Autophagy–virus interplay in plants: from antiviral recognition to proviral manipulation

Autophagy is a conserved self-cleaning and renewal system required for cellular homeostasis and stress tolerance. Autophagic processes are also implicated in the response to ‘non-self’ such as viral pathogens, yet the functions and mechanisms of autophagy during plant virus infection have only recently started to be revealed. Compelling evidence now indicates that autophagy is an integral part of antiviral immunity in plants. It can promote the hypersensitive cell death response upon incompatible viral infections or mediate the selective elimination of entire particles and individual proteins from compatible viruses in a pathway similar to xenophagy in animals. Several viruses, however, have evolved measures to antagonize xenophagic degradation or utilize autophagy to suppress disease-associated cell death and other defence pathways like RNA silencing. Here, we highlight the current advances and gaps in our understanding of the complex autophagy–virus interplay and its consequences for host immunity and viral pathogenesis in plants.     

iLIR@viral: A web resource for LIR motif-containing proteins in viruses

Macroautophagy/autophagy has been shown to mediate the selective lysosomal degradation of pathogenic bacteria and viruses (xenophagy), and to contribute to the activation of innate and adaptative immune responses. Autophagy can serve as an antiviral defense mechanism but also as a proviral process during infection. Atg8-family proteins play a central role in the autophagy process due to their ability to interact with components of the autophagy machinery as well as selective autophagy receptors and adaptor proteins. Such interactions are usually mediated through LC3-interacting region (LIR) motifs. So far, only one viral protein has been experimentally shown to have a functional LIR motif, leaving open a vast field for investigation. Here, we have developed the iLIR@viral database (http://ilir.uk/virus/) as a freely accessible web resource listing all the putative canonical LIR motifs identified in viral proteins. Additionally, we used a curated text-mining analysis of the literature to identify novel putative LIR motif-containing proteins (LIRCPs) in viruses. We anticipate that iLIR@viral will assist with elucidating the full complement of LIRCPs in viruses.     

The autophagy pathway participates in resistance to tomato yellow leaf curl virus infection in whiteflies

Macroautophagy/autophagy plays an important role against pathogen infection in mammals and plants. However, little has been known about the role of autophagy in the interactions of insect vectors with the plant viruses, which they transmit. Begomoviruses are a group of single-stranded DNA viruses and are exclusively transmitted by the whitefly Bemisia tabaci in a circulative manner. In this study, we found that the infection of a begomovirus, tomato yellow leaf curl virus (TYLCV) could activate the autophagy pathway in the Middle East Asia Minor 1 (MEAM1) species of the B. tabaci complex as evidenced by the formation of autophagosomes and ATG8-II. Interestingly, the activation of autophagy led to the subsequent degradation of TYLCV coat protein (CP) and genomic DNA. While feeding the whitefly with 2 autophagy inhibitors (3-methyladenine and bafilomycin A₁) and silencing the expression of Atg3 and Atg9 increased the viral load; autophagy activation via feeding of rapamycin notably decreased the amount of viral CP and DNA in the whitefly. Furthermore, we found that activation of whitefly autophagy could inhibit the efficiency of virus transmission; whereas inhibiting autophagy facilitated virus transmission. Taken together, these results indicate that TYLCV infection can activate the whitefly autophagy pathway, which leads to the subsequent degradation of virus. Furthermore, our report proves that an insect vector uses autophagy as an intrinsic antiviral program to repress the infection of a circulative-transmitted plant virus. Our data also demonstrate that TYLCV may replicate and trigger complex interactions with the insect vector.     

Host factors against plant viruses

Plant virus genome replication and movement is dependent on host resources and factors. However, plants respond to virus infection through several mechanisms, such as autophagy, ubiquitination, mRNA decay and gene silencing, that target viral components. Viral factors work in synchrony with pro-viral host factors during the infection cycle and are targeted by antiviral responses. Accordingly, establishment of virus infection is genetically determined by the availability of the pro-viral factors necessary for genome replication and movement, and by the balance between plant defence and viral suppression of defence responses. Sequential requirement of pro-viral factors and the antagonistic activity of antiviral factors suggest a two-step model to explain plant–virus interactions. At each step of the infection process, host factors with antiviral activity have been identified. Here we review our current understanding of host factors with antiviral activity against plant viruses.     

Viral evasion of autophagy

Autophagy is an evolutionarily ancient pathway for survival during different forms of cellular stress, including infection with viruses and other intracellular pathogens. Autophagy may protect against viral infection through degradation of viral components (xenophagy), by promoting the survival or death of infected cells, through delivery of Toll-like receptor (TLR) ligands to endosomes to activate innate immunity, or by feeding antigens to MHC class II compartments to activate adaptive immunity. Given this integral role of autophagy in innate and adaptive antiviral immunity, selective pressure likely promoted the emergence of escape mechanisms by pathogenic viruses. This review will briefly summarize the current understanding of autophagy as an antiviral pathway, and then discuss strategies that viruses may utilize to evade this host defense mechanism.     

Viral evasion of autophagy

Autophagy is an evolutionarily ancient pathway for survival during different forms of cellular stress, including infection with viruses and other intracellular pathogens. Autophagy may protect against viral infection through degradation of viral components (xenophagy), by promoting the survival or death of infected cells, through delivery of Toll-like receptor (TLR) ligands to endosomes to activate innate immunity, or by feeding antigens to MHC class II compartments to activate adaptive immunity. Given this integral role of autophagy in innate and adaptive antiviral immunity, selective pressure likely promoted the emergence of escape mechanisms by pathogenic viruses. This review will briefly summarize the current understanding of autophagy as an antiviral pathway, and then discuss strategies that viruses may utilize to evade this host defense mechanism.     

The role of the unfolded protein response in dengue virus pathogenesis

Symptomatic dengue virus (DENV) infections range from mild fever to severe haemorrhagic disease and death. Host‐viral interactions play a significant role in deciding the fate of the infection. The unfolded protein response (UPR) is a prosurvival cellular reaction induced in response to DENV‐mediated endoplasmic reticulum stress. The UPR has complex interactions with the cellular autophagy machinery, apoptosis, and innate immunity. DENV has evolved to manipulate the UPR to facilitate its replication and to evade host immunity. Our knowledge of this intertwined network of events is continuously developing. A better understanding of the UPR mediated antiviral and proviral effects will shed light on dengue disease pathogenesis and may help development of anti‐DENV therapeutics. This review summarizes the role of the UPR in viral replication, autophagy, and DENV‐induced inflammation to describe how a host response contributes to DENV pathogenesis.     

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.     

The pros and cons of autophagic flux among herpesviruses

Autophagy has been intensively studied in herpes simplex virus type 1 (HSV-1), a human alphaherpesvirus. The HSV-1 genome encodes a well-known neurovirulence protein called ICP34.5. When the gene encoding this protein is deleted from the genome, the virus is markedly less virulent when injected into the brains of animal models. Subsequent characterization of ICP34.5 established that the neurovirulence protein interacts with BECN1, thereby inhibiting autophagy and facilitating viral replication in the brain. However, an ortholog of the ICP34.5 gene is lacking in the genomes of other closely related alphaherpesviruses, such as varicella-zoster virus (VZV). Further, autophagosomes are easily identified in the exanthem (rash) that is the hallmark of both VZV diseases—varicella and herpes zoster. Inhibition of autophagy leads to diminished VZV titers. Finally, no block is detected in studies of autophagic flux following VZV infection. Thus autophagy appears to be proviral during VZV infection while antiviral during HSV-1 infection. Because divergence to this degree is extremely unusual for 2 closely related herpesviruses, we postulate that VZV has accommodated its infectious cycle to benefit from autophagic flux, whereas HSV-1 has captured cellular immunomodulatory genes to inhibit autophagy.     

Cauliflower mosaic virus protein P6-TAV plays a major role in alteration of aphid vector feeding behaviour but not performance on infected Arabidopsis

Emerging evidence suggests that viral infection modifies host plant traits that in turn alter behaviour and performance of vectors colonizing the plants in a way conducive for transmission of both nonpersistent and persistent viruses. Similar evidence for semipersistent viruses like cauliflower mosaic virus (CaMV) is scarce. Here we compared the effects of Arabidopsis infection with mild (CM) and severe (JI) CaMV isolates on the feeding behaviour (recorded by the electrical penetration graph technique) and fecundity of the aphid vector Myzus persicae. Compared to mock-inoculated plants, feeding behaviour was altered similarly on CM- and JI-infected plants, but only aphids on JI-infected plants had reduced fecundity. To evaluate the role of the multifunctional CaMV protein P6-TAV, aphid feeding behaviour and fecundity were tested on transgenic Arabidopsis plants expressing wild-type (wt) and mutant versions of P6-TAV. In contrast to viral infection, aphid fecundity was unchanged on all transgenic lines, suggesting that other viral factors compromise fecundity. Aphid feeding behaviour was modified on wt P6-CM-, but not on wt P6-JI-expressing plants. Analysis of plants expressing P6 mutants identified N-terminal P6 domains contributing to modification of feeding behaviour. Taken together, we show that CaMV infection can modify both aphid fecundity and feeding behaviour and that P6 is only involved in the latter.     

Accumulation of 24 nucleotide transgene‐derived siRNAs is associated with crinivirus immunity in transgenic plants

RNA silencing is a conserved antiviral defence mechanism that has been used to develop robust resistance against plant virus infections. Previous efforts have been made to develop RNA silencing‐mediated resistance to criniviruses, yet none have given immunity. In this study, transgenic Nicotiana benthamiana plants harbouring a hairpin construct of the Lettuce infectious yellows virus (LIYV) RNA‐dependent RNA polymerase (RdRp) sequence exhibited immunity to systemic LIYV infection. Deep sequencing analysis was performed to characterize virus‐derived small interfering RNAs (vsiRNAs) generated on systemic LIYV infection in non‐transgenic N. benthamiana plants as well as transgene‐derived siRNAs (t‐siRNAs) derived from the immune‐transgenic plants before and after LIYV inoculation. Interestingly, a similar sequence distribution pattern was obtained with t‐siRNAs and vsiRNAs mapped to the transgene region in both immune and susceptible plants, except for a significant increase in t‐siRNAs of 24 nucleotides in length, which was consistent with small RNA northern blot results that showed the abundance of t‐siRNAs of 21, 22 and 24 nucleotides in length. The accumulated 24‐nucleotide sequences have not yet been reported in transgenic plants partially resistant to criniviruses, and thus may indicate their correlation with crinivirus immunity. To further test this hypothesis, we developed transgenic melon (Cucumis melo) plants immune to systemic infection of another crinivirus, Cucurbit yellow stunting disorder virus (CYSDV). As predicted, the accumulation of 24‐nucleotide t‐siRNAs was detected in transgenic melon plants by northern blot. Together with our findings and previous studies on crinivirus resistance, we propose that the accumulation of 24‐nucleotide t‐siRNAs is associated with crinivirus immunity in transgenic plants.     

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.     

Viruses and the autophagy machinery

Autophagy is a major intracellular pathway for degradation and recycling of long-lived proteins and cytoplasmic organelles that plays an essential role in maintenance of homeostasis in response to starvation and other cellular stresses. Autophagy is also important for a variety of other processes including restriction of intracellular pathogen replication. Our understanding of the fascinating relationship between viruses and the autophagy machinery is still in its infancy but it is clear that autophagy is a newly recognized facet of innate and adaptive immunity against viral infection. Although the autophagy pathway is emerging as a component of host defense, certain viruses have developed strategies to counteract these antiviral mechanisms, and others appear to have co-opted the autophagy machinery as proviral host factors favoring viral replication. The complex interplay between autophagy and viral infection will be discussed in this review.     

Establishing RNA virus resistance in plants by harnessing CRISPR immune system

Recently, CRISPR‐Cas (clustered, regularly interspaced short palindromic repeats–CRISPR‐associated proteins) system has been used to produce plants resistant to DNA virus infections. However, there is no RNA virus control method in plants that uses CRISPR‐Cas system to target the viral genome directly. Here, we reprogrammed the CRISPR‐Cas9 system from Francisella novicida to confer molecular immunity against RNA viruses in Nicotiana benthamiana and Arabidopsis plants. Plants expressing FnCas9 and sgRNA specific for the cucumber mosaic virus (CMV) or tobacco mosaic virus (TMV) exhibited significantly attenuated virus infection symptoms and reduced viral RNA accumulation. Furthermore, in the transgenic virus‐targeting plants, the resistance was inheritable and the progenies showed significantly less virus accumulation. These data reveal that the CRISPR/Cas9 system can be used to produce plant that stable resistant to RNA viruses, thereby broadening the use of such technology for virus control in agricultural field.     

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.     

Identification of lncRNA‐155 encoded by MIR155HG as a novel regulator of innate immunity against influenza A virus infection

Long noncoding RNAs (lncRNAs) are single‐stranded RNA molecules longer than 200 nt that regulate many cellular processes. MicroRNA 155 host gene (MIR155HG) encodes the microRNA (miR)‐155 that regulates various signalling pathways of innate and adaptive immune responses against viral infections. MIR155HG also encodes a lncRNA that we call lncRNA‐155. Here, we observed that expression of lncRNA‐155 was markedly upregulated during influenza A virus (IAV) infection both in vitro (several cell lines) and in vivo (mouse model). Interestingly, robust expression of lncRNA‐155 was also induced by infections with several other viruses. Disruption of lncRNA‐155 expression in A549 cells diminished the antiviral innate immunity against IAV. Furthermore, knockout of lncRNA‐155 in mice significantly increased IAV replication and virulence in the animals. In contrast, overexpression of lncRNA‐155 in human cells suppressed IAV replication, suggesting that lncRNA‐155 is involved in host antiviral innate immunity induced by IAV infection. Moreover, we found that lncRNA‐155 had a profound effect on expression of protein tyrosine phosphatase 1B (PTP1B) during the infection with IAV. Inhibition of PTP1B by lncRNA‐155 resulted in higher production of interferon‐beta (IFN‐β) and several critical interferon‐stimulated genes (ISGs). Together, these observations reveal that MIR155HG derived lncRNA‐155 can be induced by IAV, which modulates host innate immunity during the virus infection via regulation of PTP1B‐mediated interferon response.     

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.     

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.