3-Methyladenine

Inhibition of autophagy by 3‐methyladenine restricts murine cytomegalovirus replication

Abstract
Cytomegalovirus (CMV) induced autophagy affects virus replication and survival of
the infected cells. The purpose of this study was to investigate the role of autophagy inhibition by 3‐methyladenine (3‐MA) on murine cytomegalovirus (MCMV)
replication and whether it is associated with caspase‐3 dependent apoptosis. The
eyecup isolated from adult C57BL/6J mice (6–8 weeks old) and mouse embryo
fibroblast cells (MEFs) were infected with MCMV K181 strain, followed by the treatment of 3‐methyladenine (3‐MA), chloroquine, or rapamycin to block or stimulate autophagy. In cultured MEFs, the ratio of LC3I/II was reduced at
24 hours post infection (hpi), but was increased at 48 hpi In the eyecup culture, LC3I/II ratio was also decreased at 4 and 7 days post infection (dpi). In addition,
caspase‐3 cleavage was increased at 48 hpi in MEFs and also elevated in MCMV
infected eyecups at 4, 7, 10, and 14 dpi. 3‐MA treatment significantly inhibited the virus replication in MEFs and eyecups. The expression of early antigen (EA) of
MCMV was also decreased in MEFs and eyecups. Meanwhile, cleaved caspase‐3 dependent cell death was promoted with the presence of 3‐MA in MCMV infected MEFs and eyecups, while RIPK1/RIPK3/MLKL pathway was inhibited by 3‐MA in eyecups. Inhibition of autophagy by 3‐MA restricts virus replication and promotes caspase‐3 dependent apoptosis in the eyecup and MEFs with MCMV infection. It
can be explained that during the early period of MCMV infection, the suppressed autophagy process directly reduced virus release, but later caspase‐3 dependent apoptosis dominated and resulted in decreased virus replication.

KEYWOR DS
3‐MA, apoptosis, autophagy, murine cytomegalovirus
Department of Pediatrics, Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology, Wuhan, Hubei, China

Correspondence
Feng Fang, Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology, No.1095, Jiefang Rd, Qiaokou District, Wuhan,
Hubei 430030, China. Email: [email protected]

Funding information
National Natural Science Foundation of China, Grant/Award Number: 81271807

⦁ | INTRODUCTION

As a fundamental cell biological pathway, autophagy plays an important role in the infection‐induced inflammation via regulatory interactions with innate immunity signaling. Autophagy in the immune system as a
whole confers measured immune responses, alterations in autophagy can lead to inflammation and tissue damage.1,2 During virus infection, autophagy not only could modulate the primary antiviral response and
prevent prolonged and excessive inflammation but also control viral
transmission, which resulted in the clearance of viral antigens.3 It has been demonstrated that the faster‐migrating form of microtubule‐ associated protein light chain 3 (LC3II) increased even at 2 hour post
infection (hpi) and the vesicles with the characteristic appearance of autophagosomes or autolysosomes can be detected at 6 hpi of human cytomegalovirus (HCMV) in cultured human fetal foreskin fibroblasts (HFF).4 As to murine cytomegalovirus (MCMV), there was also an

increase of LC3II in MCMV infected NIH‐3T3 fibroblasts from 6 hpi,5 which indicated that autophagy could be induced very early after cytomegalovirus (CMV) infection. In addition, autophagy can promote
cell survival and limit pathogenesis in HCMV infection. However, HCMV might have developed efficient strategies to block the activation of autophagy during infection, as it was also found that HCMV infection could drastically inhibit autophagosome formation in vitro6 at later time points of infection which were mediated by the interaction of TRS1
protein with Beclin 1.7 An mammalian target of rapamycin (mTOR)‐
independent inducer of autophagy, Trehalose, and another autophagy‐ inducing compound SMER28, have been demonstrated to suppress the production of cell‐associated virus and viral replication,8,9 which might provide a novel therapeutic target for the treatment of HCMV related
diseases.
Most of the world’s population are infected with HCMV, among which the sero‐prevalance ranged from 40% to 90%.10 Although the majority of CMV infection are asymptomatic infection in im-
munocompetent individuals, CMV retinitis occurs predominantly in those patients who are unable to generate a normal primary T‐cell response against the virus or in patients who are carriers of CMV but have decreased CMV‐specific T‐cell response due to disease or
immuno‐suppressive treatment, such as acquired immunodeficiency
syndrome (AIDS) and leukemia.11 Compared with Ebola and Zika virus which are able to infect the eye and cause severe eye diseases, such as optic neuritis and chorioretinal atrophy,12,13 CMV infection not only resulted in retinitis in immunocompromised individuals but also systematic CMV infection could transmit to specific eye com- partments, including the anterior segment and choroid in im- munocompetent individuals.14 During this systematic infection involved ocular infection, viral replication was firstly detected in the endothelial cells of the iris followed by the perivascular cells, which indicated that it is reasonable to consider CMV as a pathogen that
could induce long‐lived chronic and low‐level inflammatory sequelae
in the eye, even in the neural retina.14 Upon CMV infection, the host could initiate an innate immune response to activate caspase‐ dependent or—independent apoptosis to clear virus replication,
meanwhile, it has been found that the CMV encodes multiple cell death suppressor protein, such as M36, M45, and m38.5 to prevent
infection‐induced cell death including apoptosis and necroptosis.15‐18
However, the detailed mechanism involved in the regulation of host‐ virus balance during CMV infection was not fully understood.
Autophagy has been recognized as a protective determinant for cell
survival, and inhibition of autophagy by gene knockout of the key autophagy‐related proteins such as ATG5, ATG6/Beclin‐1, or ATG12, or by pharmacological agents including 3‐MA, hydroxychloroquine, chlor- oquine (CQ), and bafilomycin A1 could promote apoptosis‐induced cell
death.19,20 Therefore, we want to know whether autophagy could reg- ulate virus replication and whether caspase‐3 dependent apoptosis or necroptosis was involved in the regulation of host‐virus balance during
CMV infection especially in the posterior segment parts of the eye. CMV is a strictly species‐specific virus, making it difficult to study HCMV in vivo experiments. Murine CMV (MCMV), which is similar to HCMV in
sequence and in vivo pathogenesis, is widely utilized to mimic HCMV
infection in a mouse model. 3‐MA is a nonselective phosphoinositide 3 kinase (PI3K) inhibitor and hence block the catalytic activity of several PI3Ks beyond VPS34 to inhibit autophagy,21,22 while acute rapamycin
treatment leads to the relatively specific inhibition of mTORC1 through FK506‐binding protein 1A (FKBP1A) to induce autophagy, though chronic exposure to rapamycin promotes mTORC2 disassembly.23 Therefore, 3‐MA and rapamycin were used to inhibit or activate autophagy in our present study. Also, another autophagy in- hibitor CQ, which inhibits autophagic flux via decreasing autophagosome‐ lysosome fusion,24 was used to further investigate the effect of autop-
hagy on MCMV replication.

⦁ | MATERIALS AND METHODS

⦁ | Animals and eyecup culture

C57BL/6J mice used in this study were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were housed with a 12‐h light
cycle alternating with a 12‐h dark cycle and were given standard ro-
dent chow and water and libitum. All eyecups were isolated from adult (6–8 weeks old) mice. The eyecup culture procedure was followed as described previously.25 Briefly, after the mice were killed under anesthesia by CO2 asphyxiation, the eyes were removed and
immediately placed in phosphate‐buffered saline (PBS) on ice. Under
the dissecting microscope, the iris and lens at the posterior margin of the limbus and the cornea were carefully moved with sterilized scis- sors followed by the dissection of the retina. Finally, the remaining choroid/sclera was placed on a membrane filter and covered with
Matrigel and cultured in a 24‐well plate with Dulbecco’s modified
Eagle’s medium (DMEM) medium (Mediatech, Manassas, VA) including
10% FBS and 1 × PS (Penicillin‐streptomycin). Eyecups were infected with MCMV (5 × 103 PFU) or DMEM mocks, followed by treatment with 3‐MA (5 mM), rapamycin (500 nM) or chloroquine (CQ, 100 uM). The supernatant of eyecup culture, as well as the eyecup tissues, were
collected at 4, 7, 10, and 14 days post infection (dpi) used for plaque assay, western blot, or immunofluorescence staining.

⦁ | Virus

MCMV strain K181 used in this study was kindly gifted from Dr. Ming Zhang, Augusta University, Augusta, Georgia, USA. Six to eight weeks of age BALB/C mice were used to propagate the virus according to the procedure described previously.26 Briefly, all mice were injected intraperitoneal with 2 mg steroid, 2 days later, 1× 103 PFU of MCMV were injected intraperitoneal and, meanwhile, the steroid was intraperitoneally injected every 2 days. Two weeks later, all mice were killed, and the salivary glands were collected. Following homogenization within the DMEM medium, the salivary glands were centrifuged and the supernatant containing the virus was aliquoted. Virus titer was analyzed by plaque assay using mouse embryo fibroblast cells (MEFs). Virus stocks were stored in liquid

nitrogen. Every fresh stock virus could be diluted to the appropriate concentration in serum‐free DMEM when used for a single experiment.

⦁ | Plaque assay

MEFs were cultured in 24‐well plates with DMEM containing 5% (fetal bovine serum; Thermo scientific, Waltham, MA, USA) (at 37°C, 5% CO2) until 80% confluence. The supernatant of the eyecup culture was
collected at 4, 7, 10, and 14 dpi) and serially diluted. 100 μl of each dilution was added to the prepared MEF monolayers, followed by incubation at 37°C for 1 h. Then 1 part of 1% agarose solution was mixed with 1 part of 2 × DMEM (Life Technologies, Grand Island, NY, USA) and 0.5 ml of this agarose mixture (0.5% agarose in 1 × DMEM) was added to each well. After 5 days of culture in 37°C with 5% CO2, the MEFs were fixed with 4% formaldehyde for 2 h and stained with 0.13% crystal violet. The plaques were counted under the light microscope.

⦁ | Antibodies and reagents

Anti‐MCMV early antigen (EA) was used to identify MCMV‐infected cells in the eyecup.27 Rabbit anti‐RPE65 (specific for RPE cells), rabbit anti‐p62, and rabbit anti‐phospho‐MLKL were purchased from Abcam (Cambridge, MA, USA). Rabbit anti‐LC3I/II, p70 S6 Kinase, anti‐ cleaved caspase‐3, anti‐RIPK3, anti‐phospho‐RIPK3, anti‐RIPK1, anti‐ MLKL, goat anti‐rabbit IgG‐horseradish peroxidase (HRP), and goat anti‐mouse IgG‐HRP were purchased from cell signaling (Cell Signaling Technology, Danvers, MA, USA). Anti‐β‐actin was purchased from Sigma‐Aldrich (St. Louis, MO, USA). Antimouse IgG‐Alexa 488, anti- rabbit IgG‐Alexa 594 were purchased from Vector Laboratories
(Burlingame, CA, USA). BCA assay kit was purchased from Thermo Fisher Scientific. 3‐MA (5 mM), CQ (100 µM) and rapamycin (500 nM), which were used to inhibit and induce autophagy, were purchased from Sigma‐Aldrich. TUNEL assay kit was purchased from Roche (Roche Diagnostics, Indianapolis, IN, USA).

⦁ | Western blot analysis

Total proteins were extracted from eyecups with NP40 lysis buffer and the protein concentration was measured using a BCA assay kit according to the manufacturer’s instructions. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, fol-
lowed by electro‐blotting onto polyvinylidene difluoride membranes
(GE Healthcare, Piscataway, NJ, USA). The membranes were blocked with 5% skimmed milk for 1 h at room temperature followed by in- cubation with primary antibodies overnight (at the dilution of 1:1000 with 5% BSA) at 4°C. After three times wash with PBST, the mem-
branes were incubated with HRP‐conjugated secondary antibody for
1 h at room temperature. The protein blotting bands were visualized
using chemiluminescence (ECL; GE Healthcare) with ChemiDoc Image System (Bio‐rad, CA, USA) and the density of the blotting bands was quantified with Image J and normalized to β‐actin.

⦁ | Immunofluorescence staining

Cultured eyecups were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) for 1 h and then immersed in 25% sucrose overnight at 4°C, followed by snap‐frozen and sec-
tioned using a cryostat. For double staining with TUNEL and RPE, EA, cleaved caspase‐3, or phospho‐MLKL, TUNEL assay was initially performed according to the manufacturer’s instructions. After
washing with PBS and blocking with PBS containing 10% normal goat serum, slides were incubated with anti‐RPE65, biotinylated anti‐EA
(1:500), anti‐cleaved caspase‐3, and anti‐phospho‐MLKL antibody
overnight at 4°C. After three times wash with PBS, 100 µl Texas red labeled avidin (1:600) or antirabbit Alexa 594 (1:1000) was added per slide and incubated for 1 h at room temperature.
For double staining of RPE65 and viral EA in sections, slides were dried for 20 min at room temperature and washed for 30 min in
PBS, followed by permeabilization with 0.1% Triton X‐100 in 0.1%
sodium citrate for 2 min on ice. After blocking with PBS containing
10% normal goat serum, 2% BSA, and 0.5% Triton X‐100 for 1 h at room temperature, sections were incubated with rabbit anti‐RPE65 (1:800) and FITC labeled anti‐EA (1:500) antibodies overnight at 4oC.
After washing with PBS three times, sections were incubated with
antirabbit Alexa 594 (1:1000) for 1 h at room temperature. To visualize nuclei, all slides were mounted with an anti‐fade medium
containing 40, 6‐diamidino‐2‐phenylindole (DAPI; Vectashield, Vec-
tor Laboratories), and images were captured using a fluorescence microscope (Zeiss upright 780; Oberkochen, Germany).

⦁ | Statistical analysis

Data for plaque assay and quantified protein levels were presented as means ± SEM (standard error of the mean). Statistical significance was determined using either a 2‐tailed t‐test or analysis of var-
iance through the GraphPad Prism 8 software (GraphPad Software, Inc., San Diego, CA, USA). p Values < .05 represent a significant dif- ference. *p < .05, **p < .01, ***p < .001. ⦁ | RESULTS ⦁ | Time‐dependent regulation of autophagy upon MCMV infection It has been reported that autophagy could be induced immediately following HCMV infection, which is considered a beneficial strategy for virus survival.4 In our present study, the influence of MCMV infection on autophagy in cultured eyecup as well as MEFs was investigated FIGU RE 1 MCMV infection could induce autophagy and apoptosis in MEFs and eyecup culture. Protein levels of LC3I/II, SQSTM1/p62, and phospho‐p70 S6kinase (T389) in MEFs with mock infection (m) or MCMV (v) infection were measured by western blot (A) and quantified using Image J (B–D). Protein levels of LC3I/II, SQSTM1/p62, phospho‐p70 S6kinase (T389) in eyecups with different treatment were measured by western blot and further quantified (E–H). The cleaved caspase‐3 expression in MEFs with MCMV or mock control infection was detected by western blot (I) and was quantified with Image J (J). The cleaved caspase‐3 expression in eyecups was detected and quantified (K,L). TUNEL assay (Green), and immunofluorescence staining of cleaved caspase‐3 or phospho‐MLKL (Red) and nucleus (Blue) in MEFs infected with MCMV (MOI = 0.1) or mock (DMEM) medium at 48 hpi. (M). Bar graphs are the statistic results presented as mean ± SEM of three independently experiments (*p < .05; **p < .01; ***p < .001). DMEM, Dulbecco's modified Eagle's medium; hpi, hours post infection; MCMV, murine cytomegalovirus; MEFs, mouse embryo fibroblast cells; SEM, standard error of mean FIGU RE 2 Effect of 3‐MA or rapamycin treatment on MCMV replication in culture eyecups. (A) Plaque assay was performed to detect the virus titer in supernatant derived from MCMV infected eye cups with the treatment of 3‐MA (5 mM), or rapamycin (500 nM) at different time points. (B) Immunofluorescence staining of EA (Green), RPE (Red), and the nucleus (Blue) in eyecup frozen slides treated with 3‐MA (5 mM) or rapamycin (500 nM) at different time points. Quantification of EA expression in eyecup culture using Image J software (C). Bar graphs represent the statistic results of EA expression normalized to control; results presented as mean ± SEM of three independently experiments (*p < .05; **p < .01; ***p < .001). 3‐MA, 3‐methyladenine; EA, early antigen; MCMV, murine cytomegalovirus; RPE, retinal pigment epithelium; SEM, standard error of mean FIGU RE 3 (See caption on next page) firstly. Atg8/LC3 belongs to the ubiquitin‐like protein, which is the most widely used autophagy‐related protein. The two forms of LC3, including nonlipidated LC3‐I and lipidated LC3II are usually used as an excellent marker for autophagic structures.28 Therefore, the protein levels of LC3, SQSTM1/p62, an autophagic substrate located within autopha- gosomes, as well as phospho‐p70 S6kinase (T389), a downstream target of mTOR signaling, which is usually negatively correlated to autophagy, were quantified to testify the status of autophagy (Figure 1A). As shown in Figure 1B, LC3I/LC3II ratio was significantly decreased (LC3I decreased but LC3II increased relatively) at 24 hpi of MCMV in cul- tured MEFs, meanwhile, p62 and phospho‐p70 S6kinase protein levels also shown a decreased trend, while LC3I/II ratio was increased at 48 hpi (Figure 1C,D). These results indicated that autophagy was acti- vated at 24 hpi but inhibited at 48 hpi following MCMV infection in MEFs. In the cultured eyecup isolated from C57BL/6J mice, MCMV infection also increased LC3II expression at 4 and 7 dpi (Figure 1E), but the ration of LC3I/II was increased at 10 dpi and there were not any significant difference at 14 dpi (Figure 1E,F) between MCMV infected and mocks treated eyecups. MCMV infection did not have significant effect on the expression of p62 in cultured eyecups (Figures 1E and 1G). However, phospho‐p70 S6kinase levels were increasing with the pro- longed infection time (Figures 1E and 1H). Our data indicated that the autophagy was activated at the early stage following MCMV infection both in MEFs and eyecup culture, but the degradation of substrate p62 was incomplete in the eyecup. ⦁ | MCMV infection could induce caspase‐3 dependent cell death To determine whether MCMV infection could induce caspase‐3 de- pendent apoptosis, the cleaved caspase‐3 that was involved in both intrinsic and extrinsic apoptosis was tested by western blot. MCMV infection significantly increased cleaved caspase‐3 levels at 48 hpi but not at 24 hpi in MEFs (Figure 1I,J). Meanwhile, the cleaved caspase‐3 was also increased with MCMV infection in cultured eyecups, especially at 7 and 10 dpi (Figure 1K,L). To further in- vestigate the cell death caused by MCMV infection was apoptosis or necroptosis, double staining of cleaved caspase‐3 or the phos- phorylated mixed lineage kinase domain‐like protein (phospho‐ MLKL), which is a key substrate in the induction of necroptosis,29 and TUNEL assay were performed in MEFs. Results showed that there were some TUNEL positive cells stained with cleaved caspase‐3, although not all apoptotic cells overlapped with cleaved caspase‐3 (Figure 1M). The staining of TUNEL cells did not overlap with phospho‐MLKL staining, but phospho‐MLKL always appeared around TUNEL positive cells (Figure 1M). These results indicated that MCMV infection of MEFs can cause caspase‐3 dependent apoptosis and MLKL mediated necroptosis might also be involved in this process. ⦁ | Autophagy inhibition by 3‐MA restricts viral replication during MCMV infection In consistence with the notion that the MCMV infection, replication, and even latency are tissue or cell‐dependent because of the dif- ferent innate and/or adaptive immune environment (reviewed by Reddehase et al.30 our present study also showed that the tissue or cell‐specific effect of MCMV infection on autophagy and cell death pathways in cultured eyecup and MEFs (Figure 1). Furthermore, the eye is an immune‐privileged organ, although there are several stu- dies focused on the relationship between autophagy and CMV in- fection, it's still not known how autophagy involved in MCMV virus replication in the eye. In this study, 3‐MA, one of the most used autophagy inhibitors, was used to block autophagy following MCMV infection in eyecup culture. As shown in Figure 2A, THE virus titer was significantly decreased by the treatment of 3‐MA (p < .05) at 4, 7, 10, and 14 dpi. The interesting thing is that treatment with rapamycin slightly decreased the virus loading in eyecup culture at 4 and 7 dpi. To confirm the virus titer assay results, MCMV early antigen (EA) positive cells, which represent the active virus replication, were quantified in eyecups using immunofluorescence staining. As shown in Figure 2B, EA positively stained cells in the retinal pigment epi- thelium (RPE) or choroid layer was significantly decreased by the treatment of 3‐MA, which decreased 78.3% at 4 dpi, 88.3% at 7 dpi, 89.1% at 10 dpi and 52.8% at 14 dpi respectively compared with the mocks treated control group (Figure 2C). Meanwhile, the EA posi- tively stained cells in rapamycin‐treated eyecups were comparable with mocks treated control eyecup at 4 and 10 dpi, but significantly increased at 7 and 14 dpi (Figure 2C) with MCMV infection. In consistency with the staining of the slides, EA positively stained cells in the flat‐mount of eyecup was also significantly inhibited by 3‐MA FIGU RE 3 The effect of 3‐MA and rapamycin treatment on MCMV replication in flat‐mount of eyecups and cultured MEFs. (A) Immunofluorescence staining of EA (Green), RPE65 (Red), and DAPI (Blue) in the flat‐mount of MCMV infected eyecups with the treatment of mock (DMEM) medium, 3‐MA (5 mM), or rapamycin (500 nM). (B) Plaque assay was performed to detect the virus titer in supernatant derived from MCMV infected MEFs with the treatment of 3‐MA (5 mM) or rapamycin (500 nM) at different time points. (C) TUNEL assay (Green), immunofluorescence staining of EA (Red) and nucleus (Blue) in MEFs at 48 hpi treated with mock (DMEM) (C1) or with 3‐MA (C2) without MCMV infection, or with MCMV infection (MOI = 0.1) treated with mock (DMEM) (C3) or with 3‐MA (C4). The cellular morphology of mock (DMEM) medium or MCMV (MOI = 0.1) infected MEFs treated with mock (DMEM) medium (D1 and D3) or 3‐MA (D2 and D4) were photographed with the microscope at 48 hpi. 3‐MA, 3‐methyladenine; DMEM, Dulbecco's modified Eagle's medium; EA, early antigen; hpi, hours post infection; MCMV, murine cytomegalovirus; MEFs, mouse embryo fibroblast cells; RPE, retinal pigment epithelium FIGU RE 4 (See caption on next page) treatment but not by rapamycin treatment (Figure 3A). These results indicated that autophagy inhibitor, 3‐MA could suppress the virus replication and EA expression in eyecups following MCMV infection. To further confirm the inhibitory effect of 3‐MA on virus replica- tion, plaque assay was also performed to detect the virus replication in cultured MEFs following MCMV infection. Our results showed that 3‐MA treatment also significantly decreased the virus titer at 48, 72, and 96 h after MCMV infection in MEFs (p < .05), but there was no significant effect of rapamycin treatment on virus replication in MEFs (p > .05) (Figure 3B). In addition, there was less EA staining with the
presence of 3‐MA in MCMV infected MEFs compared with mock‐
infected MEFs (Figure 3C). These results further confirmed that 3‐MA, but not rapamycin could restrict MCMV infection.

⦁ | Inhibition of autophagy by 3‐MA promotes MCMV‐induced caspase‐3 dependent apoptosis

The relationship between autophagy and apoptosis is dependent on a different context,31 but in most instances, autophagy prefers to play an antiapoptotic but not proapoptotic role.32 As to MCMV infection, although it has been shown that autophagy could interact with apoptosis during MCMV infection of RPE cells,33 the relationship
was not fully elucidated. To explore whether cell death was involved
in the inhibition of MCMV replication by autophagy inhibitor 3‐MA, TUNEL assay was performed to quantify the role of 3‐MA on MCMV‐induced cell death. Firstly, to test the cytotoxicity of 3‐MA,
TUNEL assay was performed in MEFs. Results showed that it did have some TUNEL positive cells with 3‐MA treatment at 48 h (Figure 3C2) compared with DMEM treatment MEFs (Figure 3C1),
while there was little change in cell morphology and cell proliferation even at 48 h post treatment both with and without MCMV infection (Figure 3D2 and D4) compared with DMEM treatment cells
(Figure 3D1 and D3). Similarly, 3‐MA treated eyecups also have
some TUNEL positive cells (Figure 4A3), but rapamycin has no such effects (Figure 4A2), which were similar with DMEM treated eyecups
(Figure 4A1). However, there was no significant difference for the tissue structure and growth between the 3‐MA and mocks treated
eyecups. Therefore, though 3‐MA could promote apoptotic cell
death, it seems that there is no significant cytotoxicity of 3‐MA in MEFs and eyecups at this concentration (5 mM) and can be used in
this study.
As the effect of 3‐MA on MCMV infection induced cell death, there were more apoptotic cell death and less EA stating in MEFs with 3‐MA treatment (Figure 3C4) than without 3‐MA (Figure 3C3). TUNEL positive cells were also significantly increased by treatment of 3‐MA following MCMV infection in eyecup at 7 and 14 dpi, while
rapamycin treatment did not show any significant effects (Figure 4B‐
D). Western blot results showed that 3‐MA treatment only caused the bulk accumulation of LC3, especially LC3I in MEFs compared with the MCMV uninfected MEFs without 3‐MA treatment, though the ratio of LC3I/II was not significantly changed (Figure 5A lane 3 and 9,B). Meanwhile, 3‐MA treatment increased the protein levels of
phospho‐p70 S6 kinase and SQSTM1/p62 in MCMV infected MEFs
at 24 hpi (Figure 5A lane 4,B,C). SQSTM1/p62 was also increased at
48 hpi (Figures 5A lane 10 and 5C), as well as phospho‐mTOR with the presence of 3‐MA (Figures 5A lane 9 and 10 and 5E). In the cultured eyecups, 3‐MA treatment also increased the protein levels of phospho‐mTOR and p62, as well as LC3I/II ratio (Figure 5F lane 3, 7, 11, and 15, 5G‐I in MCMV infected eyecups. These results in- dicated that autophagy was inhibited by 3‐MA both in MEFs and eyecups. Rapamycin significantly reduced phospho‐p70 S6 kinase, phospho‐mTOR, and the LC3I/II ratio at 24 and 48 hpi in MEFs
(Figures 5A lane 5, 6, 11, and 12,B, and 5D,E). Also in eyecups culture, rapamycin treatment decreased the phospho‐mTOR,
SQSTM1/p62 LC3I/II ratio and phospho‐p70 S6 kinase and expres-
sion (Figure 5F lane 4,8,12, and 16,G‐J). All these results indicated that autophagy was induced in MEFs and eyecups by rapamycin
treatment.
Meanwhile, 3‐MA treatment significantly increased caspase‐3 cleavage in MCMV infected MEFs at 48 hpi (Figures 6A lane 10 to lane 8 and 5B) compared with MCMV infected MEFs without 3‐MA treatment, but at 24 hpi, it seems like that MCMV infection can convert 3‐MA induced increasing of caspase‐3 cleavage in MEFs
(Figure 6A lane 4 to lane 3,B). In MEFs, it seemed that 3‐MA has no
significant and consistent effects on necroptosis pathways. For ex- ample, phospho‐RIPK3 was reduced at 24 hpi but did not change a
lot at 48 hpi, while phospho‐MLKL were increased at 24 and 48 hpi
with the presence of 3‐MA (Figures 6A lane 3, 4 and 9, 10 and 6D,E). However, in MCMV infected eyecups, our results showed that the cleaved caspase‐3 and caspase‐8 were increased at 4, 7, 10, and 14 dpi (Figure 6F lane 3,6,9 and 12,G,H), while the expression of active RIPK1, phospho‐RIPK3, and MLKL were reduced in MCMV
eyecups treated with 3‐MA compared with MCMV infected eyecups

FIGU RE 4 TUNEL assay in MCMV infected eyecups with 3‐MA or rapamycin treatment. Eyecups infected with mock (DMEM) medium and treated with mock (DMEM) medium (A1), rapamycin (A2) or 3‐MA (A3) were used for TUNEL assay (Green), immunofluorescence staining of RPE65 (Red) and the nucleus (Blue) to test the cytotoxicity of rapamycin (500 nM) and 3‐MA (mM). Then eyecups infected with mock (DMEM) medium (B1) or MCMV (5 × 10^3 PFU) without 3‐MA treatment (B2) or with 3‐MA treatment (B3) were used for TUNEL assay (Green), immunofluorescence staining of EA (Red), and the nucleus (Blue). TUNEL assay was performed to test the apoptotic cell distribution in eyecup
treated with 3‐MA (5 mM) or rapamycin (500 nM) at 4, 7, 10, and 14 dpi. (C). Image J was used to analyze the percentage of positive staining with TUNEL (D). Bar graphs represent the statistic results of TUNEL positive cells normalized to control; results presented as mean ± SEM of three independently experiments (*p < .05; **p < .01; ***p < .001). 3‐MA, 3‐methyladenine; DMEM, Dulbecco's modified Eagle's medium; dpi, days post infection; EA, early antigen; MCMV, murine cytomegalovirus; RPE, retinal pigment epithelium; SEM, standard error of mean FIGU RE 5 3‐MA, rapamycin treatment, and autophagy in MEFs and eyecups. Western blot was performed to detect the protein levels of LC3I/II, SQSTM1/p62, phospho‐p70 S6kinase (T389), mTOR, and phospho‐mTOR with the treatment of 3‐MA or rapamycin in MCMV or mock (DMEM) infected MEFs (A), and the quantification was performed using Image J (B–E). Meanwhile, the expression of LC3I/II and SQSTM1/p62, phospho‐p70 S6kinase (T389), mTOR, p‐mTOR in eyecups (F) were also detected and quantified (G–J). Bar graphs represent the statistic results of protein levels normalized to control; results presented as mean ± SEM of three independently experiments (*p < .05; **p < .01; ***p < .001). 3‐MA, 3‐methyladenine; DMEM, Dulbecco's modified Eagle's medium; MCMV, murine cytomegalovirus; MEFs, mouse embryo fibroblast cells; mTOR, mammalian target of rapamycin; SEM, standard error of mean FIGU RE 6 (See caption on next page) only at 4,7, and 10 dpi (Figures 6F lane 3,6,9 and 12 and 6I‐K). However, it seems like that rapamycin has no such effects on caspase‐3 cleavage and RIPK1/RIPK3/MLKL expression both in MEFs and eyecups (Figures 6A‐G and 6I‐K). These results indicated that it is caspase‐3 dependent apoptosis but not RIPK1/RIPK3/MLKL dependent necroptosis was involved in 3‐MA mediated the suppression of MCMV replication in eyecups. However, in MCMV infected MEFs, 3‐MA mainly promotes MCMV‐induced caspase‐3 dependent apoptosis at 48 hpi. ⦁ | The role of another autophagy inhibitor CQ on MCMV replication As autophagy inhibitor 3‐MA could significantly restrict MCMV re- plication in MEFs and eyecups through regulating caspase‐3 depen- dent apoptosis, it interests us whether other autophagy inhibitor has such impacts. Therefore, another important autophagy inhibitor CQ, which inhibits autophagy via impairing autophagosome fusion with lysosomes,24 was used in this study. Our results showed that CQ treatment resulted in bulk LC3, especially LC3II accumulation in both MCMV infected and MCMV uninfected MEFs (Figure 7A,B), which indicated that CQ might also inhibit MCMV‐induced autophagy. CQ treatment also can promote caspase‐3 cleavage in MEFs (Figures 7A and 7E). The virus titer results showed that CQ not only could inhibit virus replication in MCMV infected MEFs at 24 and 48 hpi (Figure 7F), but also could inhibit MCMV replication in eyecups at 7 dpi (Figure 7G). Furthermore, it was observed that in CQ treated MEFs and eyecups infected with MCMV, there was less EA expres- sion but more TUNEL positive apoptotic cells compared with no CQ treated MEFs and eyecups (Figure 7H,I), which showed similar ef- fects with 3‐MA. However, it seems like that CQ treatment damaged the intact structure of eyecups (Figure 7I) and suppressed the pro- liferation of MEFs (Figure 7H), the mechanism needs to be explored further in the future. 4 | DISCUSSION As an important “self‐eating” system, autophagy can restrict viral infection through direct degradation of viral components by mod- ulating the intensity of the inflammatory response or by facilitating the processing of viral antigens for presentation by major histo- compatibility complex (MHC).34 However, multiple viruses can also utilize components of autophagy and the exosome machinery for the assembly of progeny virions and release of host proteins and RNAs that can affect pathogenesis.35‐37 Up to date, the interplay between MCMV replication and autophagy is still poorly understood. Previous studies using chemicals to investigate the impact of stimulation or inhibition of autophagy on HCMV replication have provided con- troversial results.8,9,38 On the one hand, there is a study shown that the induction of autophagy by HCMV infection impaired viral re- plication, and inhibition of autophagy by a virus expressing ATG4B- C74A enhances both viral DNA replication and progeny release,39 demonstrating the antiviral effect of autophagy and sup- porting the assumption that autophagy serves as a cellular defense against HCMV. For example, Trehalose, one of the autophagy in- ducer, induces changes in the cytoplasmic landscape and in the Rab family of regulatory proteins, limiting virus release from the cell and potentially redirecting virions to acidified, compartments in which they are degraded, while the antiviral activity of SMER28 appears to be independent of cellular trafficking pathways and interferes with the HCMV life cycle at an earlier point, reducing early protein ac- cumulation in both human foreskin fibroblasts (HFFs) and human aortic endothelial cells (HAECs) and delaying viral genome replica- tion in HAECs.9 On the other hand, activation of autophagy by ra- pamycin and methyl‐β‐cyclodextrin enhanced HCMV infectivity, whereas inhibition autophagy via Spautin 1 and ATG16L1 knockout decreased viral production, in which the pharmacological played si- milar effects on extra and intracellular viral yields, suggesting that autophagy does not influence the release of the virus.38 In addition, it has been demonstrated that HCMV can directly inhibit the formation and maturation of autophagosomes through the interaction of te- gument protein IRS1 and TRS1 with Beclin1, without affecting the synthesis of viral proteins,6,38 while the host could attenuate au- tophagy and viral replication in the early stage of HCMV infection through IL‐10.40 These studies suggest that different autophagy in- ducers or inhibitors might have various roles on viral replication, even they have the same effects on virus growth, it seems like the mechanism may be totally distinct. As to MCMV, it has been shown that autophagic vacuole accumulation was detected early during MCMV infection of RPE cells, which could protect retinal cells from MCMV infection‐induced apoptosis through mTOR‐mediated sig- naling pathway.33,41 In the present study, we found that there was less expression of early viral protein EA and less viral particles re- leased in the eyecups with 3‐MA and CQ treatment. However, at the same time, caspase‐3 dependent apoptosis was upregulated but RIPK1/RIPK3 mediated necroptosis was slightly downregulated in FIGU RE 6 The effect of 3‐MA and rapamycin treatment on cell death pathways in MEFs and eyecups culture infected with MCMV. The expression of cleaved caspase‐3, as well as RIPK1/RIPK3/MLKL pathway with 3‐MA or rapamycin treatment in MCMV or mock (DMEM), infected MEFs (A) were tested and were quantified with Image J respectively (B–E). Also, the cleaved caspase‐3, cleaved caspase‐8 as well as RIPK1/RIPK3/MLKL (F) in eyecups were detected via western blot and were quantified with Image J respectively (G–K). Bar graphs represent the statistic results of protein levels normalized to control; results presented as mean ± SEM of three independently experiments (*p < .05; **p < .01; ***p < .001). 3‐MA, 3‐methyladenine; DMEM, Dulbecco's modified Eagle's medium; MCMV, murine cytomegalovirus; MEFs, mouse embryo fibroblast cells; SEM, standard error of mean FIGU RE 7 (See caption on next page) eyecups. These results indicated that caspase‐3 dependent apoptosis was involved in 3‐MA caused inhibition of MCMV replication. Apoptosis, necroptosis, and pyroptosis are the three major ways of programed cell death (PCD) following virus infection.42 Apoptosis is an evolutionarily conserved process, which is the most extensively investigated PCD during viral infection. However, apoptosis elicited by virus infection has both negative and positive influences on viral replication. On the one hand, the host cells could use apoptosis to eliminate virally infected cells, which aborts virus infection. On the other hand, some viruses encode proteins that directly influence the function of core proteins controlling extrinsic or intrinsic apoptotic pathways.43,44 As to CMV, caspase‐dependent apoptosis has an im- portant role in controlling viruses. It has been demonstrated that CMV infection could induce extrinsic and intrinsic apoptosis, necroptosis, pyroptosis, and parthanatos during CMV retinitis.45‐47 Inhibition of apoptosis following CMV infection is mediated by a mitochondria‐localized inhibitor of apoptosis, vMIA, a viral inhibitor of caspase activation, vICA, the functional homologs of B‐cell lym- phoma 2 (Bcl‐2) and c‐FLIP proteins, as well as viral proteins in- cluding pUL38, IE1 491aa, and IE2 579aa, UL138, US21, which are encoded by MCMV.48‐52 The interplay between autophagy and apoptotic proteins is very complicated because autophagy can act both as a cell survival and cell death process.53 Although autophagy can induce cell death which is known as autophagic cell death, the main function of autophagy is to promote cell survival during normal tissue homeostasis. In general, cells initiate autophagy under stress as a pro‐survival strategy and block apoptosis. However, as stress continues beyond a threshold, cells initiate apoptotic cell death and block autophagy. It seems like that when to switch from the pro‐survival autophagic process to apoptotic state may depend on the level of stress and is regulated by mediator molecules involved in autophagy and apoptosis pathways.53,54 While the mechanisms mediating the complex counter‐regulation of apoptosis and autophagy are not yet fully un- derstood, important points of crosstalk include the interactions be- tween Beclin‐1 and Bcl‐2/Bcl‐xL and between fas‐associated protein with death domain (FADD) and Atg5, caspase‐ and calpain‐mediated cleavage of autophagy‐related proteins, and autophagic degradation of caspases.31,55 For example, inhibition or deficiency of caspase 8 resulted in excessive autophagy in fibroblasts, macrophages, and T cells,56 while autophagy itself also can counter‐balances the apoptotic response by the continuous sequestration of the large caspase‐8 subunit in autophagosomes and its subsequent elimination in lysosomes.57 The Atg7‐ caspase‐9 complex performs a dual func- tion of linking caspase‐9 to the autophagic process while keeping in check its apoptotic activity.58 Caspase‐3 could cleave Beclin‐1, which may contribute to inactivate autophagy leading towards augmented apoptosis.59 It has been demonstrated that inhibition of autophagy could promote cancer cell caspase‐dependent or caspase‐independent apoptosis,60‐62 which indicated that autophagy inhibitors as an ad- junct to chemotherapy for a variety of tumors were worth to be explored. 3‐MA, as the most widely used autophagy inhibitor, is a class III phosphatidylinositol 3‐kinase (PtdIns3K) inhibitor.63 There have been several studies shown that 5‐FU‐induced apoptosis in colon cancer cells and human skin squamous cell carcinoma can be enhanced by the inhibitor of autophagy, 3‐MA.64,65 Furthermore, there were studies that demonstrated that inhibition of autophagy by 3‐MA treatment could increase DDP, IR, 2‐DG, purvalanol, IL‐24, and hypoxia‐induced apoptosis in other cancer cells.66‐70 Interest- ingly, in our present study, we found that autophagy inhibitor, 3‐MA, not only restricted viral replication but also promoted caspase3‐ dependent apoptosis during MCMV infection in eyecups. Further- more, another autophagy inhibitor CQ also could limit MCMV re- plication in MEFs and eyecups in this study. These results posed us a challenge that whether it is inhibition of virus replication caused by autophagy inhibition that increased apoptosis or enhanced apoptosis caused by 3‐MA disrupt normal cell condition in the eyecup, which afterward restricted virus entry and reduced its replication. As we discussed above, inhibition of autophagy by 3‐MA both could control virus replication directly and enhance apoptosis. Therefore, we speculate that this depends on the infectious time. At 4 and 7 dpi of MCMV‐infected eyecups, even when caspase‐3 was not activated, virus replication had been suppressed, which suggested that during this period of MCMV infection, it was autophagy inhibition resulted in the reduced virus growth. However, when caspase‐3 dependent apoptosis was significantly activated at 10 and 14 dpi, it was more likely that the increased caspase‐3 dependent apoptosis caused by autophagy inhibition led to the interruption of virus replication. In conclusion, this study shows that autophagy inhibition by 3‐MA could both restrict virus replication and promote caspase‐3 dependent apoptosis in the eyecup culture following MCMV FIGU RE 7 The effect of another autophagy inhibitor chloroquine (CQ) on MCMV replication. MEFs infected with mock (DMEM) medium or MCMV were treated with 3‐MA or CQ. LC3I/II and SQSTM1/p62, phospho‐p70 S6kinase (T389), and cleaved caspase‐3 were detected via western blot (A). (B–E) The quantification of the protein levels. Plaque assay was used to detect the virus titer of supernatant derived from MCMV infected MEFs with the treatment of mock (DMEM) medium, rapamycin (500 nM), 3‐MA (5 mM) or CQ (100 µM) (F) at 48 hpi, or derived from MCMV infected eyecups with the same treatments as MEFs at 7 dpi (G). TUNEL assay (Green), immunofluorescence staining of EA (Red) and nucleus (Blue) at 48 h post‐MCMV or mock‐infected MEFs (H), or 7 days post‐MCMV or mock‐infected eye cups (I up panel). (I down panel) TUNEL assay (Green), immunofluorescence staining of RPE (Red) and nucleus (Blue) at 7 days post‐MCMV or mock‐infected eye cups. Bar graphs are the statistic results presented as mean ± SEM of three independently experiments (*p < .05; **p < .01; ***p < .001). 3‐MA, 3‐methyladenine; DMEM, Dulbecco's modified Eagle's medium; dpi, days post infection; EA, early antigen; hpi, hours post infection; MCMV, murine cytomegalovirus; MEFs, mouse embryo fibroblast cells; RPE, retinal pigment epithelium; SEM, standard error of mean infection. At the early stage of MCMV infection suppressed autop- hagy process directly reduced virus release, while at a later stage it was caspase‐3 dependent apoptosis that caused decreased virus replication. However, there are still lots of questions that need to be answered by future studies, such as how the virus in the eyecup was inhibited by autophagy and how apoptosis was regulated by autop- hagy during MCMV infection, which might provide a useful target for the treatment of CMV infectious diseases. ACKNOWLEDGMENT This study was funded by a grant from the National Natural Science Foundation of China, grant number 81271807. CONFLICT OF INTERESTS The authors declare that there are no conflict of interests. AUTHOR CONTRIBUTIONS Xinyan Zhang and Feng Fang conceived and designed the experi- ments. Linlin Zhang, Ting Xi, Yidan Bi, and Zhan Zhang performed the experiments. Yuan Huang, Yuanyuan Lu, and Xinglou Liu helped to analyze the data. Xinyan Zhang wrote the manuscript. All authors read and approved the final manuscript. DATA AVAILABILITY STATEMENT The data that support the findings of this study are available on request from the corresponding author. ORCID Xinyan Zhang http://orcid.org/0000-0002-5717-2714 Feng Fang http://orcid.org/0000-0002-4468-6206 REFERENCES ⦁ Deretic V, Saitoh T, Akira S. Autophagy in infection, inflammation and immunity. Nat Rev Immunol. 2013;13(10):722‐737. ⦁ Deretic V. Autophagy in infection. 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