The COVID-19 pandemic, caused by the SARS-CoV-2 virus, is the most recent example of an emergent coronavirus that poses a significant threat to human health. Virus-host interactions play a major role in the viral life cycle and disease pathogenesis, and cellular pathways such as macroautophagy/autophagy prove to be either detrimental or beneficial to viral replication and maturation. Here, we describe the literature over the past twenty years describing autophagy-coronavirus interactions. There is evidence that many coronaviruses induce autophagy, although some of these viruses halt the progression of the pathway prior to autophagic degradation. In contrast, other coronaviruses usurp components of the autophagy pathway in a non-canonical fashion. Cataloging these virus-host interactions is crucial for understanding disease pathogenesis, especially with the global challenge of SARS-CoV-2 and COVID-19. With the recognition of autophagy inhibitors, including the controversial drug chloroquine, as possible treatments for COVID-19, understanding how autophagy affects the virus will be critical going forward.
Abbreviations: 3-MA: 3-methyladenine (autophagy inhibitor); AKT/protein kinase B: AKT serine/threonine kinase; ATG: autophagy related; ATPase: adenosine triphosphatase; BMM: bone marrow macrophage; CGAS: cyclic GMP-AMP synthase; CHO: Chinese hamster ovary/cell line; CoV: coronaviruses; COVID-19: Coronavirus disease 2019; DMV: double-membrane vesicle; EAV: equine arteritis virus; EDEM1: ER degradation enhancing alpha-mannosidase like protein 1; ER: endoplasmic reticulum; ERAD: ER-associated degradation; GFP: green fluorescent protein; HCoV: human coronavirus; HIV: human immunodeficiency virus; HSV: herpes simplex virus; IBV: infectious bronchitis virus; IFN: interferon; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCoV: mouse coronavirus; MERS-CoV: Middle East respiratory syndrome coronavirus; MHV: mouse hepatitis virus; NBR1: NBR1 autophagy cargo receptor; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2 (autophagy receptor that directs cargo to phagophores); nsp: non-structural protein; OS9: OS9 endoplasmic reticulum lectin; PEDV: porcine epidemic diarrhea virus; PtdIns3K: class III phosphatidylinositol 3-kinase; PLP: papain-like protease; pMEF: primary mouse embryonic fibroblasts; SARS-CoV: severe acute respiratory syndrome coronavirus; SKP2: S-phase kinase associated protein 2; SQSTM1: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; ULK1: unc-51 like autophagy activating kinase 1; Vps: vacuolar protein sorting.
As with several other RNA viruses, coronaviruses have long been known to interact with the cellular macroautophagy/autophagy (hereafter autophagy) pathway to promote their replication [30,31]. Autophagy is a conserved cellular process involving the formation of autophagosomes which enclose cytoplasmic cargo, including long-lived proteins, protein aggregates and organelles, and deliver this cargo to lysosomes for degradation. Although autophagy is a constitutive pathway, it is upregulated when cells are under stressful conditions, such as starvation or infection by pathogens . The autophagy pathway involves multiple steps. First, the sequestering compartment of autophagy, known as the phagophore, nucleates and expands. When the phagophore closes to form the autophagosome, it traps cargo in its double-membraned structure. The autophagosome then fuses with the endosome to form the acidic amphisome. Finally, the amphisome fuses with the lysosome, allowing for the degradation of vesicular contents in what is termed the autolysosome (Figure 1).
Figure 1. Coronavirus interference in the autophagic pathway. Upon induction of the canonical autophagy pathway, ER membranes rearrange to form membranous structures known as omegasomes. These omegasomes then self-fuse into double-membrane vesicles (DMVs), which are termed autophagosomes. Infection by MHV, nsp6 of IBV, and nsp3 of CoV-NL63 promote the formation of these autophagosomes, and viral replication complexes often associate with these structures. However, coronavirus proteins may induce the formation of DMVs directly from the ER independent of the canonical autophagosome machinery, as seen for SARS-CoV nsp3, 4, and 6, EAV nsp2 and 3, and MERS-CoV nsp3 and 4 (see text.) MHV infection induces ER-derived DMVs independent of the autophagic pathway through hijacking of the host cell ERAD machinery. As the autophagic pathway progresses, the autophagosome fuses with the late endosome, then the lysosome, which results in degradation of the autophagosomal cargo. Nascent RNA of MHV colocalizes with late endosomal markers, suggesting that MHV may allow or promote the fusion of the autophagosome with the late endosome. In other cases, coronaviruses inhibit fusion of the autophagosome with the lysosome. One mechanism of blocking fusion is through direct or indirect inhibition of BECN1, a host protein known to promote this fusion. Specifically, the PLP-domain of nsp3 of CoV-NL63 binds BECN1 and STING1, which prevents BECN1 from promoting autophagosome and lysosome fusion and inhibits production of interferon. MERS-CoV inhibits BECN1-mediated fusion through a separate mechanism, by activation of SKP2, which promotes degradation of BECN1. All of these pathways converge in the late endosome or lysosome, although some coronaviruses inhibit fusion with these compartments.
The origin of the nucleating phagophore in eukaryotes is still a matter of debate. A leading hypothesis is that the initial phagophore is an endoplasmic reticulum (ER)-associated structure, termed the omegasome . The omegasome structure resides separately from the ER and forms as an autophagy-specific phagophore through the action of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex containing the human/yeast proteins PIK3C3/Vps34, PIK3R4/Vps15, NRBF2/Atg38, ATG14/Atg14 and BECN1/Vps30/Atg6 [36,37]. Next, the expansion of the phagophore occurs by means of the ULK complex including ULK/Atg1, ATG13/Atg13, RB1CC1/FIP200/Atg17 and ATG101 . In addition, mammalian Atg8-family proteins (commonly known as LC3 and GABARAP subfamilies) play a key role in the maturation of the phagophore. LC3 exists as a soluble cytoplasmic protein (LC3-I). LC3-I undergoes phosphatidylethanolamine (PE)-modifications to become LC3-II prior to its insertion into the phagophore membrane. The mammalian ATG12–ATG5-ATG16L1 complex, ATG3, and ATG7 participate in the conversion of LC3-I to LC3-II . Phagophores then recruit cytoplasmic cargo through promiscuous autophagic cargo receptors, including mammalian SQSTM1, NBR1, and CALCOCO2/NDP52, before self-fusing to form double-membraned vesicles [40,41]. These vesicles fuse with endosomes, which deliver vacuolar ATPases, inducing acidification of the vesicle and generating the amphisome . In the last step, fusion of the amphisome and the lysosome forms the so-called autolysosome, which contains the cargo designated for degradation .
Individual components of autophagy play many roles in the cell. The strict definition of autophagy is the degradation of components via the autophagosome and lysosome. Coronaviruses, similar to other viruses, likely utilizes certain components of the pathway to possibly inhibit the degradative process itself, though these components may not always be required. We begin our analysis of this complex relationship with a component of the LC3 lipidation machinery, ATG5.
Coronavirus-induced double-membraned vesicles and vesicle acidification
For all positive-strand RNA viruses studied to date, the viral replication complex associates with host intracellular membranes, although the origin and nature of these membranes vary. Some alphavirus replication complexes associate with endosomal and lysosomal membranes, while there is also evidence for the ER as the source of poliovirus-induced viral vesicles. Many viruses induce complex rearrangements of cellular membranes prior to formation of DMVs in the cytoplasm of the host cell. For some viruses, including poliovirus and hepatitis C virus, these DMVs can serve as sites for viral replication, though they may not be the primary replication sites. For coronaviruses, the replication complex of MHV associates with DMVs, as does the replication complex of the comparable equine arteritis virus (EAV). MERS-CoV and SARS-CoV proteins similarly have been associated with DMVs and other replication organelles. Although not fully understood, DMVs may arise from viral hijacking of the host autophagy pathway, and there is evidence for the involvement of autophagy proteins in DMV generation in rhinovirus- and poliovirus-infected cells.
Viral DMVs, much like autophagosomes, may fuse with late endosomes or lysosomes, suggesting that vesicle acidification may play a role in viral replication or maturation of the virion. For example, vesicle acidification promotes cleavage maturation of a poliovirus capsid protein to result in infectious viral particles. For some coronaviruses, vesicle acidification is important for release of the viral genome into the cytoplasm of the host cell during virus entry. Previous studies show little evidence to suggest a possible role for acidification of DMVs in coronavirus replication or maturation. It was thought that the colocalization of nascent MHV RNA with late endosomal markers suggested that acidification may play a role in MHV replication or maturation. This colocalization suggested fusion of DMVs, which are known sites of MHV replication, with endosomes. However, more recent work using improved imaging technology clearly depicts nascent viral RNA localizing to virus DMVs confirming their role as the primary if not only, site of viral RNA synthesis within replication organelles. It is possible that endosomal markers are re-localized to viral replication organelles.
This DMV-endosome fusion is an important step in the canonical autophagy pathway, supporting the proposed interaction between MHV and the host cell autophagy machinery. Interactions between the canonical autophagy machinery and DMVs may take place during the life cycles of other coronaviruses, though this remains to be investigated. As is true for viruses such as arteriviruses and poliovirus, viral DMVs are smaller than autophagosomes and are not necessarily functional for degradation.Coronavirus DMVs likely do not to function as degradative vesicles because DMVs act as the primary site of RNA synthesis for coronaviruses. These differences may reduce the number of proteins shared by viral replication and the host autophagy pathway, with those proteins involved in membrane curvature and “pinching” of vesicles most likely to be in common.
Roles of individual coronavirus non-structural proteins In many cases, non-structural proteins of RNA viruses induce autophagy, though some may only induce certain steps in the pathway. Specifically, autophagy-related membrane rearrangements induced and directed by individual coronavirus proteins have been studied in infectious bronchitis virus (IBV). In comparison, SARS-CoV and MERS-CoV proteins induce and direct the formation of replication organelles such as DMVs. A brief summary of these studies is listed. For SARS-CoV, individual proteins of the RNA replication complex associate with DMVs localized to the ER. Using uninfected HEK293T cells, concurrent expression of nsp3, nsp4, and nsp6, three of the sixteen non-structural proteins (nsps) of SARS-CoV, is sufficient to induce DMV formation similar to that observed in SARS-CoV-infected cells. When expressed alone, full-length nsp3 or the isolated C-terminal domain of nsp3 results in disordered and proliferating membrane structures. Expression of nsp4 alone produces no distinct phenotype, but co-expression of nsp3 with nsp4 is sufficient to promote the formation of multi-lamellar bodies described as “maze-like.” Nsp6 alone results in the production of single-membrane spherical vesicles localized to the microtubule organizing center, though this phenotype disappears when nsp6 and nsp4 are co-expressed. Only when all three constructs are expressed concurrently do they produce DMVs similar in structure and organization to those formed in SARS-CoV-infected cells. Electron tomography also showed these maze-like bodies with areas of zippered ERs and later confirmed SARS-CoV and MERS-CoV expression of nsp3 and nsp4 induce the formation of DMVs
For MERS-CoV, nsp3 and nsp4 are involved in the formation of DMVs. Co-expression of nsp3 and nsp4, either individually or within the same plasmid as a self-cleaving product, is sufficient to induce the formation of DMVs in HuH-7 cells. Similarly for SARS-CoV, where the formation of DMVs resembling those produced during SARS-CoV infection can occur during co-expression of nsp3 and nsp4 or expression of nsp3, 4, and 6, the MERS-CoV nsp6 is not a requirement for formation of DMVs and, when co-expressed with nsp3 and nsp4, does not alter DMV morphology. For the related equine arteritis virus (EAV), nsp2 and nsp3 co-expression in BHK-21 cells is sufficient for DMV formation.
Expression of nsp6 of the avian coronavirus IBV is sufficient for generation of LC3-puncta.However, the complex disordered membrane structures formed do not strongly resemble autophagosomes. The LC3 puncta, however, also colocalize with LAMP1, indicating possible endosome/lysosome fusion with these disordered membranes. These data have been confirmed in relevant avian cells, using avian LC3. IBV nsp6 induces LC3 puncta formation, but colocalizes with ER markers, whereas SARS-CoV nsp6 colocalizes tightly with LC3, suggesting that SARS-CoV nsp6 may travel to the lysosome, if the canonical autophagy pathway proceeds normally. IBV nsp6 constricts the expansion of the LC3 puncta, which is interpreted as limiting the size of autophagosomes. Interestingly, IBV itself does not induce the autophagic pathway in avian cells, although IBV nsp6 expression induces autophagic signaling in both avian and Vero cells. Furthermore, IBV nsp4 has been confirmed to be the driving protein for membrane pairing where IBV nsp3 co-expression does not alter membrane rearrangement. This same study also found that co-expression of IBV nsp3 and nsp4 and co-expression of nsp3, nsp4, and nsp6 do not result in the formation of DMVs but may require other viral proteins to induce DMVs. Taken together, these results from SARS-CoV, MERS-CoV, and IBV demonstrate a variety of roles for coronavirus nsps in the generation of DMVs and progression in the autophagic pathway
Coronaviruses and BECN1
Critical steps in the autophagy pathway rely on the action of the BECN1 protein, part of the class III PtdIns3K complex that participates in early membrane rearrangements leading to the formation of the autophagosome . Additionally, BECN1 promotes fusion of the autophagosome with the lysosome for content degradation . Proteins of negative-sense RNA viruses and DNA viruses, including Influenza A M2, HIV nef, and HSV-1 ICP34.5, target BECN1 to inhibit either nucleation of the autophagosome or fusion of the autophagosome with the lysosome to increase viral replication. BECN1 negatively regulates the innate immune response with CGAS by preventing excessive IFN (interferon) production while promoting autophagic degradation of intracellular pathogens such as HSV-1. It appears that the interaction of viral proteins with BECN1 thus serves to increase viral replication and influence the innate immune response.
Coronavirus proteins downregulate BECN1 in multiple ways. Coronaviruses have different strategies to modulate BECN1 activity. A transmembrane-containing portion of the papain-like protease domain of nsp3, PLP-TM, of HCoV-NL63, which is known to induce autophagosome formation in HeLa, HEK293T, and MCF-7 cells, binds to BECN1 to inhibit the fusion of autophagosomes with lysosomes. Specifically, the nsp3 PLP-TM domain is necessary to complex BECN1 to STING1 (stimulator of interferon response cGAMP interactor 1), which prevents the stimulation of IFN production. Though the interaction between BECN1 and the PLP2-TM domain of HCoV-NL63 is best characterized, it is possible that BECN1 may also associate with the PLP domains of the nsp3 proteins of other coronaviruses; for example, the nsp3 proteins of SARS-CoV and the porcine epidemic diarrhea virus (PEDV), because these domains are known to act as IFN antagonists, and a knockdown of BECN1 with siRNA decreases PEDV replication.
Other coronaviruses may downregulate BECN1 by indirectly downregulating its protein levels. The cellular E3 ubiquitin ligase SKP2 ubiquitinates BECN1, resulting in its degradation. SKP2 therefore acts as a negative regulator of autophagy. The kinase AKT1 activates SKP2 by phosphorylation. MERS-CoV was found to increase phosphorylation of AKT1, increasing its kinase activity. This increase in activation of AKT1 leads to phosphorylation and activation of SKP2, stalling progression of the autophagic pathway. This hypothesis supports the increase in SKP2 phosphorylation and the comparable increase in BECN1 degradation upon infection of VeroB4 cells with MERS-CoV as a mechanism for increasing viral replication. As expected, the number of autophagosomes seems to increase upon infection, despite the indication of so few autolysosomes, which is likely due to a decrease in BECN1 levels. Therefore, MERS-CoV interferes with host cell autophagy by promoting BECN1 degradation. This is consistent with the increase in BECN1 levels and decrease in MERS-CoV replication when infected cells are treated with a SKP2 inhibitor. In this model, MERS-CoV infection activates AKT1 by phosphorylation, which in turn activates SKP2, resulting in the degradation of BECN1. Loss of BECN1 inhibits the fusion of autophagosomes with lysosomes, potentially protecting viral replication complexes located on cellular double-membraned structures.
It is important to contextualize these data; while coronaviruses often inhibit BECN1 function, it does not necessarily mean that the autophagy pathway is inhibited or not involved. Other viruses inhibit upstream autophagy signaling while triggering LC3 lipidation, double-membraned vesicle formation, or utilizing other parts of the pathway. For example, picornaviruses do not require ULK1, which is upstream of BECN1, but they do require LC3 lipidation. Presumably some advantage is afforded to the viruses by non-canonically signaling to induce the membrane-reshaping machinery of autophagy.
As for many other RNA viruses, evidence suggests that coronaviruses interact with the cellular autophagy pathway to enhance virus replication. The development of ER-derived double-membraned vesicles in the host cytoplasm is so similar to autophagosome development that it suggests that coronaviruses are mimicking the cellular autophagy pathway. For many RNA viruses, these double-membrane vesicles serve as genome replication sites, and there is conclusive evidence that virus-induced DMVs are the site of coronavirus RNA synthesis.
However, the link between formation of DMVs, true autophagy, and coronavirus replication remains unclear. An example is MHV: one study determined that MHV replication requires the LC3 modification protein ATG5, whereas a later study using biologically relevant knockout cells concluded that ATG5, and thus autophagy, is not required for MHV replication. Another set of studies posited that MHV does not use canonical autophagy at all, but rather subverts the LC3 protein for a non-canonical role in formation of ERAD-associated structures. The roles of the autophagic pathway and its components appear to vary widely across coronaviruses.
Going forward, what lessons can we take from the history of coronavirus-autophagy studies in understanding the cell biology of SARS-CoV-2 infection? One lesson from the history of the field is that each coronavirus is likely to interact with this pathway in its own unique fashion. Therapeutics such as chloroquine and its derivatives, which inhibit the autophagic pathway, have been suggested as treatments for COVID-19. Therefore, understanding how this and other intracellular pathways affect SARS-CoV-2, and if it is similar to other coronaviruses in these interactions, is an important factor in confronting this and future coronavirus outbreaks.
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