Time-dependent regulation of autophagy upon MCMV infection
It has been reported that autophagy could be induced by CMV infection at very early period, which is beneficial to virus survival. Here, theinfluence of MCMV infection on autophagy in culturedeyecup as well as MEFs was investigated. Atg8/LC3 is the most widely used autophagy-related protein, belonging to a ubiquitin-like protein. The two forms of LC3 including nonlipidated and lipidated are usually referred to respectively as LC3-I and LC3-II, which are often an excellent marker for autophagic structures[17]. In our present study, the protein levels of LC3, SQSTM1/p62, an autophagic substrate located within autophagosomes,and phosphor-p70 S6kinase (T389), a downstream target of mammalian target of rapamycin(mTOR) signaling, were quantified to testify the autophagy levels. As shown in Figure 1A,LC3I/LC3II ratio was decreased (LC3I decreased but LC3II increased relatively) at 24, 72 and 96 hours post infection(hpi) of MCMVin cultured MEFs(Lane 2,6,8), meanwhile p62 and phosphor-p70 S6kinase were also reduced slightly (Lane 2,6,8), which indicated that MCMV infection activates autophagy during MCMV infection in MEFs.In the eyecup culture of C57BL/6J mice, MCMV infection also increasedLC3II expression at 4 and 7days post infection(dpi.) (Figure 1B lane 2,4). However, there’sno significantly difference between MCMV infection and mocks treated eyecups at 10 and 14 dpi(Figure 1B lane 6 and 8) with MCMV infection. Also, the expression of p62 didn’t change a lot with MCMV infection (Figure 1B lane 2,4,6 and 8).In addition, phosphor-p70 S6kinase expression was increasing over time (Figure 1B lane 2,4,6, and 8). These results indicated thatthe autophagy was activated early after MCMV infection both in MEFs and eyecup culture, while without the degradation of substrate p62 completely in the eyecup.
Tissue/cell specific effect of MCMV infection on cell death
In order to determine whether MCMV infection could induce caspase-3 dependent apoptosis or necroptosis, related proteins were detected via western blot. Results show that the cleaved caspase-3 increased over time, but MCMV infection down regulated cleaved caspase-3 protein levels at all time points detected in MEFs (Figure 1A lane 2,4,6, and 8). Interestingly, in eyecup culture the cleaved caspase-3 was increased with MCMV infection, especially at 10 dpi(Figure 1C lane 2,4,6, and 8). The necroptosis related protein levels of active RIPK1, pRIPK3(s232), and pMLKL (s345) were slightly increased at 24 and 48hpi in MEFs (Figure 5B lane 2 and 8). Similarly, in cultured eyecup MCMV infection also increased the protein levels of active RIPK1, RIPK3 and MLKL, to some extent, at 4,7,10 and 14 dpi (Figure 1C lane 2,4,6, and 8). These results suggested that caspase-3 dependent apoptosis and RIPK1/RIPK3/MLKL dependent necroptosis were induced by MCMV infection in eyecup, while in MEFs infected with MCMV, caspase-3 dependent apoptosis was inhibited and RIPK1/RIPK3/MLKL dependent necroptosis was activated.
Autophagy inhibition by 3-MA restricted viral replication upon MCMV infection
Consistent with the notion that the MCMV infection, replication and even latency are tissue or cell dependent because of the different innate and/or adaptive immune environment (reviewed by [18], our present study show that the tissue-specific effect of MCMV infection on autophagy and cell death pathway. Furthermore, the eye is an immune privileged organ, although there areseveral studies focused on the relationship between autophagy and CMV infection, 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 inhibitor, was used to block autophagy induced by MCMV infection in MEFs and eyecup culture. Plaque assay show that 3-MA treatment significantly decreased active virus particle releasing at 48,72 and 96 hours after MCMV infection in MEFs(p<0.05), but there’s no significant effect of rapamycin treatment on virus replication in MEFs(p>0.05) (Figure 1D). Meanwhile, in the eyecup culture, the virus releasing was also decreased significantly after treatment with 3-MA(p<0.05) at4, 7, 10 and 14dpi(Figure 1E). However, treatment with rapamycin just slightly decreased the virus releasing in eyecup cultureat 4 and 7 dpi (Figure 1E).
To confirm the virus titer assay results, MCMV early antigen(EA), indicating the active virus replication within MCMV-infected eyes,was detected in eyecup using immunofluorescence staining.As show in Figure 2A, EA positive stained cells in the retinal pigment epithelium(RPE) or choroid layer treated with 3-MA were significantly reduced, which decreased 78.3% at 4dpi, 88.3% at 7dpi, 89.1% at 10dpi and 52.8% at 14dpi compared with MCMV infection only(Figure 2B).While the EA positive stained cells in rapamycin treated eyecups are comparable with control eyecup at 4 and 10dpi and even increased at 7 and 14dpi(Figure 2B) during MCMV infection. Consistent with the slides staining, EA positively stained cells in the flatmount ofeyecup was inhibited significantly by 3-MA treatment but not by rapamycintreatment (Figure 3). These results indicated that autophagy inhibitor, 3-MA could suppress the production of virus particle and EA expression in eyecup with MCMV infection.
Inhibitory effect of 3-MA suppressionon MCMV replication might through caspase-3 dependent apoptosis
The interrelationship between autophagy and apoptosis is depended on different context [19]. It is well known that the autophagy could mediate cell death, no matter within or beyond the same cell. In most instances, autophagy tends to play an anti-apoptotic but not pro-apoptotic role[20]. As to MCMV infection, although it’s not well investigated, autophagy interacted with apoptosis during MCMV infection of RPE cells[21]. To explore the mechanism of inhibition of MCMV replication by autophagy inhibitor 3-MA, TUNEL assay was used to detect that whether cell death was involved in this process. To our unexpected, TUNEL positive cells were significantly increased with treatment of 3-MA under MCMV infection in eyecup especially at 7 and 14 dpi, while rapamycin treatment did notshow any significantlyeffects (Figure 4A,B). ThenWestern blot was used to determine which mode of cell death plays a leading role, and results showed that when treated with 3-MA, LC3I and p62 expression increased in MEFs (Figure 5A lane 3 and 9), also mTOR and pmTOR, as well as LC3I and p62 increased in eyecup(Figure 6A lane 3 and 6), indicated that autophagy was inhibited. When treated with rapamycin, LC3I, p62, phosphor-p70 s6 kinase were decreased and LC3II increased in MEFs (Figure 5A lane 5 and 11), also mTOR and pmTORwere reduced in eyecup(Figure 6A lane 4,8,12,and 16), indicated that autophagy was induced.
Cleaved caspase-3 significantly increasedwith 3-MA treatment, while RIPK1/RIPK3/MLKL pathway was inhibited in both MEFs(Figure 5B lane 4 and 10) and eyecup (Figure 6B lane 3,6,9 and 12), implied that it is caspase-3 dependent apoptosis but not RIPK1/RIPK3/MLKL dependent necroptosis was involved in 3-MA and autophagy mediated the suppression of MCMV replication in eyecup culture and MEFs.