolism and limits autophagy, and identify druggable host pathways for virus inhibition.

SARS-CoV-2-mediated dysregulation of metabolism and autophagy uncovers host-targeting antivirals.

Therapeutic options for the highly pathogenic human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing the current pandemic coronavirus disease (COVID-19) are urgently needed. COVID-19 is associated with viral pneumonia and acute respiratory distress syndrome causing significant morbidity and mortality. The proposed treatments for COVID-19 have shown little or no effect in the clinic so far. Additionally, bacterial and fungal pathogens contribute to the SARS-CoV-2-mediated pneumonia disease complex. The antibiotic resistance in pneumonia treatment is increasing at an alarming rate. Therefore, carbon-based nanomaterials (CBNs), such as fullerene, carbon dots, graphene, and their derivatives constitute a promising alternative due to their wide-spectrum antimicrobial activity, biocompatibility, biodegradability, and capacity to induce tissue regeneration. Furthermore, the antimicrobial mode of action is mainly physical (e.g., membrane distortion), characterized by a low risk of antimicrobial resistance. In this Review, we evaluated the literature on the antiviral activity and broad-spectrum antimicrobial properties of CBNs. CBNs had antiviral activity against 13 enveloped positive-sense single-stranded RNA viruses, including SARS-CoV-2. CBNs with low or no toxicity to humans are promising therapeutics against the COVID-19 pneumonia complex with other viruses, bacteria, and fungi, including those that are multidrug-resistant.

Carbon-Based Nanomaterials: Promising Antiviral Agents to Combat COVID-19 in the Microbial-Resistant Era.

A recent mutation analysis suggested that Non-Structural Protein 6 (NSP6) of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a key determinant of the viral pathogenicity. Here, by transcriptome analysis, we demonstrated that the inflammasome-related NOD-like receptor signaling was activated in SARS-CoV-2-infected lung epithelial cells and Coronavirus Disease 2019 (COVID-19) patients' lung tissues. The induction of inflammasomes/pyroptosis in patients with severe COVID-19 was confirmed by serological markers. Overexpression of NSP6 triggered NLRP3/ASC-dependent caspase-1 activation, interleukin-1β/18 maturation, and pyroptosis of lung epithelial cells. Upstream, NSP6 impaired lysosome acidification to inhibit autophagic flux, whose restoration by 1α,25-dihydroxyvitamin D3, metformin or polydatin abrogated NSP6-induced pyroptosis. NSP6 directly interacted with ATP6AP1, a vacuolar ATPase proton pump component, and inhibited its cleavage-mediated activation. L37F NSP6 variant, which was associated with asymptomatic COVID-19, exhibited reduced binding to ATP6AP1 and weakened ability to impair lysosome acidification to induce pyroptosis. Consistently, infection of cultured lung epithelial cells with live SARS-CoV-2 resulted in autophagic flux stagnation, inflammasome activation, and pyroptosis. Overall, this work supports that NSP6 of SARS-CoV-2 could induce inflammatory cell death in lung epithelial cells, through which pharmacological rectification of autophagic flux might be therapeutically exploited. 

/pdf/sars-cov-2-non-structural-protein-6-triggers-nlrp3-dependent-1xvcnha0.pdf

SARS-CoV-2 non-structural protein 6 triggers NLRP3-dependent pyroptosis by targeting ATP6AP1

SARS-CoV-2 evolution threatens vaccine- and natural infection–derived immunity and the efficacy of therapeutic antibodies. To improve public health preparedness, we sought to predict which existing amino acid mutations in SARS-CoV-2 might contribute to future variants of concern. We tested the predictive value of features comprising epidemiology, evolution, immunology, and neural network–based protein sequence modeling and identified primary biological drivers of SARS-CoV-2 intrapandemic evolution. We found evidence that ACE2-mediated transmissibility and resistance to population-level host immunity has waxed and waned as a primary driver of SARS-CoV-2 evolution over time. We retroactively identified with high accuracy (area under the receiver operator characteristic curve = 0.92 to 0.97) mutations that will spread, at up to 4 months in advance, across different phases of the pandemic. The behavior of the model was consistent with a plausible causal structure where epidemiological covariates combine the effects of diverse and shifting drivers of viral fitness. We applied our model to forecast mutations that will spread in the future and characterize how these mutations affect the binding of therapeutic antibodies. These findings demonstrate that it is possible to forecast the driver mutations that could appear in emerging SARS-CoV-2 variants of concern. We validated this result against Omicron, showing elevated predictive scores for its component mutations before emergence and rapid score increase across daily forecasts during emergence. This modeling approach may be applied to any rapidly evolving pathogens with sufficiently dense genomic surveillance data, such as influenza, and unknown future pandemic viruses. 

/pdf/predicting-the-mutational-drivers-of-future-sars-cov-2-3jax725a.pdf

Predicting the mutational drivers of future SARS-CoV-2 variants of concern

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the ongoing coronavirus disease 2019 pandemic. How SARS-CoV-2 regulates cellular responses to escape clearance by host cells is unknown. Autophagy is an intracellular lysosomal degradation pathway for the clearance of various cargoes, including viruses. Here, we systematically screened 28 viral proteins of SARS-CoV-2 and identified that ORF3a strongly inhibited autophagic flux by blocking the fusion of autophagosomes with lysosomes. ORF3a colocalized with lysosomes and interacted with VPS39, a component of the homotypic fusion and protein sorting (HOPS) complex. The ORF3a-VPS39 interaction prohibited the binding of HOPS with RAB7, which prevented the assembly of fusion machinery, leading to the accumulation of unfused autophagosomes. These results indicated the potential mechanism by which SARS-CoV-2 escapes degradation; that is, the virus interferes with autophagosome-lysosome fusion. Furthermore, our findings will facilitate strategies targeting autophagy for conferring potential protection against the spread of SARS-CoV-2.

/pdf/the-sars-cov-2-protein-orf3a-inhibits-fusion-of-58v538i7me.pdf

The SARS-CoV-2 protein ORF3a inhibits fusion of autophagosomes with lysosomes

Following outbreaks of severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV) in 2002 and 2012, respectively, the severe acute resp...

https://www.tandfonline.com/doi/pdf/10.1080/22221751.2021.1872353

The interplay between emerging human coronavirus infections and autophagy.

Biogenesis of autophagosomes is the early step of macroautophagy/autophagy and requires membrane acquisition mainly from ER-Golgi-sourced precursor vesicles. Matured autophagosomes fuse with lysoso...

PtdIns4P restriction by hydrolase SAC1 decides specific fusion of autophagosomes with lysosomes

O-GlcNAcylation is a form of posttranslational modification, and serves various functions, including modulation of location, stability, and activity for the modified proteins. O-linked-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential cellular enzyme that posttranslationally modifies the cellular proteins with O-GlcNAc moiety. Early studies reported that the decreased O-GlcNAcylation regulates cellular autophagy, a process relevant for hepatitis B virus replication (HBV) and assembly. Therefore, we addressed the question how O-GlcNAcylation regulates cellular autophagy and HBV replication. Inhibition of OGT activity with a small molecule inhibitor OSMI-1 or silencing OGT significantly enhanced HBV replication and HBsAg production in hepatoma cells and primary human hepatocytes (PHHs). Western blotting analysis showed that inhibition of O-GlcNAcylation-induced endoplasmic reticulum (ER) stress and cellular autophagy, two processes subsequently leading to enhanced HBV replication. Importantly, the numbers of autophagosomes and the levels of autophagic markers LC3-II and SQSTM1/p62 in hepatoma cells were elevated after inhibition of O-GlcNAcylation. Further analysis revealed that inhibition of O-GlcNAcylation blocked autophagosome-lysosome fusion and thereby prevented autophagic degradation of HBV virions and proteins. Moreover, OSMI-1 further promoted HBV replication by inducing autophagosome formation via inhibiting the O-GlcNAcylation of Akt and mTOR. In conclusion, decreased O-GlcNAcylation enhanced HBV replication through increasing autophagosome formation at multiple levels, including triggering ER-stress, Akt/mTOR inhibition, and blockade of autophagosome-lysosome fusion.

https://faseb.onlinelibrary.wiley.com/doi/pdf/10.1096/fj.202001168RR

Kefeng Lu

Papers

The SARS-CoV-2 protein ORF3a inhibits fusion of autophagosomes with lysosomes

The interplay between emerging human coronavirus infections and autophagy.

PtdIns4P restriction by hydrolase SAC1 decides specific fusion of autophagosomes with lysosomes

O-GlcNAcylation modulates HBV replication through regulating cellular autophagy at multiple levels