SIRT-1 connects autophagy and release of virus-containing vesicles during picornavirus infection

Enterovirus D68 is a re-emerging enterovirus which causes acute respiratory illness in infants. EV-D68 infection has recently been associated with Acute Flaccid Myelitis, a severe polio-like neurological disease that causes limb weakness and loss of muscle tone in infants. There is currently no FDA-approved drug or prophylactic vaccine against EV-D68. Here, we investigated the role of the histone deacetylase, SIRT-1, in autophagy and EV-D68 infection. We show that SIRT-1 plays an important role in both autophagy and EV-D68 infection. siRNA-mediated knockdown of the cellular protein blocks basal and stress-induced autophagy and reduces EV-D68 extracellular viral titers. The proviral activity of SIRT-1 does not require deacetylase activity, since transient expression of both wild-type and deacetylase-inactive SIRT-1 mutant plasmids increased EV-D68 release. In non-lytic conditions, EV-D68 is primarily released in extracellular vesicles, and SIRT-1 is required for this process. Knockdown of SIRT-1 further impedes EV-D68 release in the autophagy-deficient ATG-7 knockout cells. Knockdown of SIRT-1 also decreases titers of poliovirus (PV) and SARS-CoV-2, but not Coxsackievirus-B3 (CVB3). CVB3 is the only tested virus that fails to induce SIRT-1 translocation to the cytosol. Our data suggest a correlation between SIRT-1 translocation during viral infection and extracellular vesicle-mediated non-lytic release of infectious viral particles. SIGNIFICANCE Picornaviruses, including EV-D68, constitute a significant cause of human disease. EV-D68 infection generally causes mild respiratory tract infection in infants but has recently been implicated in a severe polio-like neurological disease, AFM. Given the lack of prophylactic vaccines or antivirals against EV-D68, identifying host factors that modulate EV-D68 infection is crucial. Here, we show that SIRT-1 regulates autophagy and EV-D68 infection. Knockdown of SIRT-1 blocked autophagy and impeded the non-lytic release of EV-D68 in extracellular vesicles. We also show that SIRT-1 modulates the release of SARS-CoV-2 and poliovirus but not Coxsackievirus-B3 virus. Our data suggest that many RNA viruses require SIRT-1 for egress and that targeting SIRT-1 could constitute a broad-spectrum antiviral strategy.


INTRODUCTION
Enterovirus D-68 (EV-D68) is a re-emerging enterovirus and a cause of acute respiratory illness in infants. EV-D68 infection has recently been associated with Acute Flaccid Myelitis, a severe polio-like neurological disease that causes limb weakness and loss of muscle tone in infants (1) (2). There is currently no FDA-approved drug or prophylactic vaccine against EV-D68. Hence, it is of utmost importance to study how EV-D68 hijacks host processes to facilitate its life cycle.
EV-D68 is a positive-sense single-stranded RNA virus belonging to the Piconarviridae family of enteroviruses. The viral genome encodes a single polyprotein that is immediately processed upon translation by viral proteases into multiple intermediate and mature structural and seven non-structural proteins (3). Picornavirus infection induces extensive rearrangement of cytosolic membranes, beginning with the formation of complex single convoluted membranes, also known as replication vesicles, on the cytosolic face of which viral RNA replication occurs. These intricate structures eventually morph into double-membrane autophagosome-like structures as the infection progresses (4). Picornavirus replication, maturation, and nonlytic release are known to require autophagy (5)(6)(7).
Autophagy is a highly regulated catabolic process that maintains cell survival by targeting superfluous cytoplasmic contents and infectious microorganisms, including pathogenic bacteria and viruses, for degradation. Despite its antimicrobial mechanism, several RNA viruses, including EV-D68, are known to subvert autophagy for their own benefit (8). Autophagy begins with the formation of a phagophore/isolation membrane, which expands and closes to form the autophagosome. The autophagosomes then fuse with endosomes, forming amphisomes, before finally fusing with lysosomes for degradation (9). The conversion of soluble LC3-I to the membrane-associated LC3II, and the degradation of the autophagy receptor, p62, are used as markers for autophagy initiation and completion (autophagic flux) (10). While the mechanistic target of rapamycin complex 1 (MTORC1) and AMP kinase (AMPK) are well-known upstream autophagy regulators by inhibiting or inducing the cellular process, a growing body of evidence recently implicates sirtuin 1 (SIRT-1) as an essential regulator of autophagy downstream of the nucleation complex (11)(12)(13).
SIRT-1 belongs to the NAD + -dependent family of histone deacetylase enzymes, which are known to control several physiological processes. The sirtuin family of enzymes contains seven members (SIRT-1 to 7), which display varying subcellular localization, with SIRT-1 being the most studied owing to its role in lifespan expansion (14) (15).
SIRT-1 has been shown to regulate cellular responses to various stresses, including the cell cycle, apoptosis, inflammation, and more (14). In 2008, SIRT-1, which is typically localized to the nucleus in cancer cells, was demonstrated to form a molecular complex with ATG-5, ATG-7, and LC3 and deacetylates these autophagy-related proteins, thereby regulating autophagy induction (13,16). In agreement with the above study, SIRT-1 was recently shown to be indispensable for starvation-induced autophagy. The authors demonstrated that during acute amino acid starvation, SIRT-1 deacetylates nuclear LC3, allowing it to interact with TP53INP2. The LC3-TP53INP2 complex then translocates to the cytosol, where LC3 interacts with ATG-7 to initiate autophagosome formation (12). These studies underscore the significance of SIRT-1 for autophagy initiation, but whether SIRT-1 is essential for the later stages of autophagy (autophagic flux) or translocates to the cytosol during starvation or other stress stimuli, such as viral infection, is unknown. SIRT-1 has also been shown to promote the Middle East respiratory syndrome coronavirus infection, but whether SIRT-1 regulates picornavirus infection is unknown (17).
Here, we report that EV-D68 infection induces SIRT-1 translocation to the cytosol. We also show that SIRT knockdown impedes the extracellular vesicle-mediated release of infectious EV-D68 particles. We further demonstrate that the pro-EV-D68 activity of SIRT-1 does not require SIRT-1 deacetylase function and is not dependent on functional autophagy. Moreover, SIRT-1 knockdown reduces extracellular titers of poliovirus (PV) and SARS-CoV-2 but not Coxsackievirus B3 (CVB3). Our results indicate that certain picornaviruses induce SIRT-1 translocation to the cytosol and require the cellular protein for efficient release.

SIRT-1 is required for autophagy and EV-D68 infection
SIRT-1 has previously been reported to regulate autophagy initiation, but whether the cellular protein is necessary for autophagic flux is unclear (12,13). We first examined the effect of SIRT-1 knockdown on basal and starvation-induced autophagy. SIRT-1 and scramble knockdown H1HeLa cells were starved or treated with Carbonyl cyanide m-chlorophenylhydrazone (CCCP) to induce or block autophagy, respectively. As shown in Fig. 1A, starvation reduces p62 expression, and CCCP treatment increased LC3 lipidation in the scramble control group as expected. In contrast, acute amino acid starvation did not significantly alter p62 expression, and CCCP treatment failed to trigger LC3II accumulation in SIRT-1 knockdown cells, suggesting that SIRT-1 is essential for basal and starvation-induced autophagy. To further confirm SIRT-1's importance for basal autophagy, we knocked down SIRT-1 and performed immunofluorescence analysis (IFA) against endogenous LC3.
SIRT-1 has been shown to be proviral in some cases, but its role in EV-D68 infection is unknown ((17). Given the significance of autophagy for the EV-D68 life cycle, we interrogated the effects of SIRT-1 knockdown on EV-D68 titers. SIRT-1 knockdown reduced both the intracellular (Fig. 1D) and extracellular (Fig. 1E) EV-D68 titers, with the knockdown being more effective in decreasing the extracellular titers than the cell-associated viral titers. Next, we assessed the effect of SIRT-1 overexpression on EV-D68 titers. Both wild-type (SIRT-1 WT) and the deacetylase inactive mutant (SIRT-1 H363Y) increased EV-D68 extracellular titers (Fig. 1F).

SIRT-1 knockdown reduces the extracellular vesicle-mediated release of infectious EV-D68 viral particles
Since SIRT-1 knockdown reduces the extracellular EV-D68 titer an order of magnitude more than the intracellular titers, we posit that SIRT-1 proviral activity promotes non-lytic viral release. Hepatitis A Virus, a member of the Piconarviridae family, has been previously reported to be released in exosomes. Hence, we asked whether EV-D68 is similarly released in extracellular vesicles (EVs) and whether SIRT-1 is essential for this process. We knocked down SIRT-1 and isolated EVs for viral titer measurement. As shown in Fig. 2A, EV-D68 appears to be released chiefly in EVs compared to the post-spin supernatant (PSS), and SIRT-1 knockdown severely impeded the extracellular vesicle-mediated release of EV-D68. We knocked down SIRT-1 to understand its impact on viral release. We also examined the expression of CD63, the most widely used marker for exosomes/multivesicular bodies by western blot. As depicted in Fig. 2B, SIRT-1 knockdown increased CD63 expression compared to the scramble control.
Given the increase in CD63 expression in SIRT-1 knockdown cells, we hypothesize that knockdown of SIRT-1 may prevent the exocytosis of CD63-positive EVs. To confirm this hypothesis, we infected SIRT-1 knockdown cells for 4 h and isolated the EVs for western blotting against CD63. As shown in Fig. 2C, SIRT-1 knockdown increased CD63 expression in the whole cell lysate. Our IFA analysis in Fig. 2D also showed the aggregation of large CD63-positive punta in SIRT-1 knockdown cells, as previously observed (18). Interestingly, for the extracellular vesicle fraction, we observed that SIRT-1 knockdown decreased the release of CD63 positive EVs during EV-D68 infection compared to the scramble control, suggesting that SIRT-1 is essential for releasing virus-loaded CD63 positive EVs (Fig. 2C). Together, these results indicate that EV-D68 is mostly non-lytically released in EVs and that SIRT-1 is indispensable for this process.

The proviral activity of SIRT-1 does not require functional autophagy
Poliovirus has previously been reported to be non-lytically released in autophagosomes.

EV-D68 infection induces exportin-1 independent SIRT-1 translocation to the cytosol
Given SIRT-1's proviral role in EV-D68 infection, we asked whether EV-D68 infection alters SIRT-1 protein expression or subcellular localization. We infected H1HeLa cells for various time points before subjecting the cells to IFA against SIRT-1. As indicated in

SIRT-1 is not required for EV-D68 binding, entry, or replication
Because SIRT-1 translocates to the cytosol at approximately the time of peak viral RNA replication (3hpi), we asked whether SIRT-1 is necessary for viral RNA replication. We first examined whether SIRT-1 is translocated to viral RNA replication sites by conducting IFA using antibodies against SIRT-1 and dsRNA, which detects viral RNA replication intermediates (21). Our data show that SIRT-1 does not colocalize with dsRNA ( Fig. 5A). In contrast, GBF-1, a host factor important for forming picornavirus replication organelles, colocalized to dsRNA (

SIRT-1 Reduces PV, not CVB3, titers
We next sought to understand whether SIRT-1 modulates the infection of other medically essential picornaviruses, including PV and CVB3. While protective vaccines are available for PV, few vaccine-derived PV infection cases exist. On the other hand, there is no vaccine or treatment against CVB3, a significant cause of myocarditis and neurological disorders in infants. Therefore, identifying host factors that influence the infection of these viruses could help control their infection. We first examined whether PV and CVB3 alter the subcellular localization of SIRT-1. To our surprise, while PV, similar to EV-D68, induces SIRT-1 translocation to the cytosol, CVB3 did not (Fig. 6A).
These results indicate that certain but not all picornaviruses require SIRT-1 for their egress from infected cells.

Knockdown of SIRT-1 reduces SARS-CoV-2 titers
SIRT-1 has previously been reported to be essential for Middle Eastern respiratory

DISCUSSION
Here, we demonstrate that SIRT-1 is essential for basal and starvation-mediated autophagy. Depletion of SIRT-1 blocks basal autophagy and attenuates starvation-induced autophagic degradation. In addition, we show that EV-D68 in pre-lytic conditions is mainly released in EVs and that SIRT-1 is essential for the extracellular vesicle-mediated release of EV-D68. Consequently, the knockdown of SIRT-1 severely decreases infectious EV-D68 particles released in EVs. Knockdown of SIRT-1 also attenuates the release of PV and SARS-CoV-2 but marginally increases CVB3 egress. Our results suggest that some viruses induce SIRT-1 translocation to the cytosol to promote their vesicular release.
Our data show that SIRT-1 plays a cellular role in modulating basal autophagy.
Depletion of SIRT-1 increased LC3II accumulation (Fig. 1A) and enhanced endogenous Our data also show that SIRT-1 regulates starvation-induced autophagy. The kinetic studies in our supplemental data revealed that starvation induces rapid SIRT-1 translocation to the cytosol as early as 1h post-treatment (Fig. S1A). This finding is consistent with the notion that SIRT-1 drives autophagosome formation during amino acid starvation. Although starvation slightly decreased SIRT-1 protein expression by western blot, pharmacological inhibition of autophagic flux failed to restore SIRT-1 expression. Moreover, SIRT-1 did not colocalize with p62 in starved cells (Fig. S1B), suggesting that the cellular protein is not a substrate for autophagy.
Previous studies have shown that SIRT-1 forms a complex with cytoplasmic autophagy-related proteins, such as ATG7, ATG5, and LC3, upon acute amino acid starvation (13). However, given SIRT1's nuclear localization, exactly how SIRT-1 might form a molecular complex with the abovementioned proteins has been unclear. Our finding that amino acid starvation induces extensive SIRT-1 translocation provides a plausible explanation for how SIRT-1 interacts with these cytosolic proteins.
While picornaviruses are mainly released by cell lysis, a growing body of evidence indicates that these viruses can be released from intact cells without cell lysis, a phenomenon called non-lytic release. For instance, PV has been shown to escape intact cells through AWOL (autophagosome-mediated exit without lysis), in which virus-containing autophagosomes fuse with the plasma membrane to release virions (5,24). We show that knockout of ATG-7 reduces EV-D68 extracellular/EV titers, which is consistent with the AWOL model ( Fig. 3B and C). Hepatitis A virus (HAV), which is non-lytic, has been reported to exit cells in an enveloped form known as eHAV (25,26).
Similar events, modulated through the autophagic pathway, have been shown for other picornaviruses (27)(28)(29). We show here for the first time that EV-D68 can also exit intact cells non-lytically in EVs. This extracellular vesicle-mediated release of EV-D68 virions is, as we show here, dependent upon the autophagy regulator SIRT-1 ( Figures 1E, 2A,   3B).
We noticed that EV-D68 infection induces SIRT-1 translocation to the cytosol starting at 3 hpi. Given that RNA replication peaks at 3hpi, we initially thought SIRT-1 might be essential for viral RNA replication. However, during EV-D68 infection, SIRT-1 did not colocalize to dsRNA (Fig.5A) and its knockdown did not significantly impact EV-D68 RNA replication (Fig. 5C) nor viral binding (Fig. 5D) or entry (Fig. 5E). Instead, our findings suggest that the effect of SIRT-1 on virus production can largely be explained by its prominent role in the re-configuring the exocytosis pathway to promote the release of virus-loaded EVs. Knockdown of SIRT-1 led to the accumulation of large CD63 positive puncta in cells (Fig. 2D), suggesting that the turnover or the release of these vesicles is blocked in the absence of SIRT-1. Interestingly, our western blot data shows a marked decrease in CD63-positive EVs in cells with reduced SIRT-1 expression (Fig. 2C). This finding, coupled with the increased CD63 in the whole-cell lysate ( Fig. 2B and C), indicates that the reduction of SIRT-1 attenuates the release of virus-loaded CD63-positive EVs.
Although EV-D68 is known to induce autophagy and block the downstream autophagic maturation, how exactly EV-D68 infection causes autophagy and the specific viral protein(s) involved is not entirely understood (30,31). Our finding that EV-D68 infection induces SIRT-1 translocation starting at 3 hpi, which coincides with the emergence of lipidated LC3, suggests that SIRT-1 may be essential for virus-induced changes in the autophagic pathway. We observed that EV-D68 infection also induces ATG-7 punta formation, which, intriguingly, partially colocalizes to SIRT-1 (Fig. 3A). Hence, SIRT-1 translocation to the cytosol during EV-D68 infection could constitute a mechanism by which EV-D68 induces the generation and rearrangement of autophagic membranes.

SIRT-1 has previously been demonstrated to be important for MERS-CoV infection (17).
Here, we demonstrate that the cellular protein is also crucial for SARS-CoV-2 infection.
Our data show that, much like in the MERS-CoV study, knockdown of SIRT-1 decreases SARS-CoV-2 release, suggesting that SIRT-1 may be a shared host factor utilized by many Betacoronaviruses.
Our data show that a portion of nuclear SIRT-1 re-localizes to the cytosol upon infection by EVD-68 and PV. (Fig. 4A, 6A). We also observe that SARS-CoV-2 infection causes SIRT-1 translocation to the cytosol (Fig 7A). These viruses require SIRT-1 for normal virus release. It will be interesting to examine whether other Betacoronaviruses alter the subcellular localization of SIRT-1 and whether SIRT-1 is important for their infection.
CVB3, which does not require SIRT-1, does not induce re-localization of the protein (Fig. 6A). This, along with the finding that SIRT-1 is found on virus-induced extracellular vesicles, suggests to us a central role for SIRT-1 in constructing virus-containing vesicles for extracellular release -but only for some viruses. Why at least one picornavirus has evolved a SIRT-1-independent mechanism for release and what the mechanism might be will help understand how to target specific viruses or broad classes of viruses to prevent their release from infected cells. While we believe SIRT-1 is a common regulator of virus release, it is not a universal one, and identifying the homolog, or paralog, protein playing a parallel role in CVB3 release will undoubtedly shed light on the differences between these otherwise similar viruses.
In summary, our data show that SIRT-1 is essential for basal-and stress-induced autophagy and EV-mediated non-lytic release of EV-D68. We also demonstrated that SIRT-1 is crucial for releasing other important public health viruses, including SARS-CoV-2 and PV, indicating that SIRT-1 may be a common host factor regulating multiple viruses' release. Understanding how SIRT-1 regulates viral egress could open avenues for therapeutic intervention against many viruses.

Cell culture, plasmids, and viruses
H1HeLa cells were purchased from ATCC and cultured in DMEM supplemented with 10% fetal bovine saline, 1x penicillin/streptomycin, and 1x sodium pyruvate. The cells were incubated at 37°C in a 5% CO 2 incubator. The wild-type (Flag-SIRT1) and mutant

Western blot
Cells were lysed using RIPA buffer supplemented with cOmplete Tablets Mini Protease Inhibitor Cocktail. The lysates were incubated on ice for at least 30 minutes before being clarified at 12000 rpm for 30 minutes. The supernatants were transferred into Eppendorf tubes, and protein concentrations were determined by Bradford assay.
Lysates were then cooked and loaded onto SDS-PAGE. Following transfer onto PVDF membranes, the membranes were blocked in 5% skim milk for 1 h, washed twice with TBST, and stained with the following primary antibodies: anti-SQSTM1/p62, anti-LC, anti-b-actin, at 1: 1000 dilutions, and anti-CD63 (1: 250) overnight. The membranes were stained with the secondary antibodies for 1 h at room temperature and imaged using the Chemidoc machine after two washes.

Immunofluorescence analysis (IFA)
The cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes and

Extracellular vesicle isolation
Extracellular vesicles were isolated using the Invitrogen Total Exosome Isolation Reagent (from cultured cells). In brief, 1 ml of cell culture supernatants were clarified at 2000 x g for 30 minutes. The supernatants were then transferred to Eppendorf tubes, and 500 μl of the exosome isolation buffer was added and incubated at 4°C overnight.
At the end of the incubation, the tubes were centrifuged for 1 h at 10000 x g. The supernatants were transferred to new Eppendorf tubes, and the pellets were resuspended in PBS for western blot or plaque assay.

RNA isolation and qPCR
TRIzol was used to isolate total RNA according to the manufacturer's instructions, and cDNA was synthesized using the Thermo Scientific RevertAid H Minus First Strand cDNA Synthesis Kit. KiCqStart SYBR qPCR Ready Mix was used to perform qPCR using the Fast Dx Real-Time PCR Instrument (Applied Biosystems). Primers specific to the 5′ untranslated region (5′ TAACCCGTGTGTAGCTTGG-3′ and 5′ -ATTAGCCGCATTCAGGGGC-3′) were used to amplify EV-D68, and gene expression was normalized to GAPDH and plotted as relative expression compared to the 0h infection-only time point.

Plaque assay
The cells were washed twice with PBS for cell-associated titer determination and scraped in 1ml PBS, after which they were subjected to three free-thaw cycles and added to H1HeLa cells for 30 minutes. Cells were then overlaid with a 1:1 ratio of 2x MEM and 2% agar for 48 h before staining plaques with crystal violet. For extracellular titers, 1 ml of supernatants were collected and treated as the cell-associated titer without being subjected to free-thaw cycles.

Virus entry assays
Scramble and SIRT-1 knockdown cells were pre chilled on ice for 30 minutes for viral binding assay. The cells were then infected with EV-D68 (MOI =30) for 1 h on ice. The inoculum was removed, and the cells were washed twice with PBS, scraped, freeze-thawed 3 times, and stored at -80°C for plaque assay.
For viral entry, scramble and SIRT-1 knockdown cells were similarly pre chilled on ice for 30 minutes and then infected with EV-D68 for 30 minutes on ice. The unbound viral particles were washed off with PBS before shifting the cells to 37°C, allowing viral entry for 1 h. The cells were finally washed with PBS, scraped into Eppendorf tubes, and prepared for plaque assay.

SARS-CoV-2 titer determination by plaque assay
Plaque assays were performed as described previously (33). Briefly, 12-well plates were seeded with 2 x 10 5 VeroT cells/well one day before processing. On the day of processing, media was removed from the 12-well plates, and 200 µl of dilutions of virus stock or collected cell supernatants in DMEM were added to each well. Plates were incubated at 37°C (5% CO 2 ) for 1 hour with rocking every 15 minutes. Following incubation, 2 ml of plaque assay media, DMEM containing 0.1% agarose (UltraPure™) and 2% FBS (Gibco), was added to each well and incubated for 48 h at 37°C (5% CO 2 ).

siRNA Knockdown Protocol for SARS-CoV-2 infection
siRNA knockdown assays were performed as described previously (34). Briefly, A549/hACE2 cells were seeded in 24-well cell culture plates one day before siRNA

Statistical analysis
GraphPad Prism software (Version 7.03) was used for all statistical analyses, and values represent the mean ± standard error of the mean (SEM) of at least 3 independent repeats. Student t-test for comparison and a p-value of < 0.05. was considered statistically significant.

ACKNOWLEDGEMENTS
We thank Sohha Ariannejad for assistance and the members of the Jackson, Frieman, and Coughlan labs for thoughtful discussion. This work was funded by NIH/NIAID grants R01141359, R01104928 to W.T.J. and R21158134 to W.T.J. and M.B.F.