SIRT-1 is required for release of enveloped picornaviruses

Reviewer #1 (Public Review):

The enteroviruses comprise a medically important genus in the large and diverse picornavirus family, and are known to be released without lysis from infected cells in large vesicles containing numerous RNA genome-containing capsids - a feature allowing for en bloc transmission of multiple viral genomes to newly infected cells that engulf these vesicles. SIRT-1 is an NAD-dependent protein deacetylase that has numerous and wide ranging effects on cellular physiology and homeostasis, and it is known to be engaged in cellular responses to stress and autophagy.

Jassey et al. show that RNAi depletion of SIRT-1 impairs the release of enterovirus D-68 (EV-D68) in EVs recovered from the supernatant fluids of infected cells using a commercial exosome isolation kit. The many functions attributed to SIRT-1 in the literature reflect its capacity to deacetylate various cell proteins engaged in transcription, DNA repair, and regulation of metabolism, apoptosis and autophagy. However, Jassey et al. make the surprising claim that the proviral role of SIRT-1 in promoting enterovirus release is not dependent on its deacetylase activity. Fig. S1C is crucial to this suggestion, as it is said to show that reconstituting expression with a catalytically-inactive mutant can rescue virus release from SIRT-1 depleted cells. However, no information is provided concerning the levels of endogenous and ectopically-expressed SIRT-1 proteins in this experiment, making it very difficult to interpret the results. Is the mutant SIRT-1 protein expressed at a higher level than the non-mutant protein? Is there a 'sponging' effect with these transfections that lessens the siRNA efficiency and reduces knockdown of the endogenous protein? Fig. S1B and Fig. 4C convincingly show that EX527, a small molecule inhibitor of the deacetylase activity of SIRT-1, inhibits extracellular release of the virus. This suggests that the deacetylase activity of SIRT-1 is in fact required for the proviral effect of SIRT-1. This is a fundamentally important question that will require more investigation.

Fig. 6 shows how SIRT-I knockdown impacts the release of enterovirus D68 in EVs recovered from cell culture supernatant using a commercial 'Total Exosome Isolation Kit'. The authors should describe the principle this kit exploits to isolate 'exosomes' (affinity isolation?) and specify which antibodies it involves (anti-phosphatidylserine, anti-CD63, others?) This could impact the outcome of these experiments, and moreover is important to include in the long-term scientific record. The authors are appropriately cautious in describing the vesicles they presume to be isolated by the kit as simply 'extracellular vesicles', since there are multiple types of EVs with very different mechanisms of biogenesis, of which 'exosomes' are but one specific type. It would have been more elegant had the authors shown that SIRT-1 is required for EV-D68 release in detergent-sensitive vesicles with low buoyant density in isopycnic gradients, and to characterize the size and number of viral capsids in these vesicles by electron microscopy.

Fig. 6 shows that SIRT-1 depletion upregulates CD63 expression, but has no apparent impact on the release of CD63-positive 'EVs' from uninfected cells. EV-D68 infection also upregulates CD63 expression in SIRT-1 replete cells, and in this case, increases the release of CD63-positive EVs. The combination of infection and SIRT-1 depletion massively upregulates CD63 expression, but appears to eliminate the enhanced release of CD63-positive EVs resulting from infection alone. These are interesting results, from which the authors infer CD63 is associated with EVs containing EV-D68. But, do we know this? Can a CD63 pulldown immunoprecipitate EV-D68 capsid proteins or viral RNA? CD63 is strongly associated with exosomes released from cells through the multi-vesicular body pathway, which are distinct from the LC3-positive EVs released by secretory autophagy that have previously been associated with enteroviruses. The authors suggest that 'knockdown of SIRT-1 may prevent the exocytosis of CD63-positive EVs", but this is a very broad claim (and not really demonstrated by Fig. 6): it requires a clearer definition of what the authors mean by 'exocytosis' and a much more detailed analysis of the size and buoyant density of EVs released in a SIRT-1-dependent process.

The authors suggest that almost all EV-D68 released from infected cells is released without cell lysis in EVs. However, they generally show data from only a single time point following infection (5 or 6 hrs post-infection). It would have been interesting to see a more complete temporal analysis, and to know whether a high proportion of virus continues to be released in EVs, or if it is swamped out ultimately by lytic release of nonenveloped virus.

Fig. 1D indicates that a small fraction of SIRT-1 leaks from the nucleus in EV-D68 infected cells. The authors suggest this is due to targeted nuclear export, rather than simply leaky nuclear pores which are well known to exist in enterovirus-infected cells. The authors present similar fluorescent microscopy data showing inhibition of TFEB export in leptomycin-B treated cells in Fig. S2A in support of their claim that this is specific SIRT-1 export, but these data are far from convincing - there is equivalent residual TFEB and SIRT-1 in the cytoplasm of the treated cells. Quantitative immunoblots of cytoplasmic and nuclear cell fractions might prove more compelling.

Finally, the authors should be more specific in describing the viruses they have studied (EV-D68 and PV). It would be preferable to describe these as 'enteroviruses' (including in the title of the manuscript), rather than more broadly as 'picornaviruses'. There is no certainty that the requirement for SIRT-1 in non-lytic release of virus extends to hepatoviruses or other picornaviral genera, for which mechanisms of nonlytic release may be quite different.

Reviewer #2 (Public Review):

The authors aimed to connect SIRT-1 to EV-D68 virus release through mediating ER stress. They are successful in robustly connecting these pathways experimentally and show a new role for SIRT-1 in EV-D68 infection. These results extend to additional viruses, suggesting role(s) for SIRT-1 in diverse virus infection.

The authors note that EV-D68 does not significantly impact SIRT-1 protein levels (Fig 1E and F), though this has been described for other picornaviruses (Xander et al., J Immunol 2019; Han et al., J Cell Sci 2016; Kanda et al Biochem Biophys Res Commun 2015). This may be of interest to note in the manuscript.

The data regarding CVB3 (Fig S4) are especially interesting because they show no discernable impact on infection. The manuscript should describe this further and perhaps speculate on potential reasons. Could it be due to inefficient knockdown?

SIRT-1 (and other sirtuins) have been linked to an innate interferon response. Are any of the phenotypes observed here due to IFN responses? The use of H1HeLa cells would suggest this is not the case.