Basal Autophagy Is Required for Herpes simplex Virus-2 Infection

Autophagy is a conserved catabolic process of the cell, which plays an important role in regulating plethora of infections. The role of autophagy in Herpes simplex virus-2 (HSV-2) infection is unknown. Here, we found that HSV-2 does not allow induction of an autophagic response to infection, but maintains basal autophagy levels mostly unchanged during productive infection. Thus, we investigated the importance of basal autophagy for HSV-2 infection, using pharmacological autophagy suppression or cells genetically deficient in an autophagy-essential gene (ATG5). Interference with basal autophagy flux in cells significantly reduced viral replication and diminished the infection. These results indicate that basal autophagy plays an indispensable role required for a productive infection. Importantly, this study draws a sharp distinction between induced and basal autophagy, where the former acts as a viral clearance mechanism abrogating infection, while the latter supports infection.


Results
Basal autophagy levels are maintained unchanged during HSV-2 infection. In response to HSV-1 infection, we previously found that autophagy levels may be slightly inhibited, or in most cases remain unchanged, during productive HSV-1 infection 29 . The autophagic response to HSV-2 infection was not previously assessed. Thus we monitored autophagy flux in cells during HSV-2 infection using sequestosome1 (or p62) immunoblotting. p62 is a protein that is degraded mainly by autophagy and thus its levels represent a reliable indicator of autophagy flux in cells [30][31][32] . Id est, accumulation of p62 marks suppression of the autophagy flux, whereas its depletion reflects autophagy induction [30][31][32] . We found that HSV-2 infection does not cause any significant changes in autophagy flux in host cells (Fig. 1a-d). This result indicated that HSV-2, similarly to HSV-1, prevents autophagy induction in response to infection, but meanwhile maintains the basal autophagy activity of the host mostly unhampered.

Pharmacological suppression of autophagy inhibits HSV-2 infection.
Having found that HSV-2 maintains the basal autophagic activity of host cells, we hypothesized that this basal autophagic activity could play a role in infection. To investigate such a role of autophagy in HSV-2 infection, we used pharmacological means to suppress cellular autophagy levels. First, we used chloroquine to suppress autophagy. We found that chloroquine treatment of the cells led to reduced viral yields in cells (data not shown). Various chemical agents have been classically used in the field to suppress autophagy, however some of these agents suffer from side effects that make it difficult to interpret whether the results are directly caused by autophagy. For example, 3-methyladenine, a commonly used autophagy inhibitor, was also reported to enhance basal autophagy despite suppressing starvation-induced autophagy 33 . In addition, chloroquine and vinblastine were shown to cause autophagy-complicating nonspecific effects such as mammalian target of rapamycin (mTOR) inhibition 34 . In a comparative study testing various side effects of autophagy inhibitors, bafilomycin A1 (BFN), a well-known autophagy inhibitor that blocks fusion of autophagosomes with lysosomes 35 , was shown to have least nonspecific effects, and no effect on mTOR activity, in treated cells in contrast to other agents tested 34 . Thus, we decided to confirm the chloroquine preliminary finding with a more specific autophagy inhibitor, BFN.
We treated human corneal epithelial (HCE) cells, known to be targeted by both HSV-1 and HSV-2 in vivo and in vitro 9,10 , with BFN. We then confirmed BFN-mediated suppression of autophagy through p62 and LC3 immunoblotting. Suppression of basal autophagy by BFN was confirmed by the accumulation of p62 along with LC3-II (Fig. 2a). To rule out any possible effects of BFN on viral entry, we incubated the cells with HSV-2 for 2 hrs, and the cells were then untreated or treated with BFN. Using HSV-2-GFP virus, we monitored the progress of infection microscopically. We observed a significant drop in virus levels in BFN-treated, relative to mock-treated, cells (Fig. 2b,c). To confirm this finding, we employed a quantitative FACS-based assay to measure virus yields in cells. We found that BFN-mediated block of autophagy suppressed viral yields and HSV-2 infection (Fig. 2d,e). To further confirm, we determined virus levels in the cells via qPCR virus genome quantification. qPCR assay confirmed that BFN treatment significantly reduces HSV-2 infection (Fig. 2f). Moreover, the possibility of cell death upon BFN treatment was ruled out (Fig. 2g). These results demonstrated that suppression of basal autophagy in host cells interferes with viral replication and the progress of infection.
Monitoring HSV-2 infection under genetic deficiency of autophagy. Due to the limitations of pharmacological assays and the difficulty to completely rule out nonspecific effects of chemicals, we sought to confirm the effect of basal autophagy on HSV-2 infection using genetic means. Therefore, we used mouse embryonic fibroblasts (MEFs) that are incapable of executing autophagy due to a genetic knockout of the autophagy-essential gene ATG5 19,36 . Out of the 31 autophagy-related (ATG) genes, 18 genes (including ATG5 and ATG7) are essential for development and maturation of autophagosomes 37 . ATG7 −/− MEFs, for instance, showed reduced degradation of long-lived proteins and cytoplasmic organelles even at basal levels. These cells also exhibited impairment of autophagosome formation and maturation, and impairment of both basal and starvation-induced autophagy 38 .
Before using the ATG5 −/− cells in our virus assays, we wanted to validate their autophagy deficiency. We monitored basal autophagosomal levels in normal feeding conditions and induced autophagosomal levels under nutrient loss (starvation). Using GFP-LC3-expressing MEFs, we were able to detect low basal levels of autophagy in wild-type (WT) cells, but much less levels in ATG5 −/− cells (Fig. 3a-c). In addition, ATG5 −/− cells were also defective in mounting an autophagic response to starvation (Fig. 3a-c). These results validated the autophagy-deficient nature of these cells.
Then we infected WT or ATG5 −/− MEFs with HSV-2-GFP at various multiplicities of infection (MOIs) and assessed the infection with fluorescence microscopy. We found that while WT MEFs showed signs of robust HSV-2 infection, including viral gene expression, viral spread, and syncytia formation (Fig. 4a), ATG5 −/− cells showed extremely reduced infection signs at all the MOIs tested (Fig. 4b). These findings suggested that autophagy deficiency interferes with HSV-2 infection.

Autophagy deficiency abrogates HSV-2 infection.
Having observed low infection of cells deficient in autophagy, we sought to confirm quantitatively the effect of autophagy deficiency on viral levels in infected cells. We thus utilized various quantitative assays of infection: FACS assay, qPCR viral genome quantification and virus titer determination by plaque assay. Compared to WT cells, ATG5 −/− cells showed significantly lower viral yields and seemed highly resistant to infection (Fig. 5a,b). Even at higher MOIs, ATG5 −/− MEFs still showed significant block of infection ( Fig. 5a,b). qPCR assay also showed that ATG5 knockout in cells dramatically diminishes the infection (Fig. 5c). Finally, we determined virus titer from WT or ATG5 −/− cells using plaque assay. While WT cells secreted approximately 10 2 -10 3 plaque-forming units (PFU)/monolayer, ATG5 −/− cells secreted approximately 10 6 -10 7 PFU/ monolayer (Fig. 5d). Additionally, in order to rule out the possibility that autophagy deficiency may interfere with virus entry into cells, we assessed internalized virus levels in WT or ATG5 −/− cells. We found that autophagy deficiency does not affect virus entry into the cells (Fig. 5e). Together, our results indicate that basal autophagic activity of the host cells supports HSV-2 infection, which is consistent with results of pharmacological autophagy inhibition (Fig. 2).

In-depth validation of the role of basal autophagy in HSV-2 infection.
We then sought to evaluate the role of autophagy in HSV-2 infection under various productive infection conditions and in multiple infection systems. First, we tested the effect of autophagy inhibition on infection in other cell types. We found that autophagy suppression caused a significant reduction of infection in a retinal pigment epithelial cell line (Fig. 6a,b) or in human foreskin fibroblast cells (Fig. 6c,d). Second, using ATG5 −/− cells, we assessed the importance of autophagy for infection at various time points and under infection with various MOIs. We found that autophagy deficiency was always associated with diminished viral replication levels and infection under all these condition (Fig. 6e,f). Finally, this requirement of autophagy for infection was independent of the virus strain used (Fig. 6g). Collectively, these results confirm that basal autophagy is an important factor required for a robust HSV-2 infection.

Discussion
Autophagy plays a pivotal role as a quality control mechanism and in regulating infections and coordinating the cellular response to a pathogen attack. Induction of autophagy was previously shown to enhance HSV-1 viral clearance and diminish viral growth 23,39 . HSV-1 establishes virulence through inhibiting cellular autophagy 22,23 , or at least preventing its activation 29 , during infection. In this study, we show that HSV-2 prevents autophagy induction but maintains the basal levels of the pathway (Fig. 1). Although  reports suggested that HSV-1 may inhibit autophagy via ICP34.5-mediated beclin1 binding while HSV-2 ICP34.5 was not tested for its ability to bind beclin1, our results may suggest that HSV-2 ICP34.5 also binds beclin1 to a certain extent; however this hypothesis remains to be experimentally investigated. Notably, a closely related α -herpesvirus, varicella-zoster virus (VZV), which lacks the ICP34.5 gene can  allow robust induction of autophagy responses to infection 40,41 . Moreover, in similarity to HSV-2 results shown here, autophagy suppression downregulated VZV virus replication and growth in cells 40 . Thus, these findings together suggest that these different viruses may benefit from the autophagy pathway to varying extents and perhaps the viral autophagy-modulating genes serve to optimize or fine-tune the autophagy flux to levels that bolster each virus's growth, according to the differential reliance of each virus on autophagy. The present study provides insights that may support this concept which requires further work to be firmly established.
Two modes of autophagy exist under different conditions, which play distinct functions 37,42 . Under certain autophagy-inducing conditions such as nutrient deficiency and some viral infections, autophagy may be induced above its constitutive levels. Induced autophagy qualifies the cell to adapt to drastic conditions and generate required energy from alternative sources, or to combat infections [12][13][14]39,42,43 . On the other hand, basal autophagy performs various homeostatic and quality control functions such as preventing protein aggregation or limiting reactive oxygen species (ROS) production 37,38 . Basal autophagy takes place at low or modest levels under normal conditions and regardless of the feeding conditions, as in embryogenesis 44 . A prominent example of basal autophagy is autophagy in neuronal cells and the brain. Autophagy in the brain proceeds at its basal levels, without significant induction even under nutritional deficiency (due to the fact that the brain can quickly compensate nutrient deficiency by relying on nutritional supply from other organs). Disruption of such basal autophagy leads to neurodegeneration, indicating that the role of basal autophagy is important for normal neuronal cell functioning and survival 45 . Moreover, suckling and non-suckling ATG7 −/− mice all died at the same time, within 1 day of birth 38 , suggesting that the reason of death is not due to nutrient deprivation and that it may be due to disruption of a cellular function performed by basal autophagy. The absence of induced autophagy in HSV-1 and HSV-2 infection and persistence of basal autophagy suggest a role for this basal autophagy that supports infection (as we show here for HSV-2).
For possible reasons for the contribution of basal autophagy to infection, some scenarios may be proposed based on lessons learned from other viruses. First, the virus may in fact be protected in the autophagosomes from more severe innate immune defense mechanisms 43 . Second, autophagosomal inclusion of the virus might support a step in the viral life cycle such as using the vesicle membranes for viral envelopment [46][47][48] . Third, autophagic vesicles may facilitate intracellular trafficking of the virions. For example, virions can be released from the cell via fusion of the lysosomes with the plasma membrane 46 . Fourth, autophagy performs many homeostatic functions, which might support infection. Future work is to determine the exact mechanism by which constitutive autophagic activity (or machinery) supports HSV infection.
Another possibility for the reason of resistance of ATG5 −/− cells to HSV-2 infection may be attributed to an aberrant proinflammatory cytokine response to infection in these cells. For example, HSV-1 blocks NALP3 (or NLRP3) inflammasome activity during the course of infection 49 . Inflammasomes induce maturation and secretion of proinflammatory cytokines 50 , which represent powerful antiviral effectors [51][52][53] . Autophagy-deficient cells (lacking LC3B or beclin1) showed increased NALP3 inflammasome activity and caspase-1 levels, and exhibited enhanced inflammatory responses (e.g. interleukin (IL)-1β , and IL-18) to lipopolysaccharide in vitro and in vivo 54 . Autophagy could also limit inflammasome activity directly through targeting inflammatory cytokines such as IL-1β for autolysosomal degradation 55 . Additionally, several autophagy proteins target components of the antiviral type-I interferon (IFN) response. Atg5-Atg12 blocks caspase recruitment domain (CARD)-mediated signaling and the downstream production of type I IFN 56,57 . Also, Atg9 was shown to regulate STING (Stimulator of Interferon Genes) cytoplasmic localization and thus control IFN-I responses 58,59 . It is thus possible to hypothesize that autophagy-deficient cells inherently possess elevated cytokine levels that help confer on these cells resistance to HSV-2 infection.
In addition to the role of autophagy in regulating an innate immune response to infection, housekeeping functions of autophagy may also provide a possible reason for supporting HSV-2 infection. First, autophagy-mediated removal of damaged organelles such as mitochondria (mitophagy) prevents accumulation of reactive oxygen species (ROS) (Ref. 60). ROS may induce NALP3 inflammasomes 61 , type-I IFN responses 62 , or cytokine production in response to infection 63 . Compounds that enhance ROS production suppress HSV infection 64 , while factors that relieve herpesvirus-induced oxidative stress 65 significantly enhance infection 66,67 . Second, protein aggregation under autophagy-deficient conditions 38 could also present a reason for infection halt. Third, dysregulation, in ATG5 −/− cells, of metabolism and turnover of metabolites known to regulate infection 68,69 may contribute to their resistance to HSV-2 infection. Finally, a distinct role of autophagy machinery in enodosomal trafficking and endolysosomal functions was recently reported 70 . Perhaps, impairment in autophagy-deficient cells of intracellular endosomal trafficking of viral, or infection-regulating cellular, components participates to infection suppression in these cells.
In conclusion, this study demonstrates the importance of basal autophagy in HSV-2 infection. Induced and basal autophagy may serve distinct functions, and their dysregulation causes morbidities and mortalities 19,71,72 and influences susceptibility to various pathogens 73,74 . Importantly, this study draws a difference between induced and basal autophagy in HSV infections, and will incite future mechanistic studies of the basal autophagy-mediated support of herpesvirus infection. Viruses. HSV-2 (strain 333) and HSV-2 (333)-GFP viruses were provided by J. Vieira (University of Washington). HSV-2 strain G was provided by P. Spear (Northwestern University, Chicago, IL). Viruses were propagated on Vero cells according to standard procedures.
Infection. The cells were incubated with the virus in phosphate-buffered saline (PBS) supplemented with 0.1% glucose and 1% serum at 37 °C-5% CO 2 conditions. After 2 hrs, the virus was removed and fresh medium was added to the cells.
Immunoblotting. The cells were harvested, and lysed in RIPA buffer (Sigma, Catalog number R0278) containing protease-phosphatase inhibitors. Cell lysates were electrophoresed on denaturing SDS-PAGE gel (Novex), and proteins were transferred onto a PVDF membrane, followed by blocking of non-specific binding, incubation with primary antibody and horse radish peroxidase (HRP)-conjugated secondary antibody. The membrane was developed by incubation with Femto-Sensitivity ECL (Thermo), and bands were imaged using ImageQuant LAS4000 digital imager (GE).
Plasmids. pEX-GFP-hLC3WT plasmid (referred to as GFP-LC3) previously described 75  Starvation. To induce autophagy by starvation, the cells were washed in PBS for three times to remove residual medium, and then were cultured in Hanks' Balanced Salt Solution (Gibco) for 2-3 hr.
Confocal fluorescence microscopy. To monitor LC3-GFP punctae, confocal microscopy (Zeiss 710 microscope, Zeiss) was used. After treatment, the cells were washed, fixed in paraformaldehyde, and used in imaging. Image acquisition was performed using ZEN software (Zeiss), and image analysis was performed using MetaMorph software (Zeiss).
Virus titer (plaque formation) Assay. Confluent Vero cell monolayers were infected with serially diluted virus for 2 hrs. Then the cells were washed and covered with methylcellulose (Sigma)-containing Scientific RepoRts | 5:12985 | DOi: 10.1038/srep12985 DMEM. The cells were grown for 72 hrs, then fixed and stained with crystal violet. Plaques were counted, and plaque counts were used to calculate viral titers.
Cell viability (MTT) assay. Cell viability was assessed using MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay. MTT was purchased from Sigma. The assay was performed as previously described 76 . Statistical analyses. Experiments were independently replicated for at least three times. Quantification figures show mean values, and error bars represent standard error of the mean. Statistical significance was determined via Student's t-test (minimum p-value for significance 0.05). Unless otherwise indicated, the data were statistically significant (p-values < 0.05).