Abstract
Autophagy is a constitutive cellular process of degradation required to maintain homeostasis and turn over spent organelles and aggregated proteins. For some viruses, the process can be antiviral, degrading viral proteins or virions themselves. For many other viruses, the induction of the autophagic process provides a benefit and promotes viral replication. In this Review, we survey the roles that the autophagic pathway plays in the replication of viruses. Most viruses that benefit from autophagic induction block autophagic degradation, which is a ‘bend, but don’t break’ strategy initiating but limiting a potentially antiviral response. In almost all cases, it is other effects of the redirected autophagic machinery that benefit these viruses. This rapid mechanism to generate small double-membraned vesicles can be usurped to shape membranes for viral genome replication and virion maturation. However, data suggest that autophagic maintenance of cellular homeostasis is crucial for the initiation of infection, as viruses have evolved to replicate in normal, healthy cells. Inhibition of autophagic degradation is important once infection has initiated. Although true degradative autophagy is probably a negative for most viruses, initiating nondegradative autophagic membranes benefits a wide variety of viruses.
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Introduction
Autophagy is a process by which proteins, lipids and even whole organelles are sequestered and targeted to lysosomes for degradation1,2,3. Autophagy is critical for many physiological processes, including cell development and differentiation, embryogenesis, erythrocyte maturation, stemness and the removal of damaged and dysfunctional cellular contents, ensuring cellular quality control3,4. Defects in autophagy have been implicated in pathophysiological conditions such as Alzheimer’s disease, Parkinson’s disease and Crohn’s disease5,6,7,8. Although the landscape of autophagy literature is expansive, this section aims to introduce the key factors that are targeted by viruses. For a more comprehensive understanding, we recommend reading more general reviews on autophagy9,10.
The simplest description of nonspecific general macroautophagy, usually shortened to autophagy, involves the creation of cellular sequestration membranes known as phagophores, which acquire the lipidated autophagy protein LC3 (refs. 11,12). As shown in Fig. 1, these phagophores engulf cytosolic contents with assistance from both general and cargo-specific adapter proteins, the best-studied of which is sequestosome 1 (SQSTM1; formerly known as p62)13,14,15. The phagophore self-anneals, forming a double-membraned vesicle known as an autophagosome. Autophagosomes fuse with endosomes, which introduce cargo, endosomal markers and vacuolar ATPases that acidify the vesicle, which is now known as an amphisome16. This acidification is required for fusion with lysosomes to form the autolysosome, in which contents are degraded and recycled for use by the cell, completing the process of autophagic flux17.
Cup-shaped phagophore membranes containing LC3-phosphatidylethanolamine (known as LC3-II) are the first physical hallmarks of autophagy. These phagophores capture cytosolic cargo using both specific and generalized cargo receptors and self-fuse to form double-membraned autophagosomes. Fusion with endosomes generates acidic amphisomes. The amphisomes then fuse with lysosomes to form single-membraned autolysosomes, in which autophagic degradation of delivered cargo occurs.
The induction of the autophagic process is regulated by the master nutrient and energy sensors adenosine monophosphate-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR), which signal to the first of two major autophagy signalling complexes shown in Fig. 2 (ref. 18). The first complex, consisting of the Unc-51-like kinase 1 (ULK1) and accessory proteins RB1CC1, ATG13 and ATG101, receives the signal from mTOR and transfers it to the second specific complex, consisting of Beclin-1, VPS34, VPS15 and ATG14 (ref. 19). The latter complex contains a class III phosphoinositide kinase catalytic subunit (VPS34) and regulatory subunit (VPS15)20. The phosphorylation of Beclin-1 is a major regulatory trigger for the generation of phosphatidylinositol 3-phosphate, which is crucial for the formation of the isolation membrane or phagophore21. The LC3 protein, one of the best-studied and defined markers of autophagosomal membranes, is cleaved at the C terminus by the ATG4 protease, removing five amino acids and leaving a C-terminal glycine22. The ATG7 protein, homologous to an E1 ubiquitin ligase, and ATG3, which is E2-like, conjugate the LC3 protein to the lipid phosphatidylethanolamine, which both triggers localization of LC3 to nascent autophagosomal membranes and induces their curvature23,24. The conjugation of ATG5–ATG12–ATG16L1, which ATG7 and ATG10 mediate, drives the elongation and closure of the phagophore to form the autophagosome23,25,26. The ATG5–ATG12–ATG16L1 complex functions as an E3 ligase for the lipidation of LC3 and is recruited to the phagophore through the membrane binding property of ATG16L1 and its interaction with the phagophore-associated protein, WIPI-2 (refs. 26,27). Loading of cargo into autophagosomes is mediated by receptors that bind LC3 and cargo simultaneously, including the general cargo adapters SQSTM1, NBR1 and ALFY13.
Signals relayed through general cellular regulators Akt and the mammalian target of rapamycin (mTOR) complex initiate modulation of the autophagic pathway. Specific autophagic regulation begins with the complex containing Unc-51-like kinase 1/2 (ULK1/2) kinases, regulated by RB1CC1, ATG13 and ATG101. ULK signals to the Beclin-1 complex, which also contains the type III phosphoinositide 3-OH (PI3) kinase VPS34, ATG14 and Ambra1. This complex is positively modulated by UVRAG binding and is negatively regulated by Rubicon binding. Beclin, and other independent factors such as TBK1, regulate the steps of LC3 lipidation. First, ATG4 cleaves the C terminus of LC3, exposing a glycine residue. Then, ATG7 and ATG3, which are E1 and E2-like enzymes, respectively, conjugate LC3 to phosphatidylethanolamine. This anchors LC3 to membranes, promoting curvature. The ATG5– ATG12– ATG16L complex promotes elongation of the nascent autophagosome. Cargo receptors interact with LC3 to bring varied cargo to the autophagosome interior before it closes. SQSTM1 (p62), NBR1 and ALFY are the best-studied receptors for macroautophagy. Mitophagy is known to use NDP52 and OPTN. Many endoplasmic reticulum (ER)-resident proteins, shown, have been implicated in ER-phagy. Specific lipophagy receptors have not been elucidated.
There is a major second level of regulation: postautophagosome formation. This comes into play when autophagosomes fuse with late endosomes and lysosomes for cargo degradation and recycling (Fig. 3). Endosomal fusion, although not well characterized, is thought to involve Rab7. This process delivers endosomal proteins, including LAMP1 and vacuolar ATPases, leading to the formation of amphisomes16,28. Amphisome–lysosome fusion is mediated by SNARE protein complexes, the best studied of which consists of STX17, synaptosome associated protein 29 (SNAP-29) and VAMP8, and several associated tethering factors, including the homotypic fusion and protein sorting (HOPS)-tethering complex and pleckstrin homology and RUN domain containing M1 (PLEKHM1)29,30,31. Controlling autophagosome fusion and maturation events is an important regulatory step often exploited by viruses.
Two major vesicular maturation steps lead to cargo degradation and autophagic flux. Endosomes fuse with autophagosomes, delivering vacuolar ATPases that acidify the vesicles into amphisomes. Rab7 is thought to play a role in this fusion event as well as the subsequent event. Amphisomes fuse with lysosomes in a tightly regulated step involving the SNAP-29–STX17–VAMP8 SNARE complex, as well as the multifactor homotypic fusion and protein sorting (HOPS) complex, pleckstrin homology and RUN domain containing M1 (PLEKHM1) and EPG5. vATPase, vacuolar ATPase.
Autophagic processes are often induced during viral infection. The autophagosomes formed during viral infection can target viruses or their components to the lysosomes for degradation through a process known as xenophagy or virophagy32,33. Autophagy also provides microbial antigenic peptides for presentation to CD4+ T cells, forming an essential part of the host immune response34. Despite the inherently antiviral nature of a process that degrades cytosolic contents, several viruses, in particular RNA viruses, have developed mechanisms to avoid, subvert or co-opt the cellular process for their benefit35. Typically, viruses that do not need autophagic membranes, and may be susceptible to autophagic degradation, often block autophagy initiation by targeting Beclin-1. By contrast, viruses that require autophagic signalling or membranes induce the cellular process but often block the downstream autophagic maturation, suggesting that autophagic flux may be detrimental to these viruses. A few viruses appear indifferent to the autophagic process. In addition to manipulating nonspecific bulk autophagy, many viruses also induce organelle-specific autophagy, including mitophagy (autophagic degradation of mitochondria), lipophagy (autophagic degradation of lipids) and endoplasmic reticulum (ER)-phagy (autophagic degradation of the ER), for distinct purposes, such as blocking apoptosis, evading host antiviral immune responses and providing ATP to power viral RNA replication. These forms of autophagy share many features with nonselective macroautophagy, as they both require core autophagic components and entail the formation of autophagosomes that envelop the organelles and target them for lysosomal degradation. Unlike nonselective autophagy, however, organelle-specific autophagy includes additional molecular players that are typically not involved in macroautophagy and that may, in part, contribute to the selectivity of the process. For instance, during mitophagy induction, the accumulation of the mitochondrial health sensor, PINK1, in depolarized mitochondria triggers the translocation of the E3 ubiquitin ligase, Parkin, to the mitochondria, leading to the autophagic degradation of the mitochondria36,37. In ER-phagy, the ER-resident proteins FAM134B, ATL3, RTN3L and others act as cargo receptors and recruit the LC3 degradation machinery to the ER38 (Fig. 2). In lipophagy, lipid-droplet-containing autophagosomes fuse with lysosomes for degradation, although the specific cargo receptors that ensure selectivity for lipophagy are unclear39.
In this Review, we explore the complex albeit fascinating interplay between viruses that cause human disease and autophagy, focusing on the strategies that viruses use to avoid or manipulate autophagic components and membranes for their benefit.
Autophagy as a ‘Goldilocks’ process
Biologists are often trained to think of signalling pathways and their downstream consequences as being ‘on’ or ‘off’. There is good reason for the existence of this paradigm. Some of the best-studied pathways, such as the mitogen-activated protein kinase (MAPK) signalling pathway, have redundant kinase cascades. These cascades have evolved to tightly regulate the binary nature of the output signal40,41,42,43. Autophagy, however, is regulated in a different fashion. The default state of the pathway is ‘on’, as a certain level of autophagy is required for cellular homeostasis, which is ‘just right’ for the cell. This level can be tweaked and drastically increased in circumstances where it is desired, such as amino acid starvation. However, when such upregulation occurs, the resources required for this process can be quickly exhausted. In these circumstances, the major cargo adaptors, such as SQSTM1 and NBR1, are degraded along with their cargo, more rapidly than they can be replaced15,44. Specific phospholipids required for production of autophagic membranes can also have their pools depleted. The LC3 protein itself, essential for the formation of the initial phagophore, is only partially de-lipidated from LC3-II to LC3-I and recycled; the LC3 population on the inner autophagosomal membrane is degraded, limiting new LC3-II generation45,46. Understanding the just right nature of the process is critical to understanding its relationship to viruses. This leads to strange paradoxes, in which viral proteins induce autophagic induction, whereas others, sometimes from the same virus, inhibit degradative flux, leading to what we describe as ‘not-ophagy’, which we define as ‘engagement with the autophagic machinery without autophagic degradation’.
Viral evasion of macroautophagy
Macroautophagy has often been described as a potent antimicrobial mechanism against intracellular pathogens. Many of the earliest papers on autophagy and viruses, some of which predated our understanding of the molecular genetics of the process, described autophagy as a probable antiviral mechanism47,48,49. Accordingly, viruses have evolved myriad strategies to avoid, manipulate or even repurpose the cellular process of autophagy for their benefit. Viruses generally avoid autophagy in two ways: (1) blocking autophagosome formation (initiation of autophagy) and (2) preventing autophagic flux (autophagosome maturation).
Preventing autophagosome formation
Viruses known to inhibit autophagic initiation are shown in Fig. 4. One of the first virally encoded proteins discovered to block autophagosome formation is the herpes simplex virus 1 multifunctional protein and neurovirulence factor, ICP35.4. During herpes simplex virus 1 (HSV-1) infection, ICP35.4 blocks the initiation of autophagy by directly interacting with Beclin-150. Beclin-1 is a crucial member of the VPS34–Beclin-1 complex, which is required for isolation membrane or phagophore formation21. ICP35.4 also interacts with TANK-binding kinase 1 (TBK1), a pivotal component of the Toll-like receptor signalling pathway, obstructing TBK1 interaction with its downstream target IRF3, thereby blocking the type 1 interferon response51. As TBK1 is also essential for autophagic flux52, ICP34.5 interaction with TBK1 could potentially obstruct autophagosome–lysosome fusion in HSV-1-infected neuronal cells. However, this is unlikely as HSV-1 blocks the initiation of autophagy. In non-neuronal cells, interestingly, replication of the ICP34.5 mutant, which cannot interact with Beclin-1, was unaffected when autophagy was blocked, suggesting autophagy restricts HSV-1-infection in a cell-type-dependent manner53.
Activation of the phosphoinositide 3-OH (PI3) kinase VPS34 is induced by two different hepatitis viruses, hepatitis B virus (HBV) and Newcastle disease virus (NDV). For hepatitis C virus (HCV), timing is important, with activation of Rubicon early in infection and UVRAG later in infection. Multiple viruses inhibit Beclin-1 function, including human immunodeficiency virus (HIV), herpes simplex virus (HSV) and influenza A virus (IAV), African swine fever virus (ASFV), barley stripe mosaic virus (BSMV) and foot-and-mouth disease virus (FMDV). These viruses each exhibit different secondary inhibitions of activation: HIV also inhibits STAT1 signalling, HSV inhibits TBK1 activity and IAV inhibits the mammalian target of rapamycin (mTOR) complex. Cauliflower mosaic virus (CMV) activates mTOR, which inhibits autophagy induction.
Although HSV-1 blocks autophagic initiation in neuronal cells, the selective autophagy receptor, Optineurin, has been shown to restrict HSV-1 infection in vitro and in vivo by targeting the viral tegument protein VP16 and the fusion glycoprotein gB for autophagic degradation. Optineurin-deficient mice succumbed to lethal central nervous system infection and demonstrated significant cognitive decline, suggesting a protective role for autophagy against HSV-1-induced neuronal damage54. A similar neuroprotective role of autophagy during Sindbis virus (SINV) infection, orchestrated by another autophagy receptor, p62, has been previously reported55.
Recently, the Us3 gene product, an α-herpesvirus specific Ser/Thr kinase, has also been reported to block autophagic initiation during HSV-1 infection. Us3 phosphorylates Beclin-1 at S234 and S295 (ref. 56). Beclin-1 phosphorylation at S234 and S295 augments its interaction with 14-3-3 and vimentin intermediate filament proteins, which block autophagy in cancer cells57. Other well-studied herpesviruses, such as human cytomegalovirus and Kaposi’s sarcoma-associated herpesvirus, also restrict autophagy by targeting Beclin-1 using various mechanisms58,59. Given the deleterious effects of autophagy on the replication of these viruses, targeting Beclin-1 appears to constitute a common strategy that herpesviruses use to restrict autophagy at an early step.
In addition to herpesviruses, the human immunodeficiency virus 1 (HIV-1) Nef protein has also been reported to block autophagic initiation by enhancing the interaction of Beclin-1 with its negative regulator, B cell lymphoma 2 (Bcl2), thereby impairing autophagosome formation during HIV-1 infection60. The HIV-1 transactivator protein Tat has also been documented to attenuate interferon-γ-induced autophagosome formation in macrophages by blocking STAT1 phosphorylation61. In most cases, inducing autophagy reduces HIV-1 infection62,63,64.
Some animal and plant viruses have also been documented to block autophagic initiation. For example, A179L, the African swine fever virus (ASFV) Bcl2 homologue, blocks autophagy during ASFV infection by directly interacting with Beclin-1 through its BH3 homology domain65. Induction of autophagy before or during ASFV infection reduces viral titres, suggesting that autophagy downregulates ASFV infection. The plant virus cauliflower mosaic virus (CMV) impairs autophagic initiation by activating MTOR. The CMV multifunctional protein P6 induces MTOR phosphorylation and blocks salicylic-acid-induced autophagy in Arabidopsis thaliana66. Another plant virus, barley stripe mosaic virus (BSMV), also inhibits autophagic induction through its γb protein, which interacts with Beclin-1 to impair early steps in the process. Silencing autophagy enhances BSMV spread and symptoms in plants, consistent with an antiviral effect67. Together, these findings suggest that Beclin-1 is a primary target for viruses that block autophagic initiation.
Blocking autophagic flux
Although many herpesviruses do not require the machinery of autophagy for their replication and therefore block autophagy initiation, picornaviruses and many other RNA viruses, including flaviviruses and coronaviruses, need autophagic signalling or membranes for productive infection (Figs. 5 and 6). Infection with these viruses, therefore, triggers autophagic signalling, and they use the autophagy-pathway-derived membranes for their benefit. As these viruses are cytoplasmic and could be potentially targeted for autophagic degradation, they have evolved multiple mechanisms to stop autophagic flux. Blocking autophagic flux may benefit these viruses in multiple ways: first, it could maximize virus replication by inducing the accumulation of autophagosomes; second, it could prevent the degradation of host or viral factors important for viral infection; and third, it could promote viral egress.
Several RNA viruses, which carry out most of their viral production cycle in the cytosol, inhibit cargo loading into vesicles by cleaving autophagic cargo receptors. Proteases from the picornaviruses poliovirus (PV), enterovirus D68 (EV-D68) and coxsackievirus B3 (CVB3) all cleave the primary cargo receptor sequestosome 1 (SQSTM1), also known as p62. EV-D68 and CVB3 have also been shown to cleave another receptor, NBR1. Three flaviviruses, dengue virus (DENV), Zika virus (ZIKV) and West Nile virus (WNV) cleave the cargo receptor FAM134B. The prevention of cargo loading may prevent some viral products from being taken up in autophagosomes when it is not to the virus’ advantage. Alternatively, these mechanisms may simply provide more cargo space for viral proteins, including virions.
Although specific virophagy is understudied, two viruses, Zika virus (ZIKV) and Sindbis virus (SINV), inhibit virophagy-specific receptors. For other viruses, degradation is inhibited in a more general fashion. Hepatitis B virus (HBV) inhibits multiple proteins involved in autophagic flux regulation, including the vacuolar ATPase (vATPase) that acidifies endosomes (and amphisomes after fusion with autophagosomes). HBV also inhibits Rab7, part of the homotypic fusion and protein sorting (HOPS) fusion complex, and synaptosome associated protein 29 (SNAP-29), a SNARE protein, both of which mediate lysosomal fusion. SNAP-29 is also targeted by enterovirus D68 (EV-D68), coxsackievirus B3 (CVB3) and human parainfluenza virus type-3 (HPIV3). The CVB3 3C protease also cleaves pleckstrin homology and RUN domain containing M1 (PLEKHM1). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibits the HOPS complex. The only viruses with a relationship to autophagy that do not appear to inhibit flux are the flavivirus family. For most viruses, a ‘bend, but don’t break’ relationship to autophagy, in which flux is inhibited, is clearly an advantage to the virus. FANCC, Fanconi anaemia protein C; SMURF1, SMAD-ubiquitin regulatory factor 1.
Picornaviruses, such as poliovirus, enterovirus D68 (EV-D68), coxsackievirus B3 (CVB3) and enterovirus 71(EV-71), among others, manipulate the cellular autophagic process for their benefit. These viruses rely on the autophagic machinery and membranes for various phases of their life cycle. Consequently, knockdown of essential autophagy-related proteins attenuates the replication of these viruses. Although the significance of autophagic induction for picornavirus infection is incontestable, these viruses typically block autophagic flux in multiple ways.
For instance, during EV-D68 or CVB3 infection, the viral protease 3C cleaves the SQSTM1 protein, the major adaptor protein linking ubiquitinated cargo to the autophagic machinery for degradation68,69. By cleaving SQSTM1, these viruses attenuate cargo loading, thereby inhibiting virophagy. We and others have observed SQSTM1 cleavage in poliovirus-infected and rhinovirus-infected cells, indicating that these viruses use similar strategies to impair cargo loading and block autophagic degradation70. The 3C protease also cleaves SNAP-29. SNAP-29 is a Qbc SNARE that is essential for autophagosome–lysosome fusion by linking the Qa SNARE on the autophagosome to the R SNARE on the lysosome. As such, cleaving SNAP-29 impairs autophagosome–lysosome fusion, preventing cargo degradation. Another SNARE protein that plays essential roles in autophagosome–lysosome fusion is the SNAP-47 orphan SNARE, whose protein levels are reduced during EV-D68 infection through unknown mechanisms68.
In addition to cleaving SQSTM1 and SNAP-29, the 3C protease of CVB3 also cleaves the tethering protein, PLEKHM1, which also participates in autophagome–lysosome fusion69. The human parainfluenza virus type-3 (HPIV3) has also been reported to block autophagic flux, which increases viral production. Mechanistically, the viral phosphoprotein interacts with SNAP-29 and impairs the formation of the SNARE complex, thereby blocking autophagosome–lysosome fusion71.
Hepatitis B virus (HBV), a hepatotropic liver pathogen and a significant cause of viral-induced hepatocellular carcinoma, also induces autophagosome formation but blocks autophagosome–lysosome fusion by decreasing SNAP-29 and Rab7 protein levels and impairing lysosomal acidification by interacting with vacuolar-type H+-ATPase, which is essential for the acidification of lysosomes72,73,74.
Dengue virus (DENV), a vector-borne flavivirus and the aetiologic agent of dengue haemorrhagic fever and dengue shock syndrome, also impedes autophagic flux late during infection. However, the mechanism by which DENV blocks autophagic flux was not clarified in this study75.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the aetiologic agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic, has also been reported to block autophagic flux. The SARS-CoV-2 accessory protein, ORF3a, is the primary viral protein that impairs autophagic flux. During SARS-CoV-2 infection, ORF3a interacts and sequesters VPS39, a component of the HOPS complex, preventing the interaction of the HOPS complex with the autophagosomal SNARE proteins syntaxin-17 or Rab7 and blocking autophagic flux76,77.
The gammaherpesvirus Epstein–Barr virus (EBV) activates autophagic signalling during its lytic cycle but blocks autophagosome–lysosome fusion. Paradoxically, the expression of ZEBRA, the first EBV protein expressed on EBV lytic cycle activation, induces autophagic flux, whereas the expression of the complete set of lytic genes impairs autophagic flux by presumably decreasing Rab7 levels78. Subsequently, the authors found that another early lytic gene, BFRF1, is responsible for autophagic flux blockade during the lytic cycle. Loss of BFRF1 augments Rab7 protein levels, suggesting that BFRF1 blocks autophagic flux by decreasing Rab7 protein expression79.
Influenza A virus (IAV) also blocks autophagic flux by preventing autophagosome–lysosome fusion80. IAV infection induces extensive GFP–LC3 puncta formation, which failed to colocalize with LysoTracker. Overexpression of the M2 protein of IAV is sufficient to cause autophagosome accumulation. Although precisely how M2 blocks autophagosome–lysosome fusion is unclear, immunoprecipitation analysis revealed that M2 interacts with Beclin-1, which forms an integral part of VPS34 complex 2, an essential component for autophagic flux. Therefore, by interacting with Beclin-1, M2 disturbs the formation of VPS34 complex 2 and impairs autophagic flux.
The picornavirus foot-and-mouth disease virus (FMDV), an aetiologic agent of foot-and-mouth disease in animals, uses a similar mechanism to block autophagic flux. The 3C protein of FMDV physically interacts with Beclin-1, preventing Beclin-1 from mediating autophagosome–lysosome fusion. Overexpression of Beclin-1 overrides the effect of 3C in impairing autophagosome–lysosome fusion and reduces FMDV titres, indicating that blocking autophagic flux is essential for FMDV infection81.
Finally we come to the flaviviruses, which are also known to benefit from cellular autophagy, but in a unique fashion. These viruses use autophagy-pathway-derived membranes for translation, RNA replication and viral release. Hepatitis C virus (HCV) is one of the most widely studied flaviviruses in terms of its interaction with cellular autophagy. However, although it is clear that HCV induces the autophagic machinery, whether or not the virus causes complete or incomplete autophagy is still contentious. HCV was first shown to induce the accumulation of autophagosomes to promote viral RNA replication through the unfolded protein response in 200882. Since then, several groups have demonstrated that HCV infection causes complete autophagy83,84. In 2015, a study showed that HCV in fact regulates autophagy temporally, with different effects at different stages of infection85. The authors show that HCV causes incomplete autophagy at early time points during infection, whereas complete (degradative) autophagy is induced at later time points. This temporal regulation of autophagy depends on Rubicon and UVRAG proteins, which negatively and positively regulate autophagic signals. Whereas Rubicon is induced early and prevents autophagic flux, UVRAG, which promotes autophagic flux, is induced later during infection, thereby allowing autophagic flux induction. It was recently reported that acute, but not chronic HCV infection, induces autophagic flux, which is consistent with in vitro data regarding the temporal regulation of flux86. Still, HCV was shown to impede autophagosome–lysosome fusion by altering the subcellular localization of Arl8b, a lysosome localized GTPase that plays essential roles in autophagic flux87,88. Given that the HCV multifunctional protein NS5A was shown to be targeted for autophagic degradation, impairing autophagic flux would be expected to promote HCV RNA replication89. Although there are mixed data, the flaviviruses stand out as the only family for which it is suggested that autophagic degradation may benefit the viruses.
Viral exploitation of autophagic membranes
The result of inducing autophagosome formation, but blocking flux, is the accumulation of autophagic membranes. As highlighted above, several positive-strand RNA viruses, which require membranes to assemble viral RNA replication organelles, induce autophagosome accumulation during their infection. The double-membrane autophagosomes generated during infection with these viruses could serve as scaffolds for viral replication, protect virions from innate immune sensing, promote capsid maturation and facilitate viral release.
Picornaviruses, which are known to benefit from the autophagic machinery, are adept at exploiting the pathway for their benefit. Poliovirus, a prototypical member of the Picornaviridae family, was first shown in 1965 to induce the formation of double-membrane vesicles that resemble autophagosomes90. For several years, as molecular tools for autophagy were developed, researchers in the field extensively studied these membranes. It was 40 years after the publication of images showing that poliovirus forms double-membrane vesicles that it was finally shown that poliovirus-induced vesicles display several hallmarks of autophagy, including their colocalization with the autophagy markers LAMP1 and LC3 (ref. 91). Co-expression of viral proteins 2BC and 3A was sufficient to induce double-membraned vesicles with many of the hallmarks of autophagosomes, similar to what was observed during bona fide poliovirus infection91,92. The induction of the autophagic machinery increased poliovirus titres, whereas inhibition of autophagy either through 3-methyladenine (3MA) treatment or small interfering RNA (siRNA)-mediated knockdown of essential autophagy-related proteins reduced poliovirus titres, indicating the importance of autophagy for poliovirus replication. In a subsequent study, it was demonstrated that poliovirus induction of the autophagic machinery, in contrast to starvation-induced autophagy, does not require canonical autophagic signalling complexes. Depletion of the members of the ULK1 initiation complex, including RB1CC1, ATG13 and ATG101, did not affect poliovirus-induced LC3 lipidation or poliovirus titres93. These findings suggest that poliovirus manipulates the autophagic pathway downstream of the initiation complex, which is something that has become a theme in the field.
Autophagic membranes play critical roles in poliovirus nonlytic release through a phenomenon called autophagosome-mediated exit without lysis (AWOL), for which virus-containing autophagosomes fuse with the plasma membrane to release virions, avoid innate immune detection and facilitate cell-to-cell spread. Depleting LC3 through siRNA-mediated knockdown reduced nonlytic intercellular spread, and the induction of autophagy enhanced viral spread and increased pathogenicity in mice94. These virus-filled, autophagosome-derived phosphoserine-rich vesicles deliver multiple virions to target cells95. Autophagic membranes also play a vital role in poliovirus capsid maturation, which entails processing the viral protein VP0 to generate VP2 and VP4. Whereas inhibiting autophagosome formation attenuates viral RNA replication, blocking vesicle acidification through bafilomycin or ammonium chloride treatments does not affect viral RNA replication but strongly inhibits the final maturation cleavage of VP0. Together, these findings demonstrate that poliovirus and other picornaviruses use autophagy-derived membranes for multiple purposes, including genomic RNA replication, capsid maturation and nonlytic release and suggest that targeting the autophagic machinery could attenuate poliovirus infection and reduce its pathogenicity96.
EV-D68 was recently shown to similarly rely on autophagic signalling (and the resultant membranes) for successful infection68. EV-D68 infection of H1HeLa cells rearranges membranes and triggers the accumulation of double-membrane vesicles. The induction of LC3 lipidation was observed as early as 3 h post-EV-D68 infection, which coincides with peak viral RNA replication, indicating that EV-D68 infection induces autophagic membranes. Like poliovirus, inducing autophagy promoted EV-D68 infection and blocking autophagy via ATG7 knockdown reduced EV-D68 infection. Unlike poliovirus, the specific EV-D68 protein or proteins that induce autophagic membranes are yet to be determined. Other human and animal picornaviruses, including Seneca Valley virus and human rhinovirus-2, have been shown to require the cellular processes of autophagy for infection97,98,99.
It appears to be common among enteroviruses (but not other picornaviruses) that autophagic membranes facilitate virus release in extracellular vesicles. Infection of neuronal progenitor and stem cells with CVB3 increases the shedding of microvesicles, which harbour infectious viral particles and associate with GFP–LC3, indicating that the autophagic machinery aids CVB3 release. CVB3 RNA replication also requires functional autophagic pathways. The induction of autophagy increases CVB3 RNA replication, whereas blocking autophagy reduces CVB3 RNA levels100. Consistent with these findings, authors noted the deletion of ATG5 in pancreatic acinar cells, decreased viral titres and reduced CVB3 pathogenicity in mice101. EV-71 also exploits autophagic vesicles and membranes for its replication. EV-71 infection of rhabdomyosarcoma and neuroblastoma (SK-N-SH) cells triggers GFP–LC3 puncta formation and LC3 lipidation, and pharmacological inhibition or induction of autophagy decreased and increased EV-71 titres102,103. In a subsequent study, the authors demonstrated that EV-71-encoded proteins VP1 and 2C colocalize with LC3 and inhibition of autophagy reduced EV-71 viral load and pathogenicity in suckling mice104.
Multiple lines of evidence, with the earliest being a study reported in 2008, have demonstrated that the suppression of autophagy diminishes HCV replication, indicating that the autophagic membranes or vesicles induced during HCV infection support viral production82. Consistent with a role for autophagic membranes in promoting HCV RNA replication, an HCV double-stranded RNA replication intermediate, associated with autophagosomes, was detected by electron microscopy. In addition, several HCV replicase proteins, including the NS5B RNA-dependent RNA polymerase, were found to colocalize with autophagosomes105,106. Autophagic proteins have also been implicated in the translation of the incoming HCV RNA. Depletion of Beclin-1, ATG4 and ATG12 robustly reduced HCV internal ribosome entry site–dependent translation107. However, the role of autophagy itself in promoting HCV translation has been contested84. Instead, these authors argue that the entire autophagy process from induction to completion is required to promote HCV RNA replication through inhibition of the antiviral immune response.
RNA viruses from different families share similar strategies. DENV infection causes autophagosome formation in mouse embryonic fibroblasts (MEFs) and Huh-7 cells. These autophagosomes later mature by fusing with endosomes and lysosomes, as indicated by the colocalization of GFP–LC3 punctate dots with LAMP1. The induction of autophagy through rapamycin treatment increased DENV titres. Inhibiting autophagy, by contrast, reduced DENV titres, indicating that autophagy promotes DENV replication108. A further study demonstrated that a DENV replicase protein, NS1, colocalized with LC3 and a double-stranded RNA replication intermediate, indicating that DENV RNA replication may occur on autophagic membranes. Interestingly, blocking autophagosome–lysosome fusion increased both intracellular and extracellular DENV titres, which is consistent with data from other RNA viruses showing that a block in autophagic flux can promote virus replication and cell exit109,110.
For other viruses, autophagy is a double-edged sword. Zika virus (ZIKV), a flavivirus associated with microcephaly, a severe fetal abnormality that impairs normal brain development, was shown to induce autophagy in fetal neural stem cells111,112. ZIKV infection of these cells results in LC3 lipidation accompanied by a concomitant decrease in p62 protein levels, indicating that ZIKV induces degradative autophagy in these cells. Inducing autophagy increased ZIKV RNA levels, whereas blocking autophagy initiation (3MA treatment) or flux (bafilomycin treatment) reduced ZIKV RNA replication112. Autophagy has also been implicated in ZIKV transmission from mother to child, and inhibition of autophagy attenuated vertical transmission in pregnant mice113. Interestingly, whereas ZIKV infection induces nonselective autophagic flux, the virus-encoded NS4B and NS5 proteins block virophagy (the selective autophagic degradation of viral components) by downregulating Fanconi anaemia protein C (FANCC), which was shown to be essential for virophagy and mitophagy but not for starvation-induced autophagy112,114. However, whether ZIKV reduces the protein levels of SMAD-ubiquitin regulatory factor 1 (SMURF1), a virophagy receptor that targets HSV-1 and SINV for degradation, is unclear115. Together, these findings indicate that vector-borne flaviviruses induce autophagic flux. However, how these viruses or their components avoid autophagic degradation has yet to be entirely understood. The finding that ZIKV induces autophagic flux, while simultaneously blocking virophagy, could partly explain how these viruses avoid autophagic degradation.
IAV, for example, subverts the autophagy pathway for its benefit. IAV infection of Madin–Darby canine kidney cells leads to the induction of LC3-II. Pharmacological and genetic inhibition of autophagy reduced IAV yields and the accumulation of IAV matrix (M) proteins M1 and M2. As M2 is essential for IAV assembly, inhibition of autophagy could disrupt IAV assembly and reduce IAV yields116. To further confirm how IAV regulates autophagy and determine the significance of autophagy for IAV infection, a study recently demonstrated that IAV M2 and nucleoprotein (NP) proteins are sufficient to induce autophagy by inhibiting the Akt–mTOR signalling117. Furthermore, these viral proteins colocalize with LC3 puncta and increase viral ribonucleoprotein (vRNP) export and infectious viral particle formation. Induction of autophagy through starvation or rapamycin treatment increased progeny virus production, and inhibition of autophagy decreased viral production. The NP-mediated induction of HSP90AA1, an essential host factor for influenza virus genome replication, is also dependent on autophagic components, as overexpression of NP failed to induce HSP90AA1 in ATG5 knockout cells117. These findings are congruent with the positive role of autophagy during IAV infection and suggest that, as with the other viruses discussed above, modulation of autophagy could constitute a plausible anti-influenza virus strategy.
Unique viruses have unique relationships with autophagy. During HBV infection, the viral regulatory protein HBx, binds and stabilizes the class III phosphoinositide kinase to induce autophagy. Blocking autophagy through 3MA treatment or VPS34 or ATG5 knockdowns reduces HBV DNA replication118,119. Autophagy is also implicated in HBV envelopment. Genetic or pharmacological inhibition of autophagy severely impeded HBV assembly, without affecting its release. By contrast, the induction of autophagy enhances HBV production120.
Measles virus (MeV), the causative agent of measles, a highly contagious childhood disease characterized by maculopapular rash, also subverts autophagic pathways for its benefit. MeV-induced autophagy requires the immunity-associated GTPase family M (IRGM), as knockdown of the cellular-autophagy-associated protein reduced autophagic machinery induction in MeV-infected cells121. Fascinatingly, multiple steps of the viral life cycle, including entry, viral RNA replication and syncytia formation, successively induce the autophagic machinery, which enhances MeV infectivity by attenuating cell death and promoting viral particle formation122. Knockdown of ATG5 or ATG7, or inhibition of autophagic flux with chloroquine, decreases MeV titres, indicating that MeV requires complete autophagy for its replication121,122.
Autophagic membranes also support EBV lytic gene expression and viral production. Inhibition of autophagy reduced lytic gene expression and EBV virus production78,123. EBV uses autophagosomes for intracellular transport, particle envelopment and exit, and the lipidated LC3, LC3-II, not LC3-I, could be detected in viral particles78,123. Human cytomegalovirus (HCMV) also uses autophagic membranes for assembly, and many autophagy proteins could be detected in purified HCMV particles. Similar to EBV, inhibition of autophagy impedes HCMV production124.
Varicella zoster virus (VZV) also benefits from autophagic membranes. Infection with VZV induces autophagosome formation, which contributes to viral glycoprotein biosynthesis and processing125. Interestingly, both lipidated LC3 and Rab11, a marker for amphisomes, copurified with VZV particles, suggesting a role for amphisomes in VZV exocytosis126.
The highly pathogenic avian IAV (H5N1), which primarily infects alveolar epithelial cells in the lungs, has been suggested to induce autophagic cell death in MEFs. IAV H5N1 infection of MEFs leads to cell death, and inhibition of autophagic signalling attenuated the cell death caused by IAV H5N1, suggesting that autophagy promotes the pathogenicity of IAV H5N1 (ref. 127). However, given that the autophagic machinery is essential for influenza virus infection, restoration of cell viability in autophagy-deficient cells is most probably due to reduced viral replication in these cells.
Newcastle disease virus (NDV), a highly contagious animal virus that infects birds, also usurps the machinery of autophagy for its replication. Live NDV infection, but not ultraviolet (UV)-inactivated virus infection, induces autophagic signals in U251 glioma cells through the class III phosphoinositide 3-OH kinase (PI3K)–Beclin-1 pathway, indicating that NDV replication is required to induce autophagic signals128. Rapamycin-mediated induction of autophagy increased NDV titres, whereas genetic or pharmacological inhibition of autophagy reduced viral titres128,129. Similar results were observed in avian reovirus (ARV)-infected cells, where ARV infection induced hallmarks of autophagy. Pharmacological induction of autophagy increased ARV viral titres, while inhibition of autophagic flux reduced titres130.
Finally, FMDV has been reported to rely on autophagic pathways for its infection. In 2012, a study showed that FMDV infection induces autophagy, which, in turn, is vital for its replication as viral titres were reduced in autophagy-deficient MEFs131. The FMDV-induced autophagic signals, unlike starvation-induced autophagy, does not require class III PI3K activity, nor does it need active viral replication, as infection with UV-inactivated virus induces LC3 lipidation. This finding suggests that FMDV-induced autophagy may result from viral entry, as previously reported for vesicular stomatitis virus and HCMV132,133. Several FMDV proteins were found to colocalize with autophagy-related proteins and inhibition of autophagy was shown to decrease FMDV titres134,135. The plant virus bamboo mosaic virus (BaMV) induces the expression of autophagy-related proteins, including the plant autophagosomal marker, ATG8f, and genetic or pharmacological disruption of autophagy reduces BaMV coat protein accumulation136.
Viral exploitation of the autophagic machinery
Several viruses use components of the autophagic machinery, particularly lipidated LC3, without the formation of recognizable autophagosomes. For example, the coronavirus mouse hepatitis virus uses the ER-associated degradation (ERAD) pathway to shape LC3-associated double-membraned vesicles as replication organelles, although the specific mechanism of ERAD engagement by the virus is unknown137,138,139. This has led to other examples of utilization of parts of the ER machinery without inducing the generation of ‘true’ autophagosomes. For example, a recent study suggested the LC3 lipidation observed during IAV infection is mostly driven by a phenomenon known as conjugation of ATG8 to single membranes (CASMs), which depends on the proton channel activity of M2 (refs. 140,141,142). The work demonstrated that IAV induces LC3 lipidation in the absence of typical autophagic signals, using the ATG16L1 WD40 mutant, which is not required for canonical autophagy. However, the significance of LC3 lipidation in IAV infection is unclear, as its absence does not affect influenza virus titres. Interestingly, expression of the poliovirus 2BC protein induces lipidated LC3 that decorates single-membraned vesicles; however, co-expression of the viral 3A protein induces autophagosome-like vesicles. If a similar mechanism holds true for IAV, it could indicate that a second signal may be required to convert CASM-derived structures to autophagosomes91,92,143.
Viral subversion of organelle-specific autophagy
Although macroautophagy makes up the ‘bulk’ of autophagy studies of viral infection, recent work has begun to highlight the role of various organelle-specific autophagy pathways in virus replication, which we highlight here. Although studies have clearly identified selective receptors and mechanisms for clearing specific organelles, membranes and lipids, selective and nonselective autophagy are a continuum. It can be difficult to clearly distinguish or describe them as autophagy-receptor-dependent and autophagy-receptor-independent processes. Here, we describe circumstances in which viruses interact with proteins or mechanisms specific to organelle-selective autophagy.
Mitophagy
As the powerhouse of cells and critical determinants of cell fate, mitochondria play essential roles in many cellular processes, including oxidative phosphorylation, innate immunity, cell survival and death. Thus, the quality control of the damaged mitochondria through mitophagy is essential for cell survival144. Chronic HBV infection induces oxidative stress, resulting in mitochondrial injury145. Damaged or dysfunctional mitochondria are significant sources of reactive oxygen species (ROS), which are detrimental to cell survival. Given that HBV is adapted to replicating in viable cells, removing damaged mitochondria is essential for its infection. HBV infection was demonstrated to induce mitochondrial fragmentation, mitochondrial perinuclear clustering and subsequent Parkin translocation to the mitochondria. Mitophagosomes can also be readily observed in HBV-infected cells. Expression of the pleiotropic HBx protein alone is sufficient to induce hallmarks of mitophagy. However, unlike the transient expression of HBx, HBV infection impairs mitophagolysosome formation, indicating that HBV induces incomplete mitophagy. Functionally, mitophagy is thought to promote HBV infection by blocking apoptotic cell death and maintaining cell viability146.
HCV, which is similarly adapted to replicating in liver cells, also induces hallmarks of mitophagy, including mitochondrial fission and perinuclear clustering, and translocation of the Parkin protein to mitochondria. Knockdown of mitophagy through Parkin silencing attenuated HCV replication147. Subsequent findings from the same group showed that mitophagy prevents apoptosis and impairs innate immune signalling during HCV infection148. In contrast to findings of the above study, using cells from transgenic mice expressing the HCV polyprotein and human hepatocyte chimeric mice, a study showed that HCV inhibits mitophagy. The authors demonstrated that the HCV structural protein, Core, interacts with Parkin, preventing its translocation to mitochondria and inhibiting mitophagy149. The authors argue that this inhibition of mitophagy during HCV infection could exacerbate liver injury, by inducing the production of ROS. Overexpression of HCV Core attenuated mitophagy induction in AB12–A2 HCV subgenomic replicon cells, in agreement with the inhibition of mitophagy150. In total, these results suggest that, as with autophagy, HCV also temporally regulates mitophagy. One possibility for temporal regulation is through the HCV Core protein, the expression of which is known to be delayed compared to other HCV proteins151. For instance, mitophagy may proceed normally during early HCV infection when HCV Core levels are low. As the infection progresses and HCV Core levels begin to rise, mitophagy is blocked, leading to ROS build-up and disruption of liver cell homeostasis, leading to end-stage liver disease development.
Besides the hepatotropic viruses, HPIV3 was also demonstrated to cause mitophagy. However, unlike the hepatitis viruses, the HPIV3-induced mitophagy is not dependent on the well-characterized Parkin or PINK1 pathways. Instead, during HPIV3 infection, the viral M protein acts as a receptor by interacting with the mitochondrion-localized protein, Tu translation elongation factor mitochondrial (TUFM) and LC3, leading to the formation and accumulation of mitophagosomes152. Interestingly, whereas HPIV3 infection induces incomplete mitophagy, overexpression of M protein alone paradoxically induces mitophagic flux and blocks the type I interferon response. These findings suggest that the HPIV3-induced mitophagy, similar to HBV-induced and HCV-induced mitophagy, attenuates the type I interferon signalling.
Lipophagy
HCV also induces lipophagy, the autophagic degradation of lipid droplets. Lipid droplets are the primary organelles regulating the storage and catabolism of neutral lipids and triacylglycerols and are therefore essential for energy production. Although lipids were thought to be primarily degraded through lipolysis, the discovery of lipophagy in 2009 shows an additional mechanism by which lipids are degraded39. Steatosis, the accumulation of fatty deposits in the liver, is a common feature of chronic HCV infection. Using patient biopsy samples, researchers discovered an inverse correlation between autophagy and microvesicular steatosis. Furthermore, using HCV subgenomic replicon systems, they showed that HCV selectively induces lipophagy and that blocking autophagy causes cholesterol accumulation153. These findings suggest that functional autophagy is essential for maintaining hepatocyte homeostasis and preventing end-stage liver disease formation.
DENV infection also induces lipophagy. However, in contrast to HCV, which triggers lipophagy to prevent fatty deposit accumulation, DENV induces the autophagic degradation of lipids to provide energy through β-oxidation to power viral RNA replication. In DENV-infected cells, GFP–LC3 colocalizes with a subset of lipid droplets, and the lipid droplet area is reduced in DENV-infected cells, indicating that DENV induces lipophagy. Pharmacological and genetic inhibition of autophagy increased lipid droplet area in DENV-infected cells and reduced DENV RNA replication154.
ER-phagy
DENV and ZIKV also subvert ER-phagy, also known as reticulophagy, a constitutive pathway pivotal to maintaining ER quality control and homeostasis155,156. Like other RNA viruses, members of the Flaviviridae family, including DENV and ZIKV, rearrange the ER and use the ER-derived membranes to (1) construct their replication organelles, (2) assemble their genome, and (3) facilitate their maturation via the secretory pathway. These disturbances to the ER trigger ER-phagy, which targets the ER for autophagic degradation157. Given the importance of the ER-derived membranes for flavivirus replication, ER-phagy would be expected to reduce flavivirus replication. DENV, ZIKV and West Nile virus counteract ER-phagy by cleaving the ER-phagy receptor, FAM134B, using their encoded NS3 proteases, thereby preserving their replication organelles156. Depletion of FAM134B enhances viral replication, indicating that in contrast to general autophagy, which is proviral for many flaviviruses, ER-phagy is antiviral.
Timing of autophagic induction
Many RNA viruses that induce autophagic signals tend to block the downstream autophagosome–lysosome fusion. For many of these viruses, induction of autophagic signals through starvation or rapamycin treatment enhances viral infection, whereas inhibition of autophagy conversely attenuates their infection. Given the multiple strategies used by these viruses to block autophagic degradation, we hypothesize that inducing autophagic flux during viral infection would be detrimental to these viruses. We established starvation before infection and starvation after infection protocols to induce autophagy before or immediately after viral adsorption using EV-D68 and other picornaviruses as models. In agreement with our hypothesis, it was recently showed that inducing autophagic flux through starvation instantly after viral adsorption attenuates the replication of many picornaviruses70.
In contrast to EV-D68 infection, which induces autophagosome formation at 3 h postinfection, autophagosomes can be detected as early as 1 h poststarvation. By 2 h poststarvation, the autophagosomes had disappeared owing to flux. These results suggest that the starvation after infection protocol uses resources through rapid autophagosome formation, depriving the virus of those critical resources required for formation of replication organelles, impairing viral RNA replication.
Conclusions: bend, but don’t break
Although this Review is structured around the stages of autophagy, very few viruses induce autophagy in the classic sense, meaning bulk protein degradation. Viruses generally induce what we call here not-ophagy or the formation of autophagic membrane structures without accompanying degradation. For viruses, autophagy or not-ophagy is the question. We have highlighted the fascinating interplay between viruses and autophagy, as well as the strategies that human, animal and plant viruses use to block autophagy (both at the initiation and maturation steps) or subvert autophagic membranes or signalling for their benefit. We have also highlighted common regulatory mechanisms; for example, given the significance of Beclin-1 for autophagy initiation, it is unsurprising that many viruses that block autophagy initiation inhibit Beclin-1 activity, making Beclin-1 an ideal candidate for modulating autophagy interaction with multiple viruses.
The members of the Flaviviridae family appear to be unique in studies thus far in using autophagic degradation for energy production and innate immune evasion. Perhaps their membranous web for RNA replication, or particular unique features of their capsid formation and acquisition of viral envelopes, make it necessary for flaviviruses to specifically induce autophagic degradation. It seems probable that for most viruses, degradative autophagy would be so detrimental to their infection that they have developed multiple strategies to impair autophagic flux. The published data certainly suggest that redirected autophagic components and vesicles, not autophagy as a catabolic mechanism, are essential for viral infection.
Although several mechanisms have been proposed for how viruses induce autophagy, we do not know whether viral infections predominantly induce the basal or stress-induced autophagic pathway. Our findings that stress-induced autophagy attenuates picornavirus infection, and that the upstream autophagy regulators are not essential for poliovirus replication, suggest that picornaviruses probably hijack basal autophagy. It is interesting to speculate that viruses known to allow autophagic flux, for example, a subset of flaviviruses, trigger stress-induced autophagy, whereas those that inhibit autophagic flux induce basal autophagy. Assays to clearly distinguish basal from stress-induced autophagy, which to date are not well-defined, could help to address this question.
Autophagy benefits many viruses, who use the ‘nuts and bolts’ of the pathway to shape membranes and facilitate viral replication and escape. These viruses push a potentially virucidal pathway forward without allowing the degradation to complete, which is a bend, but don’t break strategy. By understanding the specific mechanisms by which viruses usurp and carefully control the autophagic pathway, we may be able to use the enemy’s weapon to control the enemy.
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Acknowledgements
The authors thank the members of the Jackson, Frieman and Coughlan labs for weekly discussion sessions and M. Patricia (2017) for inspiring the title. This work was funded by National Institutes of Health (NIH)/National Institute of Allergy and Infectious Diseases (NIAID) grants R01AI141359 and R01AI104928 to W.T.J. The authors apologize to any authors whose work was missed or given short mention because of space limitations.
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Jassey, A., Jackson, W.T. Viruses and autophagy: bend, but don’t break. Nat Rev Microbiol 22, 309–321 (2024). https://doi.org/10.1038/s41579-023-00995-y
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DOI: https://doi.org/10.1038/s41579-023-00995-y
