Intracellular Vesicle Acidification Promotes Maturation of Infectious Poliovirus Particles

The autophagic pathway acts as part of the immune response against a variety of pathogens. However, several pathogens subvert autophagic signaling to promote their own replication. In many cases it has been demonstrated that these pathogens inhibit or delay the degradative aspect of autophagy. Here, using poliovirus as a model virus, we report for the first time bona fide autophagic degradation occurring during infection with a virus whose replication is promoted by autophagy. We found that this degradation is not required to promote poliovirus replication. However, vesicular acidification, which in the case of autophagy precedes delivery of cargo to lysosomes, is required for normal levels of virus production. We show that blocking autophagosome formation inhibits viral RNA synthesis and subsequent steps in the virus cycle, while inhibiting vesicle acidification only inhibits the final maturation cleavage of virus particles. We suggest that particle assembly, genome encapsidation, and virion maturation may occur in a cellular compartment, and we propose the acidic mature autophagosome as a candidate vesicle. We discuss the implications of our findings in understanding the late stages of poliovirus replication, including the formation and maturation of virions and egress of infectious virus from cells.     

How Positive-Strand RNA Viruses Benefit from Autophagosome Maturation

The autophagic degradation pathway is a powerful tool in the host cell arsenal against cytosolic pathogens. Contents trapped inside cytosolic vesicles, termed autophagosomes, are delivered to the lysosome for degradation. In spite of the degradative nature of the pathway, some pathogens are able to subvert autophagy for their benefit. In many cases, these pathogens have developed strategies to induce the autophagic signaling pathway while inhibiting the associated degradation activity. One surprising finding from recent literature is that some viruses do not impede degradation but instead promote the generation of degradative autolysosomes, which are the endpoint compartments of autophagy. Dengue virus, poliovirus, and hepatitis C virus, all positive-strand RNA viruses, utilize the maturation of autophagosomes into acidic and ultimately degradative compartments to promote their replication. While the benefits that each virus reaps from autophagosome maturation are unique, the parallels between the viruses indicate a complex relationship between cytosolic viruses and host cell degradation vesicles.     

That which does not degrade you makes you stronger

Several years ago, an explosion of research into pathogens and autophagy showed that viruses have a wide variety of relationships to this conserved homeostatic pathway. Often, autophagy acts as a host defense mechanism, degrading viruses before they can escape the host cell, and, as such, autophagy is suppressed or avoided by those viruses. A subset of viruses, however, induces and subverts the autophagic machinery to promote their own replication. Many of these viruses inhibit the degradative step in the autophagic pathway, presumably to prevent degradation of cytosolic virions before they exit the cell. Recently, we published a study showing that poliovirus (PV), a well-studied model virus, induces true autophagic degradation. The remainder of our study provided surprising clues about the role of autophagy in promoting virus production. The purpose of this punctum is to discuss the significance of our findings to a general understanding of the autophagic pathway and its relationship to a common class of cellular pathogens.     

Mouse infection by Legionella, a model to analyze autophagy

Autophagy is a conserved membrane traffic pathway that equips eukaryotic cells to capture cytoplasmic components within a double-membrane vacuole, or autophagosome, for delivery to lysosomes. Although best known as a mechanism to survive starvation, autophagy is now recognized to combat infection by a variety of microbes.(1-3) Not surprisingly, to establish a replication niche in host cells, some intracellular pathogens have acquired mechanisms either to evade or subvert the autophagic pathway. Because they are amenable to genetic manipulation, these microbes can be exploited as experimental tools to investigate the contribution of autophagy to immunity. Here we discuss the mouse macrophage response to L. pneumophila, the facultative intracellular bacterium responsible for an acute form of pneumonia, Legionnaire's disease.     

Impact of cellular autophagy on viruses: Insights from hepatitis B virus and human retroviruses

Autophagy is a protein degradative process important for normal cellular metabolism. It is apparently used also by cells to eliminate invading pathogens. Interestingly, many pathogens have learned to subvert the cell’s autophagic process. Here, we review the interactions between viruses and cells in regards to cellular autophagy. Using findings from hepatitis B virus and human retroviruses, HIV-1 and HTLV-1, we discuss mechanisms used by viruses to usurp cellular autophagy in ways that benefit viral replication.     

Perturbation of autophagic pathway by hepatitis C virus

Autophagy removes long-lived proteins and damaged organelles and is important for maintaining cellular homeostasis. It can also serve in innate immunity to remove intracellular pathogens. As such, viruses have evolved different mechanisms to subvert this innate immune response. We have recently demonstrated that hepatitis C virus (HCV) can also suppress autophagic protein degradation by suppressing the fusion between autophagosomes and lysosomes. This suppression causes the accumulation of autophagosomes and enhances HCV RNA replication.(1) Our further analysis indicated that the induction of autophagosomes by HCV is dependent on the unfolded protein response (UPR). Our studies thus delineate a molecular pathway by which HCV induces autophagosomes. The prolonged perturbation of the autophagic pathway by HCV likely plays an important role in HCV pathogenesis.     

The two faces of autophagy: Coxiella and Mycobacterium

In the world of pathogen-host cell interactions, the autophagic pathway has been recently described as a component of the innate immune response against intracellular microorganisms. Indeed, some bacterial survival mechanisms are hampered when this process is activated. Mycobacterium tuberculosis infection of macrophages, for example, is impaired upon autophagy induction and the bacterial phagosomes are redirected to autophagosomes. On the other hand, pathogens like Coxiella burnetii are benefited by this cellular response and subvert the autophagy process resulting in a more efficient replication. We study at the molecular level these two different faces of the autophagy process in pathogen life in order to elucidate the intricate routes modulated by the microorganisms as survival strategies.     

Bacterial pathogens and the autophagic response

The host cell recognition and removal of invading pathogens are crucial for the control of microbial infections. However, several microorganisms have developed mechanisms that allow them to survive and replicate intracellularly. Autophagy is an ubiquitous physiological pathway in eukaryotic cells, which maintains the cellular homeostasis and acts as a cell quality control mechanism to eliminate aged organelles and unnecessary structures. In addition, autophagy has an important role as a housekeeper since cells that have to get rid of invading pathogens use this pathway to assist this eradication. In this review we will summarize some strategies employed by bacterial pathogens to modulate autophagy to their own benefit and, on the other hand, the role of autophagy as a protective process of the host cell. In addition, we will discuss here recent studies that show the association of LC3 to a pathogen-containing compartment without a classical autophagic sequestering process (i.e. formation of a double membrane structure).     

Manipulation of autophagy by bacteria for their own benefit

Autophagy is the host innate immune system's first line of defense against microbial intruders. When the innate defense system recognizes invading bacterial pathogens and their infection processes, autophagic proteins act as cytosolic sensors that allow the autophagic pathway to be rapidly activated. However, many intracellular bacterial pathogens deploy highly evolved mechanisms to evade autophagic recognition, manipulate the autophagic pathway, and remodel the autophagosomal compartment for their own benefit. Here current topics regarding the recognition of invasive bacteria by the cytosolic innate immune system are highlighted, including autophagy and the mechanisms that enable bacteria to evade autophagy. Also highlighted are some selective examples of bacterial activities that manipulate the autophagic pathways for their own benefit.     

Autophagy during Early Virus–Host Cell Interactions

Autophagy refers to the conserved, multi-step mechanism that delivers cytosolic cargoes to vesicles of the endo-lysosomal system for degradation. It maintains cellular homeostasis by ensuring the continuous degradation of misformed/senescent intracellular components and the associated recycling of nutrients. Autophagy also represents an important cell-intrinsic defense mechanism against invasion by intracellular pathogens, including viruses. Autophagy might oppose viral invasion by targeting viral particles or viral components for degradation. It can also promote the interaction of viral constituents with receptors specialized in the activation of innate immunity pathways or facilitate the activation of anti-viral adaptive immunity. In response to such pressures, viruses have evolved various sophisticated strategies to avoid anti-viral autophagic responses or to manipulate the autophagic machinery to promote their own replication. This review focuses on our current knowledge of autophagy-related events that take place at early stages during interaction of viruses with host cells as well as on their associated consequences in terms of virus replication and cell fate.     

The Two Faces of Autophagy: Coxiella and Mycobacterium

In the world of pathogen-host cell interactions, the autophagic pathway has beenrecently described as a component of the innate immune response against intracellularmicroorganisms. Indeed, some bacterial survival mechanisms are hampered when thisprocess is activated. Mycobacterium tuberculosis infection of macrophages, for example,is impaired upon autophagy induction and the bacterial phagosomes are redirected toautophagosomes. On the other hand, pathogens like Coxiella burnetii are benefited bythis cellular response and subvert the autophagy process resulting in a more efficient replication.We study at the molecular level these two different faces of the autophagy processin pathogen life in order to elucidate the intricate routes modulated by the microorganismsas survival strategies.     

Pepstatin A alters host cell autophagic machinery and leads to a decrease in influenza A virus production

Autophagy is a survival mechanism that can take place in cells under metabolic stress and through which cells can recycle waste material. Disturbances in autophagic processes appear to be associated with a number of human pathologies, including viral infections. It has been hypothesized that viruses can subvert autophagy in order to penetrate the host cell and replicate. Because it has been suggested that autophagy is involved in influenza A virus replication, we analyzed the effects of two inhibitors of lysosomal proteases on the cellular control of influenza A virus replication. In particular, we used biochemical and morphological analyses to evaluate the modulation of influenza A/Puerto Rico/8/34 H1N1 virus production in the presence of CA074 and Pepstatin A, inhibitors of cathepsin proteases B and D, respectively. We found that Pepstatin A, but not CA074, significantly hindered influenza virus replication, probably by modulating host cell autophagic/apoptotic responses. These results are of potential interest to provide useful insights into the molecular pathways exploited by the influenza in order to replicate and to identify further cellular factors as targets for the development of innovative antiviral strategies.     

Pathogens and polymers: microbe-host interactions illuminate the cytoskeleton

Intracellular pathogens subvert the host cell cytoskeleton to promote their own survival, replication, and dissemination. Study of these microbes has led to many discoveries about host cell biology, including the identification of cytoskeletal proteins, regulatory pathways, and mechanisms of cytoskeletal function. Actin is a common target of bacterial pathogens, but recent work also highlights the use of microtubules, cytoskeletal motors, intermediate filaments, and septins. The study of pathogen interactions with the cytoskeleton has illuminated key cellular processes such as phagocytosis, macropinocytosis, membrane trafficking, motility, autophagy, and signal transduction.     

Manipulation or capitulation: virus interactions with autophagy

Autophagy is a homeostatic process that functions to balance cellular metabolism and promote cell survival during stressful conditions by delivering cytoplasmic components for lysosomal degradation and subsequent recycling. During viral infection, autophagy can act as a surveillance mechanism that delivers viral antigens to the endosomal/lysosomal compartments that are enriched in immune sensors. Additionally, activated immune sensors can signal to activate autophagy. To evade this antiviral activity, many viruses elaborate functions to block the autophagy pathway at a variety of steps. Alternatively, some viruses actively subvert autophagy for their own benefit. Manipulated autophagy has been proposed to facilitate nearly every stage of the viral lifecycle in direct and indirect ways. In this review, we synthesize the extensive literature on virus-autophagy interactions, emphasizing the role of autophagy in antiviral immunity and the mechanisms by which viruses subvert autophagy for their own benefit.     

Cytoplasmic bacteria can be targets for autophagy

Autophagy is an important constitutive cellular process involved in size regulation, protein turnover and the removal of malformed or superfluous subcellular components. The process involves the sequestration of cytoplasm and organelles into double-membrane autophagic vacuoles for subsequent breakdown within lysosomes. In this work, we demonstrate that the intracellular pathogen Listeria monocytogenes can also be a target for autophagy. If infected macrophages are treated with chloramphenicol after phagosome lysis, the bacteria are internalized from the cell cytoplasm into autophagic vacuoles. The autophagic vacuoles appear to form by fusion of small cytoplasmic vesicles around the bacteria. These vesicular structures immunolabel with antibodies to protein disulphide isomerase, a marker for the rough ER. Internalization of metabolically arrested cytoplasmic L. monocytogenes represents an autophagic process as the vacuoles have double membranes and the process can be inhibited by the autophagy inhibitors 3-methyladenine and wortmannin. Additionally, the rate of internalization can be accelerated under starvation conditions and the vacuoles fuse with the endocytic pathway. Metabolic inhibition of cytoplasmic bacteria prevents them from adapting to the intracellular niche and reveals a host mechanism utilizing the autophagic pathway as a defence against invading pathogens by providing a route for their removal from the cytoplasm and subsequent delivery to the endocytic pathway for degradation.     

Endoplasmic reticulum-associated protein degradation

The quality control system in the endoplasmic reticulum of eukaryotic cells ensures that newly synthesized proteins that fail to fold into the correct conformation or unassembled orphan subunits of oligomeric proteins are rapidly eliminated by proteolytic degradation. This entails the export of proteins from the endoplasmic reticulum to the cytosol followed by their destruction by the cytosolic ubiquitin/proteasome pathway. While this mechanism effectively prevents the cellular accumulation of non-functional or unwanted endogenous proteins, it renders the cell vulnerable to certain viruses and toxins that are able to subvert this degradative mechanism for their own advantage.     

Host cell autophagy contributes to Plasmodium liver development

Autophagy plays an important role in the defence against intracellular pathogens. However, some microorganisms can manipulate this host cell pathway to their advantage. In this study, we addressed the role of host cell autophagy during Plasmodium berghei liver infection. We show that vesicles containing the autophagic marker LC3 surround parasites from early time‐points after invasion and throughout infection and colocalize with the parasitophorous vacuole membrane. Moreover, we show that the LC3‐positive vesicles that surround Plasmodium parasites are amphisomes that converge from the endocytic and autophagic pathways, because they contain markers of both pathways. When the host autophagic pathway was inhibited by silencing several of its key regulators such as LC3, Beclin1, Vps34 or Atg5, we observed a reduction in parasite size. We also found that LC3 surrounds parasites in vivo and that parasite load is diminished in a mouse model deficient for autophagy. Together, these results show the importance of the host autophagic pathway for parasite development during the liver stage of Plasmodium infection.     

Masters of manipulation: Viral modulation of the immunological synapse

In order to thrive, viruses have evolved to manipulate host cell machinery for their own benefit. One major obstacle faced by pathogens is the immunological synapse. To enable efficient replication and latency in immune cells, viruses have developed a range of strategies to manipulate cellular processes involved in immunological synapse formation to evade immune detection and control T‐cell activation. In vitro, viruses such as human immunodeficiency virus 1 and human T‐lymphotropic virus type 1 utilise structures known as virological synapses to aid transmission of viral particles from cell to cell in a process termed trans‐infection. The formation of the virological synapse provides a gateway for virus to be transferred between cells avoiding the extracellular space, preventing antibody neutralisation or recognition by complement. This review looks at how viruses are able to subvert intracellular signalling to modulate immune function to their advantage and explores the role synapse formation has in viral persistence and cell‐to‐cell transmission.     

In search of Brucella abortus type IV secretion substrates: screening and identification of four proteins translocated into host cells through VirB system

Type IV secretion systems (T4SS) are specialized protein complexes used by many bacterial pathogens for the delivery of effector molecules that subvert varied host cellular processes. Brucella spp. are facultative intracellular pathogens capable of survival and replication inside mammalian cells. Brucella T4SS (VirB) is essential to subvert lysosome fusion and to create an organelle permissive for replication. One possible role for VirB is to translocate effector proteins that modulate host cellular functions for the biogenesis of the replicative organelle. We hypothesized that proteins with eukaryotic domains or protein-protein interaction domains, among others, would be good candidates for modulation of host cell functions. To identify these candidates, we performed an in silico screen looking for proteins with distinctive features. Translocation of 84 potential substrates was assayed using adenylate cyclase reporter. By this approach, we identified six proteins that are delivered to the eukaryotic cytoplasm upon infection of macrophage-like cells and we could determine that four of them, encoded by genes BAB1_1043, BAB1_2005, BAB1_1275 and BAB2_0123, require a functional T4SS for their delivery. We confirmed VirB-mediated translocation of one of the substrates by immunofluorescence confocal microscopy, and we found that the N-terminal 25 amino acids are required for its delivery into cells.