Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Constitutive Activation of the B Cell Receptor Underlies Dysfunctional Signaling in Chronic Lymphocytic Leukemia.

In cancer biology, the functional interpretation of genomic alterations is critical to achieve the promise of genomic profiling in the clinic. For chronic lymphocytic leukemia (CLL), a heterogeneous disease of B-lymphocytes maturing under constitutive B cell receptor (BCR) stimulation, the functional role of diverse clonal mutations remains largely unknown. Here, we demonstrate that alterations in BCR signaling dynamics underlie the progression of B cells toward malignancy. We reveal emergent dynamic features-bimodality, hypersensitivity, and hysteresis-in the BCR signaling pathway of primary CLL B cells. These signaling abnormalities in CLL quantitatively derive from BCR clustering and constitutive signaling with positive feedback reinforcement, as demonstrated through single-cell analysis of phospho-responses, computational modeling, and super-resolution imaging. Such dysregulated signaling segregates CLL patients by disease severity and clinical presentation. These findings provide a quantitative framework and methodology to assess complex and heterogeneous leukemia pathology and to inform therapeutic strategies in parallel with genomic profiling.

Insane in the Membrane: Glial Extracellular Vesicles Transmit Polyomaviruses.

Extracellular vesicles (EVs) are major vehicles for transporting viruses en bloc among hosts. While RNA viruses make up the great majority of transmission by EVs, in a recent article in mBio (mBio 10:e00379-19, 2019, https://mbio.asm.org/content/10/2/e00379-19.long), Morris-Love and colleagues revealed that a double-stranded DNA (dsDNA) virus, JC polyomavirus (JCPyV), a major cause of progressive multifocal leukoencephalopathy (PML), can be released from and transmitted to other glia in EVs. This mode of transmission appears to be highly infectious, independent of the free virus attachment and entry receptors LSTc and 5-HT2, and protected from neutralizing antibodies. This novel form of JCPyV transmission may potentially explain its dissemination into the central nervous system (CNS) and its increased virulence.

25-Hydroxycholesterol Production by the Cholesterol-25-Hydroxylase Interferon-Stimulated Gene Restricts Mammalian Reovirus Infection.

Following the initial detection of viral infection, innate immune responses trigger the induction of numerous interferon-stimulated genes (ISGs) to inhibit virus replication and dissemination. One such ISG encodes cholesterol-25-hydroxylase (CH25H), an enzyme that catalyzes the oxidation of cholesterol to form a soluble product, 25-hydroxycholesterol (25HC). Recent studies have found that CH25H is broadly antiviral; it inhibits infection by several viruses. For enveloped viruses, 25HC inhibits membrane fusion, likely by altering membrane characteristics such as hydrophobicity or cholesterol aggregation. However, the mechanisms by which 25HC restricts infection of nonenveloped viruses are unknown. We examined whether 25HC restricts infection by mammalian reovirus. Treatment with 25HC restricted infection by reovirus prototype strains type 1 Lang and type 3 Dearing. In contrast to reovirus virions, 25HC did not restrict infection by reovirus infectious subvirion particles (ISVPs), which can penetrate either directly at the cell surface or in early endosomal membranes. Treatment with 25HC altered trafficking of reovirus particles to late endosomes and delayed the kinetics of reovirus uncoating. These results suggest that 25HC inhibits the efficiency of cellular entry of reovirus virions, which may require specific endosomal membrane dynamics for efficient membrane penetration.IMPORTANCE The innate immune system is crucial for effective responses to viral infection. Type I interferons, central components of innate immunity, induce expression of hundreds of ISGs; however, the mechanisms of action of these antiviral proteins are not well understood. CH25H, encoded by an ISG, represents a significant constituent of these cellular antiviral strategies, as its metabolic product, 25HC, can act in both an autocrine and a paracrine fashion to protect cells from infection and has been shown to limit viral infection in animal models. Further investigation into the mechanism of action of 25HC may inform novel antiviral therapies and influence the use of mammalian reovirus in clinical trials as an oncolytic agent.

Infection with hepatitis C virus depends on TACSTD2, a regulator of claudin-1 and occludin highly downregulated in hepatocellular carcinoma.

Entry of hepatitis C virus (HCV) into hepatocytes is a complex process that involves numerous cellular factors, including the scavenger receptor class B type 1 (SR-B1), the tetraspanin CD81, and the tight junction (TJ) proteins claudin-1 (CLDN1) and occludin (OCLN). Despite expression of all known HCV-entry factors, in vitro models based on hepatoma cell lines do not fully reproduce the in vivo susceptibility of liver cells to primary HCV isolates, implying the existence of additional host factors which are critical for HCV entry and/or replication. Likewise, HCV replication is severely impaired within hepatocellular carcinoma (HCC) tissue in vivo, but the mechanisms responsible for this restriction are presently unknown. Here, we identify tumor-associated calcium signal transducer 2 (TACSTD2), one of the most downregulated genes in primary HCC tissue, as a host factor that interacts with CLDN1 and OCLN and regulates their cellular localization. TACSTD2 gene silencing disrupts the typical linear distribution of CLDN1 and OCLN along the cellular membrane in both hepatoma cells and primary human hepatocytes, recapitulating the pattern observed in vivo in primary HCC tissue. Mechanistic studies suggest that TACSTD2 is involved in the phosphorylation of CLDN1 and OCLN, which is required for their proper cellular localization. Silencing of TACSTD2 dramatically inhibits HCV infection with a pan-genotype effect that occurs at the level of viral entry. Our study identifies TACSTD2 as a novel regulator of two major HCV-entry factors, CLDN1 and OCLN, which is strongly downregulated in malignant hepatocytes. These results provide new insights into the complex process of HCV entry into hepatocytes and may assist in the development of more efficient cellular systems for HCV propagation in vitro.

Catch and Release of Cytokines Mediated by Tumor Phosphatidylserine Converts Transient Exposure into Long-Lived Inflammation.

Immune cells constantly survey the host for pathogens or tumors and secrete cytokines to alert surrounding cells of these threats. In vivo, activated immune cells secrete cytokines for several hours, yet an acute immune reaction occurs over days. Given these divergent timescales, we addressed how cytokine-responsive cells translate brief cytokine exposure into phenotypic changes that persist over long timescales. We studied melanoma cell responses to transient exposure to the cytokine interferon γ (IFNγ) by combining a systems-scale analysis of gene expression dynamics with computational modeling and experiments. We discovered that IFNγ is captured by phosphatidylserine (PS) on the surface of viable cells both in vitro and in vivo then slowly released to drive long-term transcription of cytokine-response genes. This mechanism introduces an additional function for PS in dynamically regulating inflammation across diverse cancer and primary cell types and has potential to usher in new immunotherapies targeting PS and inflammatory pathways.

Lipid Tales of Viral Replication and Transmission.

Positive-strand RNA viruses are the largest group of RNA viruses on Earth and cellular membranes are critical for all aspects of their life cycle, from entry and replication to exit. In particular, membranes serve as platforms for replication and as carriers to transmit these viruses to other cells, the latter either as an envelope surrounding a single virus or as the vesicle containing a population of viruses. Notably, many animal and human viruses appear to induce and exploit phosphatidylinositol 4-phosphate/cholesterol-enriched membranes for replication, whereas many plant and insect-vectored animal viruses utilize phosphatidylethanolamine/cholesterol-enriched membranes for the same purpose; and phosphatidylserine-enriched membrane carriers are widely used by both single and populations of viruses for transmission. Here I discuss the implications for viral pathogenesis and therapeutic development of this remarkable convergence on specific membrane lipid blueprints for replication and transmission.

Phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses.

A central paradigm within virology is that each viral particle largely behaves as an independent infectious unit. Here, we demonstrate that clusters of enteroviral particles are packaged within phosphatidylserine (PS) lipid-enriched vesicles that are non-lytically released from cells and provide greater infection efficiency than free single viral particles. We show that vesicular PS lipids are co-factors to the relevant enterovirus receptors in mediating subsequent infectivity and transmission, in particular to primary human macrophages. We demonstrate that clustered packaging of viral particles within vesicles enables multiple viral RNA genomes to be collectively transferred into single cells. This study reveals a novel mode of viral transmission, where enteroviral genomes are transmitted from cell-to-cell en bloc in membrane-bound PS vesicles instead of as single independent genomes. This has implications for facilitating genetic cooperativity among viral quasispecies as well as enhancing viral replication.

An interaction between glutathione and the capsid is required for the morphogenesis of C-cluster enteroviruses.

Glutathione (GSH) is the most abundant cellular thiol playing an essential role in preserving a reduced cellular environment. Cellular GSH levels can be efficiently reduced by the GSH biosynthesis inhibitor, L-buthionine sulfoximine (BSO). The aim of our study was to determine the role of GSH in the growth of two C-cluster enteroviruses, poliovirus type 1 (PV1) and coxsackievirus A20 (CAV20). Our results show that the growth of both PV1 and CAV20 is strongly inhibited by BSO and can be partially reversed by the addition of GSH. BSO has no effect on viral protein synthesis or RNA replication but it strikingly reduces the accumulation of 14S pentamers in infected cells. GSH-pull down assays show that GSH directly interacts with capsid precursors and mature virus made in the absence of BSO whereas capsid precursors produced under GSH-depletion do not bind to GSH. In particular, the loss of binding of GSH may debilitate the stability of 14S pentamers, resulting in their failure to assemble into mature virus. Immunofluorescence cell imaging demonstrated that GSH-depletion did not affect the localization of viral capsid proteins to the replication complex. PV1 BSO resistant (BSOr) mutants evolved readily during passaging of the virus in the presence of BSO. Structural analyses revealed that the BSOr mutations, mapping to VP1 and VP3 capsid proteins, are primarily located at protomer/protomer interfaces. BSOr mutations might, in place of GSH, aid the stability of 14S particles that is required for virion maturation. Our observation that BSOr mutants are more heat resistant and need less GSH than wt virus to be protected from heat inactivation suggests that they possess a more stable capsid. We propose that the role of GSH during enterovirus morphogenesis is to stabilize capsid structures by direct interaction with capsid proteins both during and after the formation of mature virus particles.

Influenza a virus assembly intermediates fuse in the cytoplasm.

Reassortment of influenza viral RNA (vRNA) segments in co-infected cells can lead to the emergence of viruses with pandemic potential. Replication of influenza vRNA occurs in the nucleus of infected cells, while progeny virions bud from the plasma membrane. However, the intracellular mechanics of vRNA assembly into progeny virions is not well understood. Here we used recent advances in microscopy to explore vRNA assembly and transport during a productive infection. We visualized four distinct vRNA segments within a single cell using fluorescent in situ hybridization (FISH) and observed that foci containing more than one vRNA segment were found at the external nuclear periphery, suggesting that vRNA segments are not exported to the cytoplasm individually. Although many cytoplasmic foci contain multiple vRNA segments, not all vRNA species are present in every focus, indicating that assembly of all eight vRNA segments does not occur prior to export from the nucleus. To extend the observations made in fixed cells, we used a virus that encodes GFP fused to the viral polymerase acidic (PA) protein (WSN PA-GFP) to explore the dynamics of vRNA assembly in live cells during a productive infection. Since WSN PA-GFP colocalizes with viral nucleoprotein and influenza vRNA segments, we used it as a surrogate for visualizing vRNA transport in 3D and at high speed by inverted selective-plane illumination microscopy. We observed cytoplasmic PA-GFP foci colocalizing and traveling together en route to the plasma membrane. Our data strongly support a model in which vRNA segments are exported from the nucleus as complexes that assemble en route to the plasma membrane through dynamic colocalization events in the cytoplasm.

Enteroviruses harness the cellular endocytic machinery to remodel the host cell cholesterol landscape for effective viral replication.

Cholesterol is a critical component of cellular membranes, regulating assembly and function of membrane-based protein/lipid complexes. Many RNA viruses, including enteroviruses, remodel host membranes to generate organelles with unique lipid blueprints on which they assemble replication complexes and synthesize viral RNA. Here we find that clathrin-mediated endocytosis (CME) is harnessed by enteroviruses to traffic cholesterol from the plasma membrane (PM) and extracellular medium to replication organelles, where cholesterol then regulates viral polyprotein processing and facilitates genome synthesis. When CME is disrupted, cellular cholesterol pools are instead stored in lipid droplets, cholesterol cannot be trafficked to replication organelles, and replication is inhibited. In contrast, replication is stimulated in cholesterol-elevated cells like those lacking caveolins or those from Niemann-Pick disease patients. Our findings indicate cholesterol as a critical determinant for enteroviral replication and outline roles for the endocytic machinery in both the enteroviral life cycle and host cell cholesterol homeostasis.

Viral reorganization of the secretory pathway generates distinct organelles for RNA replication.

Many RNA viruses remodel intracellular membranes to generate specialized sites for RNA replication. How membranes are remodeled and what properties make them conducive for replication are unknown. Here we show how RNA viruses can manipulate multiple components of the cellular secretory pathway to generate organelles specialized for replication that are distinct in protein and lipid composition from the host cell. Specific viral proteins modulate effector recruitment by Arf1 GTPase and its guanine nucleotide exchange factor GBF1, promoting preferential recruitment of phosphatidylinositol-4-kinase IIIbeta (PI4KIIIbeta) to membranes over coat proteins, yielding uncoated phosphatidylinositol-4-phosphate (PI4P) lipid-enriched organelles. The PI4P-rich lipid microenvironment is essential for both enteroviral and flaviviral RNA replication; PI4KIIIbeta inhibition interferes with this process; and enteroviral RNA polymerases specifically bind PI4P. These findings reveal how RNA viruses can selectively exploit specific elements of the host to form specialized organelles where cellular phosphoinositide lipids are key to regulating viral RNA replication.

Hijacking components of the cellular secretory pathway for replication of poliovirus RNA.

Infection of cells with poliovirus induces a massive intracellular membrane reorganization to form vesicle-like structures where viral RNA replication occurs. The mechanism of membrane remodeling remains unknown, although some observations have implicated components of the cellular secretory and/or autophagy pathways. Recently, we showed that some members of the Arf family of small GTPases, which control secretory trafficking, became membrane-bound after the synthesis of poliovirus proteins in vitro and associated with newly formed membranous RNA replication complexes in infected cells. The recruitment of Arfs to specific target membranes is mediated by a group of guanine nucleotide exchange factors (GEFs) that recycle Arf from its inactive, GDP-bound state to an active GTP-bound form. Here we show that two different viral proteins independently recruit different Arf GEFs (GBF1 and BIG1/2) to the new structures that support virus replication. Intracellular Arf-GTP levels increase approximately 4-fold during poliovirus infection. The requirement for these GEFs explains the sensitivity of virus growth to brefeldin A, which can be rescued by the overexpression of GBF1. The recruitment of Arf to membranes via specific GEFs by poliovirus proteins provides an important clue toward identifying cellular pathways utilized by the virus to form its membranous replication complex.