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Autophagy constitutes a major catabolic process for the quality control of internal proteins and organelles of eukaryotic cells, and is emerging as an essential part of the host antiviral defense. Many studies have shed light on the importance of autophagy in homeostasis, but it is not well understood how viruses co-opt the cellular autophagic pathway to establish virulence in vivo. Our recent study presents direct in vivo evidence for the key role of the anti-autophagic aspect of the virally encoded Bcl-2 proteins in the chronic infection of oncogenic γ-herpesviruses and proposes that cellular autophagy may have a substantial effect on viral persistence and may influence the in vivo fitness of viruses. This discovery expands upon known antiviral activities of the autophagy machinery and also suggests new approaches for treating some virally induced diseases.
The autophagy pathway is a highly conserved signaling system that controls diverse growth, differentiation, and homeostatic processes. In contrast to the ‘self-destruct’ apoptotic program, cellular autophagy (‘self-eating’) involves the lysosome-dependent bulk degradation of cytosolic proteins and organelles. As part of its integral role in maintaining cellular homeostasis, autophagy also defends cells from assaults by various pathogens. The cytoplasmic sequestration of autophagy renders it a unique capacity to target intact intracellular pathogens like bacteria and animal viruses for lysosomal degradation. In addition, autophagy processing delivers signaling for viral recognition, type I interferon (IFN) secretion, as well as endogenous MHC Class II antigen presentation. As such, neuronal overexpression of the autophagy protein, Beclin 1, confers resistance to Sindbis virus infections. Similarly, disruption of autophagy in plants leads to increased replication of tobacco mosaic virus in infected plants. Thus, autophagy, in addition to apoptosis, constitutes an important host antiviral response that must be successfully countered for certain viruses to be pathogenic.
γ-herpesviruses (γ-HVs) have developed a unique mode of interaction with their hosts wherein they establish a lifelong persistent infection, which is frequently associated with the onset of various malignancies. One critical virulence factor involved in γ-HVs persistence and oncogenicity are the viral homologs of the Bcl-2 protein (referred to as vBcl-2) encoded by all γ-HVs, including Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), herpesvirus saimiri (HVS), and murine γ-herpesvirus 68 (γ-HV68). The traditional view of vBcl-2 focused on its ability to counteract the host's apoptotic response, allowing for the completion of viral replication and the spread of progeny virus during acute infection. However, the studies of vBcl-2 in the case of γ-HV68 do not support this view, as vBcl-2 has no apparent role during acute infection in vitro or in vivo. Instead, the loss of vBcl-2 severely impairs the virus's ability to establish chronic infection in the host. Recently, a novel role of the cellular form of Bcl-2 (cBcl-2) in preventing autophagy was uncovered. We and others found that the vBcl-2s of γ-HVs, including KSHV and γ-HV68, mimic cBcl-2 to concomitantly attenuate autophagy through a direct interaction with Beclin 1. Indeed, vBcl-2s appear to confer a more robust inhibition of autophagy than their cellular counterpart. Additionally, our studies have shown that γ-HV68 vBcl-2 exhibits an even higher binding affinity to Beclin 1 than pro-apoptotic Bcl-2 proteins. These findings raise the strong possibility that Beclin 1 is a major target of vBcl-2 and that viruses, at least in some contexts, may eminently exploit the anti-autophagic aspect of vBcl-2 to facilitate chronic infection and/or pathogenesis of γ-HVs. Yet, despite these advances in our understanding of the molecular biology of vBcl-2, the exclusive role of vBcl-2-mediated inhibition of host autophagy during γ-HVs infection remains, for the moment at least, unknown.
Crystallographic analyses have shown that the two functions of vBcl-2, anti-apoptosis and anti-autophagy, engage the same structural cassette, the hydrophobic BH3-binding groove on the surface of vBcl-2. Yet, our comprehensive loss-of-function mutagenesis of vBcl-2 clearly indicates that these two activities of vBcl-2 are structurally and functionally separable: the Δα1 mutant that is impaired in autophagy inhibition retains intact its ability to inhibit apoptosis, whereas the ΔBH2 mutant behaves in the opposite manner. On the basis of these data, we further examined whether the two activities encoded by vBcl-2 have distinct roles during γ-HVs infection. γHV68 is a useful model for studying the in vivo biphasic lifecycle (lytic and latent) and pathogenesis of γ-HVs infection, and is amenable to genetic manipulation after being cloned as a bacterial artificial chromosome (BAC). vBcl-2 mutations that abrogate the anti-apoptotic and/or anti-autophagic activity, respectively, were introduced into the γ-HV68 genome. The replication profiles of these mutant viruses were then examined in different phases of viral infection, including acute infection, latency establishment, latency maintenance, and viral reactivation. Although γ-HV68 vBcl-2 efficiently blocks apoptosis in cell culture and in transgenic models, the vBcl-2 mutant viruses grew as well as wild type during the acute phase of infection in the lung. Furthermore, no evident differences were detected between wild-type and mutant viruses in their ability to seed a latent infection in the spleen. However, we observed that the mutant virus that lacks the anti-autophagic property of vBcl-2 was highly attenuated to maintain a latent reservoir in splenocytes, a prerequisite for subsequent reactivation and transmission. In line with this, targeted elimination of vBcl-2's anti-apoptotic activity specifically dampened viral reactivation frequency ex vivo. Our results thus indicate that vBcl-2-elicited anti-autophagy and anti-apoptosis confer distinct actions in different stages of γ-HV68 infection, with autophagy clearly implicated in restricting long-term viral latency.
So, how does autophagy thwart viral persistency? At present, this question remains unanswered. On the basis of the scavenging activities associated with host autophagy, however, one can predict that autophagy probably strengthens aspects of the host adaptive immune response, such as antigen presentation, or innate immunity, such as viral recognition and the stimulation of the interferon system. Cellular autophagy may also restrict cell proliferation of latently infected cells to prevent the replenishment of a latent viral pool. From a viral perspective, during a long-term persistent infection wherein the viral genome is replicated in tight conjunction with host chromosomal DNA, reshaping cellular autophagy is less likely used by viruses to replicate themselves, but is more likely to have a role in antagonizing host antiviral immune responses to allow persistence to endure. Although it is unclear whether the antiviral mode of autophagy we discovered with γHV68 is predictive of what may be seen with other persistent viruses, the fact that several human oncogenic herpesviruses encode autophagy modulators important for in vivo viral pathogenesis suggests that the evasion of autophagy may be a shared strategy, and that technologies that interfere with viral undermining of autophagy could have considerable promise in treating virally associated diseases.