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.     

Autophagy is involved in the early step of Japanese encephalitis virus infection

Japanese encephalitis virus (JEV), an enveloped Flavivirus with a positive-sense RNA genome, causes acute encephalitis with high mortality in humans. We used a virulent (RP-9) and an attenuated (RP-2ms) JEV strain to assess the role of autophagy in JEV infection. By monitoring the levels of lipidated LC3, we found that autophagy was induced in human NT-2 cells infected with RP-2ms, especially at the late stage, and to a lesser extent with RP-9. The induction of autophagy by rapamycin increased viral production, whereas the inhibition of autophagy by 3-methyladenine reduced viral yields for both RP-9 and RP-2ms. The viral replication of RP-9 and RP-2ms was also reduced in cells with downregulated ATG5 or Beclin 1 expression, suggesting a proviral role of autophagy in JEV replication. To determine the step of JEV life cycle affected by autophagy, we used an mCherry-LC3 fusion protein as the autophagosome marker. Little of no colocalization of LC3 puncta with dsRNA was noted, whereas the input JEV particles were targeted to autophagosomes stained positive for early endosome marker. Overall, we show for the first time that the cellular autophagy process is involved in JEV infection and the inoculated viral particles traffic to autophagosomes for subsequent steps of viral infection.     

Cross-talking between autophagy and viral infection in mammalian cells

Autophagy is a cellular process in degradation of long-lived proteins and organelles in the cytosol for maintaining cellular homeostasis, which has been linked to a wide range of human health and disease states, including viral infection. The viral infected cells exhibit a complicated cross-talking between autophagy and virus. It has been shown that autophagy interacts with both adaptive and innate immunity. For adaptive immunity, viral antigens can be processed in autophagosomes by acidic proteases before major histocompatibility complex (MHC) class II presentation. For innate immunity, autophagy may assist in the delivery of viral nucleic acids to endosomal TLRs and also functions as a part of the TLR-or-PKR-downstream responses. Autophagy was also reported to suppress the magnitude of host innate antiviral immunity in certain cases. On the other hand, viruses has evolved many strategies to combat or utilize the host autophagy for their own benefit. In this review we discussed recent advances toward clarifying the cross-talking between autophagy and viral infection in mammalian cells.     

Axin expression delays herpes simplex virus‐induced autophagy and enhances viral replication in L929 cells

Axin, a negative regulator of the Wnt signaling pathway, plays a critical role in various cellular events including cell proliferation and cell death. Axin‐regulated cell death affects multiple processes, including viral replication. For example, axin expression suppresses herpes simplex virus (HSV)‐induced necrotic cell death and enhances viral replication. Based on these observations, this study investigated the involvement of autophagy in regulation of HSV replication and found axin expression inhibits autophagy‐mediated suppression of viral replication in L929 cells. HSV infection induced autophagy in a time‐ and viral dose‐dependent manner in control L929 cells (L‐EV), whereas virus‐induced autophagy was delayed in axin‐expressing L929 cells (L‐axin). Subsequent analysis showed that induction of autophagy by rapamycin reduced HSV replication, and that inhibiting autophagy by 3‐methyladenine (3MA) and beclin‐1 knockdown facilitated viral replication in L‐EV cells. In addition, preventing autophagy with 3MA suppressed virus‐induced cytotoxicity in L‐EV cells. In contrast, HSV replication in L‐axin cells was resistant to changes in autophagy. These results suggest that axin expression may render L929 cells resistant to HSV‐infection induced autophagy, leading to enhanced viral replication.     

Human cytomegalovirus controls a new autophagy-dependent cellular antiviral defense mechanism

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that remains the major infectious cause of birth defects, as well as being an important opportunistic pathogen. Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved process responsible for the degradation of cytoplasmic macromolecules, and the elimination of damaged organelles via a lysosomal pathway. This process is also triggered in organisms by stressful conditions and by certain diseases. Previous observations have suggested that autophagy (also known as xenophagy in this case) may contribute to innate immunity against viral infections. Recent studies on HSV-1, another herpesvirus, have shown that HSV-1 is able to avoid this cellular defense by means of a viral protein, ICP34.5, which antagonizes the host autophagy response. However, it was not known whether HCMV was also able to counteract autophagy. Here, we show that HCMV infection drastically inhibits autophagosome formation in primary human fibroblasts. Autophagy was assessed by GFP-LC3 redistribution, LC3-II and p62 accumulation and electron microscopy. Inhibition of autophagy occurred early in the infection by a mechanism involving viral protein(s). Indeed, only infected cells expressing viral proteins displayed a striking decrease of autophagy; whereas bystander, non-infected cells displayed a level of autophagy similar to that of control cells. HCMV activated the mTOR signaling pathway, and rendered infected cells resistant to rapamycin-induced autophagy. Moreover, infected cells also became resistant to the stimulation of autophagy by lithium chloride, an mTOR-independent inducer of autophagy. These findings suggest that HCMV has developed efficient strategies for blocking the induction of autophagy during infection.     

Downregulation of autophagy by herpesvirus Bcl-2 homologs

The critical role of the cellular autophagy pathway in viral infection and pathogenesis has become increasingly apparent. Mounting evidences suggest that viruses have developed different strategies to meticulously modulate intracellular autophagy for their own benefits, thereby either promoting efficient viral replication or facilitating viral persistence. While our understanding of these strategies is still in its incipient stage, recent advances demonstrate that gamma herpesvirus Bcl-2 homolog (vBcl-2), which protects virus-infected cells from apoptosis, also suppresses cellular autophagy pathway through its direct interaction with the autophagy protein Beclin1. Interestingly, vBcl-2 has evolved to harbor the enhanced anti-autophagic activity compared to its host counterpart, suggesting an important role of cellular autophagy in response to viral infection and virus-associated pathogenesis. Thus, a detailed study of vBcl-2-mediated regulation of autophagy signal transduction pathway may lead to a better understanding of not only how virus escapes from host innate immunity but also how autophagy regulates viral infection and environmental stresses.     

Critical Role of Autophagy in the Processing of Adenovirus Capsid-Incorporated Cancer-Specific Antigens

Adenoviruses are highly immunogenic and are being examined as potential vectors for immunotherapy. Infection by oncolytic adenovirus is followed by massive autophagy in cancer cells. Here, we hypothesize that autophagy regulates the processing of adenoviral proteins for antigen presentation. To test this hypothesis, we first examined the presentation of viral antigens by infected cells using an antibody cocktail of viral capsid proteins. We found that viral antigens were processed by JNK-mediated autophagy, and that autophagy was required for their presentation. Consistent with these results, splenocytes isolated from virus-immunized mice were activated by infected cells in an MHC II-dependent manner. We then hypothesize that this mechanism can be utilized to generate an efficient cancer vaccine. To this end, we constructed an oncolytic virus encompassing an EGFRvIII cancer-specific epitope in the adenoviral fiber. Infection of cancer cells with this fiber-modified adenovirus resulted in recognition of infected cancer cells by a specific anti-EGFRvIII antibody. However, inhibition of autophagy drastically decreased the capability of the specific antibody to detect the cancer-related epitope in infected cells. Our data suggest that combination of adenoviruses with autophagy inducers may enhance the processing and presentation of cancer-specific antigens incorporated into capsid proteins.     

Viroporin-mediated calcium-activated autophagy

Autophagy is a cellular response activated by many pathogens, but the mechanism of activation is largely unknown. Recently we showed for the first time that rotavirus initiates the autophagy pathway through a calcium-mediated mechanism. Expression of the rotavirus-encoded NSP4, a pore-forming protein (viroporin), elicits the release of endoplasmic reticulum (ER) lumenal calcium into the cytoplasm of the infected cell. The increased cytoplasmic calcium activates a calcium signaling pathway involving calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and 5′ adenosine monophosphate-activated protein kinase (AMPK) to trigger autophagy. Rotavirus further manipulates autophagy membrane trafficking to transport viral ER-associated proteins to viroplasms, sites of viral genome replication and immature particle assembly. Transport of viral proteins to viroplasms is required for assembly of infectious virus. Thus, NSP4, a multifunctional viral protein known to regulate infectious particle assembly, also modulates membrane trafficking by orchestrating the activation of autophagy to benefit viral replication.     

Autophagy and viral neurovirulence

As terminally differentiated vital cells, neurons may be specialized to fight viral infections without undergoing cellular self-destruction. The cellular lysosomal degradation pathway, autophagy, is emerging as one such mechanism of neuronal antiviral defence. Autophagy has diverse physiological functions, such as cellular adaptation to stress, routine organelle and protein turnover, and innate immunity against intracellular pathogens, including viruses. Most of the in vivo evidence for an antiviral role of autophagy is related to viruses that specifically target neurons, including the prototype alphavirus, Sindbis virus, and the α-herpesvirus, herpes simplex virus type 1 (HSV-1). In the case of HSV-1, viral evasion of autophagy is essential for lethal encephalitis. As basal autophagy is important in preventing neurodegeneration, and induced autophagy is important in promoting cellular survival during stress, viral antagonism of autophagy in neurons may lead to neuronal dysfunction and/or neuronal cell death. This review provides background information on the roles of autophagy in immunity and neuroprotection, and then discusses the relationships between autophagy and viral neurovirulence.     

Gammaherpesvirus 68 infection of endothelial cells requires both host autophagy genes and viral oncogenes for optimal survival and persistence

Gammaherpesvirus-associated neoplasms include tumors of lymphocytes, epithelial cells, and endothelial cells (ECs). We previously showed that, unlike most cell types, ECs survive productive gammaherpesvirus 68 (γHV68) infection and achieve anchorage-independent growth, providing a cellular reservoir for viral persistence. Here, we demonstrated autophagy in infected ECs by analysis of LC3 localization and protein modification and that infected ECs progress through the autophagosome pathway by LC3 dual fluorescence and p62 analysis. We demonstrate that pharmacologic autophagy induction results in increased survival of infected ECs and, conversely, that autophagy inhibition results in death of infected EC survivors. Furthermore, we identified two viral oncogenes, v-cyclin and v-Bcl2, that are critical to EC survival and that modify EC proliferation and survival during infection-induced autophagy. We found that these viral oncogenes can also facilitate survival of substrate detachment in the absence of viral infection. Autophagy affords cells the opportunity to recover from stressful conditions, and consistent with this, the altered phenotype of surviving infected ECs was reversible. Finally, we demonstrated that knockdown of critical autophagy genes completely abrogated EC survival. This study reveals a viral mechanism which usurps the autophagic machinery to promote viral persistence within nonadherent ECs, with the potential for recovery of infected ECs at a distant site upon disruption of virus replication.     

Autophagy and RNA virus interactomes reveal IRGM as a common target

Several intracellular pathogens have the ability to avoid or exploit the otherwise destructive process of autophagy. RNA viruses are constantly confronted with cellular autophagy, and several of them hijack autophagy during the infectious cycle to improve their own replication. Nevertheless, our knowledge of viral molecular strategies used to manipulate autophagy remains limited. Our study allowed the identification of molecular interactions between 44 autophagy-associated proteins and 83 viral proteins belonging to five different RNA virus families. This interactome revealed that the autophagy network machinery is highly targeted by RNA viruses. Interestingly, whereas some autophagy-associated proteins are targeted by only one RNA virus family, others are recurrent targets of several families. Among them, we found IRGM as the most targeted autophagy-associated protein. Downregulation of IRGM expression prevents autophagy induction by measles virus, HCV and HIV-1, and compromises viral replication. Our work combined interactomic and analytical approaches to identify potential pathogen virulence factors targeting autophagy.     

Autophagy in antiviral innate immunity

Several autonomous arms of innate immunity help cells to combat viral infections. One of these is autophagy, a central cytosolic lysosomal‐dependent catabolic process constitutively competent to destroy infectious viruses as well as essential viral components that links virus detection to antiviral innate immune signals. Ongoing autophagy can be upregulated upon virus detection by pathogen receptors, including membrane bound and cytosolic pattern recognition receptors, and may further facilitate pattern recognition receptor‐dependent signalling. Autophagy or autophagy proteins also contribute to the synthesis of antiviral innate type I interferon cytokines as well as to antiviral interferon γ signalling. Additionally, autophagy may play a crucial role during viral infections in containing an excessive cellular response by regulating the intensity of the inflammatory response. As a consequence, viruses have evolved strategies to counteract antiviral innate immunity through manipulation of autophagy. This review highlights recent findings on the cross‐talk between autophagy and innate immunity during viral infections.     

Sustained Autophagy Contributes to Measles Virus Infectivity

The interplay between autophagy and intracellular pathogens is intricate as autophagy is an essential cellular response to fight against infections, whereas numerous microbes have developed strategies to escape this process or even exploit it to their own benefit. The fine tuned timing and/or selective molecular pathways involved in the induction of autophagy upon infections could be the cornerstone allowing cells to either control intracellular pathogens, or be invaded by them. We report here that measles virus infection induces successive autophagy signallings in permissive cells, via distinct and uncoupled molecular pathways. Immediately upon infection, attenuated measles virus induces a first transient wave of autophagy, via a pathway involving its cellular receptor CD46 and the scaffold protein GOPC. Soon after infection, a new autophagy signalling is initiated which requires viral replication and the expression of the non-structural measles virus protein C. Strikingly, this second autophagy signalling can be sustained overtime within infected cells, independently of the expression of C, but via a third autophagy input resulting from cell-cell fusion and the formation of syncytia. Whereas this sustained autophagy signalling leads to the autophagy degradation of cellular contents, viral proteins escape from degradation. Furthermore, this autophagy flux is ultimately exploited by measles virus to limit the death of infected cells and to improve viral particle formation. Whereas CD150 dependent virulent strains of measles virus are unable to induce the early CD46/GOPC dependent autophagy wave, they induce and exploit the late and sustained autophagy. Overall, our work describes distinct molecular pathways for an induction of self-beneficial sustained autophagy by measles virus.     

Inhibition of retroviral Gag assembly by non-silencing miRNAs promotes autophagic viral degradation

We recently reported an unconventional mechanism by which miRNAs inhibit HIV-1 viral production. This occurs when miRNAs bind nonspecifically to the viral structural protein Gag, interfering with viral RNA-mediated Gag assembly at the plasma membrane. Consequently, misassembled viral complexes are redirected into the endocytic pathway where they are delivered to lysosomes for degradation. In this study, we demonstrate that autophagy is a critical mediator of the viral degradation pathway and that this pathway is not HIV-1 specific. Misassembled viral complexes were found to colocalize extensively with LC3 and p62 in late endosomes/lysosomes, demonstrating a convergence of autophagy with functional degradative compartments. Knocking down autophagosome formation machineries reduced this convergence, while treatment with autophagy-inducer rapamycin enhanced the convergence. Furthermore, similar autophagy-dependent nonspecific miRNA inhibition of murine leukemia virus (MLV) assembly was shown. Overall, these results reveal autophagy as a crucial regulator of the retroviral degradation pathway in host cells initiated by nonspecific miRNA-Gag interactions. These findings could have significant implications for understanding how cells may regulate retroviral complex assembly by miRNA expression and autophagy, and raise the possibility that similar regulations can occur in other biological contexts.     

Role of autophagy in innate viral recognition

Plasmacytoid dendritic cells (pDCs) detect viruses in the acidified endosomes via Toll-like receptors (TLRs) upon endocytosis of virions. Yet, pDC responses to certain single-stranded RNA viruses occur only following live viral infection. In our recent study, we presented evidence that the recognition of such viruses by TLR7 requires autophagy. We speculate that the requirement for autophagy in viral recognition reflects the necessity for transportation of cytosolic viral replication intermediates into the lysosome where TLR7 is activated. In addition, autophagy was found to be required for pDCs to produce type I interferon (IFN) in response to both ssRNA and dsDNA viruses. These results indicated that autophagy plays a key role in mediating virus detection and IFNalpha secretion in pDCs, and suggest that cytosolic replication intermediates of ssRNA viruses serve as pathogen signatures recognized by TLR7.     

Autophagy and RNA virus interactomes reveal IRGM as a common target

Several intracellular pathogens have the ability to avoid or exploit the otherwise destructive process of autophagy. RNA viruses are constantly confronted with cellular autophagy, and several of them hijack autophagy during the infectious cycle to improve their own replication. Nevertheless, our knowledge of viral molecular strategies used to manipulate autophagy remains limited. Our study allowed the identification of molecular interactions between 44 autophagy-associated proteins and 83 viral proteins belonging to five different RNA virus families. This interactome revealed that the autophagy network machinery is highly targeted by RNA viruses. Interestingly, whereas some autophagy-associated proteins are targeted by only one RNA virus family, others are recurrent targets of several families. Among them, we found IRGM as the most targeted autophagy-associated protein. Downregulation of IRGM expression prevents autophagy induction by measles virus, HCV and HIV-1, and compromises viral replication. Our work combined interactomic and analytical approaches to identify potential pathogen virulence factors targeting autophagy.     

RNase L Induces Autophagy via c-Jun N-terminal Kinase and Double-stranded RNA-dependent Protein Kinase Signaling Pathways

Autophagy is a tightly regulated mechanism that mediates sequestration, degradation, and recycling of cellular proteins, organelles, and pathogens. Several proteins associated with autophagy regulate host responses to viral infections. Ribonuclease L (RNase L) is activated during viral infections and cleaves cellular and viral single-stranded RNAs, including rRNAs in ribosomes. Here we demonstrate that direct activation of RNase L coordinates the activation of c-Jun N-terminal kinase (JNK) and double-stranded RNA-dependent protein kinase (PKR) to induce autophagy with hallmarks as accumulation of autophagic vacuoles, p62(SQSTM1) degradation and conversion of Microtubule-associated Protein Light Chain 3-I (LC3-I) to LC3-II. Accordingly, treatment of cells with pharmacological inhibitors of JNK or PKR and mouse embryonic fibroblasts (MEFs) lacking JNK1/2 or PKR showed reduced autophagy levels. Furthermore, RNase L-induced JNK activity promoted Bcl-2 phosphorylation, disrupted the Beclin1-Bcl-2 complex and stimulated autophagy. Viral infection with Encephalomyocarditis virus (EMCV) or Sendai virus led to higher levels of autophagy in wild-type (WT) MEFs compared with RNase L knock out (KO) MEFs. Inhibition of RNase L-induced autophagy using Bafilomycin A1 or 3-methyladenine suppressed viral growth in initial stages; in later stages autophagy promoted viral replication dampening the antiviral effect. Induction of autophagy by activated RNase L is independent of the paracrine effects of interferon (IFN). Our findings suggest a novel role of RNase L in inducing autophagy affecting the outcomes of viral pathogenesis.     

麻疹病毒属相关自噬的研究进展

自噬是一种复杂的细胞内生物学过程,受众多基因调控,存在复杂的调控网络,在不同组织器官、生理和病理状态所起的作用也不同。对20多个病毒科的50多种DNA或RNA病毒的研究发现自噬是把双刃剑,但在研究麻疹病毒属病毒自噬相关内容时发现自噬有利于病毒的复制与传播,并且H和F蛋白在麻疹病毒属诱导自噬方面发挥着重要作用。麻疹病毒能够通过RNA病毒诱导自噬的关键调控分子IRGM诱发自噬,并且CD46作为麻疹病毒属的受体分子,在诱导自噬发生方面发挥了至关重要的作用。此外,麻疹病毒属病毒诱发的自噬与其引起的免疫抑制之间可能存在密切关系。本文为麻疹病毒属的免疫学研究提供了参考。     

RNase L Triggers Autophagy in Response to Viral Infections

Autophagy is a programmed homeostatic response to diverse types of cellular stress that disposes of long-lived proteins, organelles, and invading microbes within double-membraned structures called autophagosomes. The 2′,5′-oligoadenylate/RNase L system is a virus-activated host RNase pathway that disposes of or processes viral and cellular single-stranded RNAs. Here we report that activation of RNase L during viral infections induces autophagy. Accordingly, infections with encephalomyocarditis virus or vesicular stomatitis virus led to higher levels of autophagy in wild-type mouse embryonic fibroblasts (MEF) than in RNase L-null MEF. Similarly, direct activation of RNase L with a 2′,5′-oligoadenylate resulted in p62(SQSTM1) degradation, LC3BI/LC3BII conversion, and appearance of autophagosomes. To determine the effect of RNase L-mediated autophagy on viral replication, we compared viral yields in wild-type and RNase L-null MEF in the absence or presence of either chemical inhibitors of autophagy (bafilomycin A1 or 3-methyladenine) or small interfering RNA (siRNA) against ATG5 or beclin-1. At a low multiplicity of infection, induction of autophagy by RNase L during the initial cycle of virus growth contributed to the suppression of virus replication. However, in subsequent rounds of infection, autophagy promoted viral replication, reducing the antiviral effect of RNase L. Our results indicate a novel function of RNase L as an inducer of autophagy that affects viral yields.