Viral FLIPping Autophagy for Longevity

Abstract

Tumor-causing γ-herpesviruses have evolved elaborate mechanisms to deal with almost every aspect of host cell defense. In this issue of Cell Host & Microbe, Leidal et al. (2012) report an oncogenic synergy between the latent KSHV proteins v-FLIP and v-cyclin during KSHV persistent infection that reshapes autophagy.

Initially discovered as a cellular response to adapt to nutrient deficiency, macroautophagy (hereafter referred to as auto-phagy) has since been recognized as a highly conserved signaling system that patrols diverse growth, differentiation, and homeostatic processes (Levine and Kroemer, 2008; Yang and Klionsky, 2010). During autophagy, intracellular cargoes are subjected to lysosome-directed transportation and degradation in double membrane-bound autophagosomes (Yang and Klionsky, 2010). In the absence of stimuli, autophagy remains latent to maintain a relatively stable intra-cellular milieu. When cells experience various stress conditions, autophagy is activated, triggering responses that alleviate stress, repair cellular damage (e.g., organelle, protein, and/or DNA damage), or eliminate affected cells via so-called autophagic cell death (Levine and Kroemer, 2008). Furthermore, recent evidence places autophagy within the heart of the cellular senescence program evoked by the oncoprotein RAS and shows that autophagy is one of its downstream effector pathways (Young et al., 2009). Hence, stressed tumor cells that hijack autophagy for survival may alternatively or additionally run the risk of terminal growth arrest. Clearly, such protective autophagic responses do not favor tumor expansion.

Autophagy also limits pathogen survival. Autophagic processing delivers signals for viral recognition, interferon production, and antigen presentation. In turn, viruses have devised elegant strategies to co-opt the cellular autophagy pathway to guarantee their invasion, survival, and propagation. This is particularly the case for the ubiquitous and persistent herpesvirus infections, where the interplay between herpesviruses and host cell autophagy is a constant battle for control (Shoji-Kawata and Levine, 2009).

γ-Herpesviruses, exemplified by Kapo-si's sarcoma-associated herpesvirus (KSHV), are sophisticated oncogenic viruses that establish lifelong latency in lymphoid or endothelial cells. These viruses can reactivate, sometimes frequently, to cause recurrent disease and virus spreading, without being cleared by the host immune system (Ganem, 2010). One critical virulence factor for KSHV latency and oncogenicity is the viral homolog of cellular FLIPs (referred to as v-FLIP). Like its cellular counterpart (FLIP), v-FLIP was originally described to inhibit Fas-mediated caspase activation. Yet the current view of v-FLIP focuses on its ability to mobilize the NF-κB pathway in KSHV latently infected cells, giving these cells a survival advantage and inducing endothelial cell spindling (Ganem, 2010). However, more recent investigations have shown that v-FLIP also plays a critical role in concomitantly blocking cellular autophagy, whereby v-FLIP targets the autophagy effector Atg3 and impairs elongation of the autophagosome membrane, thus inhibiting autophagy in a manner more potently than cellular FLIP (Lee et al., 2009). Since autophagy is key to clearing the intracellular environment, this property of v-FLIP may tilt the homeostatic balance in favor of viral chronic infection and/or pathogenesis. Yet despite these advances in our understanding of the molecular biology of v-FLIP, the precise role of v-FLIP-mediated antiautophagy during KSHV infection remains, at least for the moment, unknown. Additionally, v-FLIP is known to share the same set of transcripts with two other latent gene products, viral cy-clin (v-cyclin), which is a viral homolog of cellular D-type cyclins and drives cell proliferation, and the latency-associated nuclear antigen (LANA), a multifunctional protein that modulates viral and cellular gene expression and helps maintain viral episomes. Together, v-FLIP, v-cyclin, and LANA comprise the so-called “oncogenic cluster” of KSHV and are thought to be necessary for viral-mediated transformation through their coordinated expression. How this oncogenic network is wired in favor of viral latency remains largely unknown. An exciting report by McCormick and colleagues in this issue of Cell Host & Microbe sheds light on the matter, identifying an oncogenic cooperation between v-FLIP and v-cyclin in KSHV infection: v-cyclin steers cell proliferation whereas v-FLIP counteracts senescence induced by v-cyclin by inhibiting autophagy (Leidal et al., 2012). This observation highlights the synergy of two crucial latent factors in KSHV pathology while also providing a mechanistic explanation for autophagy evasion during latent infection of KSHV.

One major barrier to the expansion of abnormal cells with significant replicative potential is the induction of cellular senescence, or so-called “oncogene-induced senescence” (OIS) (Gorgoulis and Halazonetis, 2010). Recent work has discovered that, despite increased cell proliferation, KSHV v-cyclin-transfected cells are in fact treading on thin ice. They appear to be highly sensitive to senescence, a process that relies on intact DNA damage responses (DDRs) and subsequent activation of the tumor suppressor protein p53 (Koopal et al., 2007). Given these findings, Leidal et al. were intrigued by their observation that KSHV infection of cultured cells, whereby latency is the default program, results in potent cellular DDRs—as expected—but not in a v-cyclin-induced senescence response (Leidal et al., 2012). They propose that the virus must use a mechanism to inactivate this failsafe program in addition to the effects it exerts through v-cyclin. The authors investigated which latency gene, when expressed, overcomes the senescence barrier in primary human foreskin fibroblasts (HFFs) expressing v-cyclin. This led to the identification of v-FLIP as a specific and potent repressor of v-cyclin OIS (Figure 1). How does v-FLIP attenuate v-cyclin-triggered OIS? One possibility is that v-FLIP and v-cyclin may share the same molecular target, but have opposing effects on its activity. Alternatively, v-FLIP and v-cyclin may function in independent and opposing signaling pathways to complement the actions of each other. Leidal et al. now provide compelling evidence that autophagy constitutes such a shared target and is a critical mediator of v-cyclin-induced senescence, which is successfully countered by v-FLIP for the virus to be pathogenic.

Figure 1. Oncogenic Synergy of v-FLIP and v-Cyclin on Cellular Senescence Control in KSHV Latent Infection.

This figure presents one mechanism of how v-FLIP assists v-cyclin to bypass the cellular senescence response, allowing v-cyclin-mediated cell hyperproliferation during latent infection of KSHV. v-cyclin, a latent gene product of KSHV and a homolog of cellular D-type cyclin, drives aberrant cell-cycle progression in partnership with the cellular cyclin-dependent kinase CDK6, leading to the activation of DNA damage response (DDR) and p53 checkpoint control. As a result, autophagy is upregulated through p53-dependent negative regulation of the mTOR signaling axis. Activated autophagy then enables cellular senescence through currently unknown mechanisms (indicated by a question mark), preventing aberrant cellular proliferation. To circumvent this antiproliferative barrier, the virus expresses v-FLIP as a “second hit” to attenuate autophagy-associated cellular senescence, acting directly on the autophagy machinery by inhibiting Atg3. v-FLIP can also activate the NF-κB pathway, extending the life span of infected cells. As a result, virally infected cells continue to grow and divide in an unregulated manner as genetic errors accumulate, establishing favorable conditions for neoplastic transformation.

In this work, the authors discovered that, following induction of v-cyclin, the oncogenic stress sensed by the cells activated p53 and triggered a strong autophagic response. Subsequently, the v-cyclin-insulted cells became senescent, accompanied by the secretion of various cytokines that are thought to help establish OIS. Removing p53 or knocking down autophagy resulted in a bypass of senescence and resumed growth of v-cyclin-expressing cells, which is reminiscent of the effects of oncogenic ras. Although this might not be an unexpected finding, it nevertheless suggests that activating autophagy may be a common event for cells to elicit a “self-disabling” process induced by RAS or oncoviruses, again highlighting the importance of autophagy in counteracting malignancies. It is important to note that the authors have demonstrated that v-cyclin-induced autophagy occurs in a p53-dependent manner. The increased expression of the p53 target genes, particularly Sestrin1, switched off the mTOR signaling axis, a master inhibitor of autophagy, and consequently elicited the autophagy cascade. These results are in agreement with a negative feed-back loop that reportedly occurs during RAS-mediated senescence (Gorgoulis and Halazonetis, 2010).

Considering that v-cyclin induces a state of increased proliferation that is kept in check by a concomitant increase in autophagy and senescence in normal cells, the finding (Leidal et al., 2012) that the virus utilizes v-FLIP as a “universal inhibitor” of autophagy to release the brakes on proliferation does not really come as a surprise. McCormick and colleagues demonstrate that expression of v-FLIP successfully antagonized v-cyclin-induced autophagy and thereof cellular senescence, albeit with no discernible effect on DDRs. Using a v-FLIP mutant that no longer activates NF-κB but can still attenuate autophagy, the authors show that v-FLIP expression continues to subvert senescence induced by v-cyclin. However, eliminating the anti-Atg3 activity of v-FLIP or feeding cells anti-v-FLIP peptides reactivated antiproliferative senescence. Their results, in conjunction with the previous finding (Lee et al., 2009) that revealed a distinct role of v-FLIP in mediating the antiautophagic interaction and NF-κB activation at a molecular level, also indicate that v-FLIP-elicited antiautophagy and NF-κB activation confer distinct actions in KSHV latency, with autophagy evasion clearly implicated in restricting senescence to allow viral-driven proliferation. The outcome of such a joint action of v-FLIP and v-cyclin in reshaping autophagy may synergistically increase the chance of cancer development and drug resistance associated with KSHV infection.

After all is said and done, it is worth noting that the oncogenic interplay of v-cyclin and v-FLIP reported by Leidal et al. was largely determined in cultured cells. Whether it is reflective of what occurs in vivo and is a real behavior of the virus in human biology awaits further investigation. Furthermore, v-FLIP likely antagonizes host autophagy not only to evade OIS but also to favor viral persistence. From a viral perspective, during long-term persistent infection wherein the viral genome is replicated in tight conjunction with host chromosomal DNA, reshaping cellular autophagy may have an active role in antagonizing host antiviral immune responses, such as antigen presentation, to allow persistence. From a host perspective, since autophagy has been implicated in patrol-ling genomic stability, blunting autophagy may also render virally infected cells error prone, an environment more favorable for viral fitness and survival. Despite our growing understanding of the molecular nature of autophagy, how autophagy enables cells' self-disabling process remains a question that is currently unanswered and is certainly a future challenge. Nonetheless, the Leidal et al. (2012) work suggests that evasion of autophagy may be a shared value for oncogenic viruses and that technologies that interfere with viral undermining of host autophagy could have considerable promise in treating virally associated malignancies.