Autophagy is an evolutionarily conserved cellular process that spans the entire spectrum of eukaryotes. It serves a housekeeping role in protein turnover and in the removal of damaged or unnecessary organelles. It also serves an important function in innate host defense by eliminating intracellular pathogens. For example, it has been shown that pharmacological induction of autophagy helps in the clearance of Mycobacterium tuberculosis
) and that CD40 induction of autophagy promotes elimination of Toxoplasma gondii
from macrophages (2
). Furthermore, recent studies suggest a role for autophagy in host adaptive immune responses through the delivery of intracellular pathogenic antigens for major histocompatibility complex class II presentation to CD4 T cells (32
Successful invading pathogens have evolved various strategies to evade host autophagy. For instance, some bacterial or viral proteins, such as IcsB secreted by Shigella flexneri
) and ICP34.5 encoded by herpes simplex virus type 1 (36
), have been documented to block or evade cellular autophagy. Other pathogens adopt different approaches in battling host autophagic defense by subverting it to facilitate their own procreation. In fact, several studies have shown that the autophagosome provides an intracellular compartment that benefits the survival and multiplication of bacteria, including Porphyromonas gingivalis
, Brucella abortus
, and Coxiella burnetii
). In addition, some viruses have been reported to induce autophagosome formation to facilitate their own replication (15
Replication of positive-stranded RNA viruses requires intracellular membrane surfaces on which they assemble their replication complexes. Colocalization of viral RNA replication machineries with double-membrane vesicles in infected cells has been well documented for various positive-stranded RNA viruses, including poliovirus, rhinovirus, equine arterivirus, murine hepatitis virus, and severe acute respiratory syndrome virus (7
). Thus, it was speculated that the autophagosome, a double-membrane vesicle, may serve as a site for viral RNA replication. Recent studies on coronavirus and poliovirus seem to support this notion. It was reported that murine hepatitis virus, which belongs to the coronavirus family, colocalizes with the ubiquitin-like autophagy proteins (LC3 and Atg12p) and that viral replication is impaired in autophagy knockout (Atg5−/−
) embryonic stem cell lines (31
). Additionally, poliovirus proteins have been shown to colocalize with a cotransfected GFP-LC3 protein (11
). Stimulation of autophagy increases the poliovirus yield, and inhibition of the autophagosomal pathway decreases the viral yield (11
). Despite this exciting observation, recent data on the role of autophagy in positive-stranded RNA virus infection remain contradictory. A recent report showed that infection of rhinovirus type 2, a member of the picornavirus family, does not induce autophagosome formation (4
). That paper further reported that neither induction nor inhibition of autophagy affects viral protein production. In addition, using ATG5-deficient cells, it was recently demonstrated that ATG5 and an intact autophagic pathway are not required for the replication of murine hepatitis virus (40
). These studies challenge the view that the autophagosome is a common cellular component utilized by positive-stranded RNA viruses to facilitate their replication.
These discrepancies of the reported data prompted us to explore the role of autophagy in coxsackievirus infection in this study. We report here that CVB3 infection induces the formation of double-membrane vesicles with concurrent LC3 lipidation. We further showed that manipulation of the host autophagy pathway by pharmacological compounds or target-specific RNA interference resulted in significant alterations in the outcome of CVB3 replication. We demonstrated that inhibition of signaling pathways or autophagy genes critical for autophagosome formation reduces viral protein expression and the viral progeny titer. Conversely, increasing the cellular number of autophagosomes prior to viral infection by cellular starvation, rapamycin treatment, or lysosomal inhibition promotes viral replication. Taken together, our data suggest that the host double-membrane autophagosome is likely utilized by CVB3 to facilitate its own replication.
As alluded to earlier, the process of autophagosome formation is tightly controlled. The serine/threonine protein kinase mTOR is one of the key regulators and negatively controls autophagosome formation (5
). Class I PI3K is the traditional upstream activator of mTOR. In addition, it has been shown that mTOR can be regulated by intracellular amino acids and ATP, independent of class I PI3K activation. ATP depletion or specific inhibition of mTOR by rapamycin has been shown to activate autophagy, whereas the addition of amino acids inhibits this process. In this study, we showed that levels of mTOR phosphorylation were unchanged after CVB3 infection, suggesting that CVB3-induced autophagy is not triggered by mTOR dephosphorylation.
In contrast, eIF2α kinase positively regulates autophagy in response to nutrient deprivation and viral infection. eIF2α kinase plays a key role in translation inhibition under various stress conditions, through phosphorylation of eIF2α. Restriction of herpes simplex virus growth has been linked to such a function of eIF2α phosphorylation (26
). Recently, the eIF2α kinase signaling pathway was also shown to promote autophagy induced by nutrient deprivation or rapamycin treatment, likely through a mechanism that induces LC3 conversion (21
). Our present study demonstrates that eIF2α is phosphorylated after CVB3 infection, suggesting a potential signaling mechanism for CVB3-induced autophagosome formation. It is unclear how eIF2α phosphorylation occurs during CVB3 infection. One explanation could be that double-stranded RNA formed from a CVB3 intermediate activates double-stranded RNA-dependent protein kinase and subsequently leads to eIF2α phosphorylation.
Class III PI3K complexes also play a crucial role in the early steps of autophagosome formation. In mammalian cells, the autophagic protein Beclin-1 (a homologue of yeast ATG6) forms the class III PI3K complex with VPS34, a class III PI3K, at the trans
-Golgi network (13
). This complex promotes autophagosome formation by supplying phosphatidylinositol 3-phosphates to the preautophagosome membrane (35
). It has been shown that 3-MA blocks the formation of autophagosomes by inhibiting the activity of class III PI3K (3
). We demonstrates that 3-MA blocks CVB3 infection, which is in agreement with our previous report that inhibition of PI3K by LY294002 suppresses CVB3 replication (6
). LY294002 is an inhibitor of both class I and class III PI3K and has also been shown to be a potent autophagy inhibitor (3
Although the specific mechanism by which the autophagosome is utilized during coxsackievirus replication in host cells has not been elucidated, it is likely that, as demonstrated for poliovirus (11
), the autophagosome provides a physical scaffold where the coxsackieviral complex may reside and viral RNA synthesis may occur. Furthermore, LC3 has been identified to be a binding protein of an AU-rich RNA element in the 3′-untranslated region of fibronectin mRNA, which facilitates the recruitment of RNA to polyribosomes for translation (41
). Perhaps AU-rich element-containing viruses, such as CVB3, which carries an AU-rich element in the 3′-untranslated region (30
), also adopt LC3-docking double-membrane vesicles as the sites of viral protein translation through the recruitment of polyribosomes. It is plausible to speculate that autophagosome double-membrane vesicles not only act as sites of viral RNA synthesis but also take an active role in CVB3 protein synthesis by bridging the nascent viral RNA with polyribosomes through LC3.