Hydrogen sulfide inhibits human T‐cell leukemia virus type‐1 (HTLV‐1) protein expression via regulation of ATG4B

Hydrogen sulfide (H2S) is a redox gasotransmitter. It has been shown that H2S has a key role in host antiviral defense by inhibiting interleukin production and S‐sulfhydrating Keap1 lead to Nrf2/ARE pathway activation. However, it is yet unclear whether H2S can play an antiviral role by regulating autophagy. In this study, we found that exogenous H2S decreased the expression of human T‐cell leukemia virus type‐1 (HTLV‐1) protein and HTLV‐1 induced autophagosomes accumulation. Transmission electron microscope assays indicated that autophagosomes accumulation decreased after H2S administration. HTLV‐1‐transformed T‐cell lines had a high level of CSE (H2S endogenous enzyme) which could be induced in Hela by HTLV‐1 infection. Immunoblot demonstrated that overexpression of CSE inhibited HTLV‐1 protein expression and autophagy. And we got the opposite after CSE knockdown. Meanwhile, H2S could not restrain the autophagy when ATG4B had a mutant at its site of 89. In a word, these results suggested that H2S modulated HTLV‐1 protein expression via ATG4B. Therefore, our findings suggested a new mechanism by which H2S defended against virus infection.

151, and then Nrf2 translocates into the nucleus and combine with antioxidant response element (ARE). 25,26 This results in the expression of antioxidant genes.
Autophagy is a degradation process that transmits cytoplasmic components to lysosomes via autophagosomes. 27,28 Lipidation of LC3 is important for early stage of autophagy membrane formation. 29,30 ATG4, a kind of cysteine protease, perform as executer to process LC3 lipidation and delipidation of lipidated LC3. 31,32 There are four ATG4 isoforms in mammal cells, such as ATG4A, ATG4B, ATG4C, and ATG4D. 33 ATG4B is the key isoform for cleaving LC3/GABARAP. 34 ATG4A has the ability of processing GABARAP. Compared with ATG4B, the activity of ATG4A is weak. 35 However, ATG4D and ATG4C have almost no activity in vitro. 34,36 Adult T-cell leukemia (ATL), a hematologic malignant disease of adult, is directly related to the infection of human T-cell leukemia virus type 1 (HTLV-1). [37][38][39] It has a variety of symptoms like acute type, chronic type, or insidious type. 40,41 Chronic and insidious patients are often supported by symptomatic support treatment; Chemical and biological therapy are often used to cure acute lymphoma. However, the effect is unsatisfactory, and the median survival of the acute patients is about 2-6 months. 40,[42][43][44] Tax, HTLV-1 protein, can continuously activate autophagy and generate a lot of autophagosomes. And then, HTLV-1 is wrapped in the autophagosomes. HTLV-1 in autophagosomes not only can get away from intracellular immunity but also complete self-replication in autophagosomes. 45,46 The massive virus promoted the expression of Tax. Lots of Tax continue to activate autophagy and advance virus replication via positive feedback, resulting in the generation and development of ATL finally. 47 In this study, we demonstrated that CSE was induced by HTLV-1 infection and suppressed HTLV-1 protein expression. We showed that exogenous H 2 S inhibited the accumulation of autophagosomes during HTLV-1 infection. Furthermore, CSE and exogenous H 2 S were associated with ATG4B which is essential for autophagy early period.
Collectively, our findings may shed some new lights on HTLV-1 therapy and contribute to our understanding of H 2 S defenses against virus infection.

| Real-time polymerase chain reaction (PCR)
Total RNA was extracted from the cultured cells with TRIzol reagent (Invitrogen) as described by the manufacturer. All gene transcripts were quantified by real-time PCR with the ABI 7500 Fast real-time PCR system (Applied Biosystems). The results were analyzed using HT7800/HT7700) and take images.

| Coculture
Hela cells were cultured for 24 h. MT2 cells were seeded and cocultured with Hela cells at a ratio of 1:100 (1 × 10 4 HTLV-1 donor cells: 1 × 10 6 uninfected recipient cells ratio) and allowed to incubate for an additional 24 h before harvesting for downstream assays.

| Statistical analysis
The data are presented as the mean ± standard error from at least three independent experiments. All p-values were calculated using two-tailed Student's t tests (Graphpad) and were considered statistically no significant when p > 0.05.

| RESULTS
3.1 | H 2 S treatment suppressed HTLV1 replication, reduced autophagy level, and had no effect on cells' viability (upper line) and LC3B (below line). LC3B is a marker of autophagy. 51 The expression of LC3B in MT2 reduced after H 2 S administration for 48 h (Figure 1A), and virus protein Tax and p19 also decreased.
We saw the same results in MT4 ( Figure 1B) and C8166 ( Figure 1C). To determine whether H 2 S had an effect on cell activity, MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed. We found that H 2 S had no effect on cell viability after NaHS administration for 2 days, and the cells viability increased after H 2 S administration for 3 days (Figure 1D-F). Meanwhile, the level of Tax gene expression was inhibited by H 2 S significantly ( Figure 1G-I).
Consistently, the accumulation of autophagosomes was decreased in  And the effect of H 2 S was not associated with the decrease of cell viability.

| The inhibition of virus replication by H 2 S was related with autophagy
We determined the role of H 2 S in MT2 cells in the presence of RA (rapamycin) or 3-MA (3-methyladenine), which were believed to activate or block the autophagy. We found that RA could reduce the effect of H 2 S, and 3-MA could enhance the effect of H 2 S (Figure 2A).
Similar results were observed in MT4 and C8166 ( Figure 2B,C). It meant that H 2 S depressed the expression HTLV-1 related protein via autophagy.

| H 2 S interacted with ATG4B
To characterize the mechanism by which H 2 S regulated autophagosome accumulation, we tested whether H 2 S was able to interact with ATG4B involved in canonical autophagy. ATG4B is a conservative cysteine proteinase among species, 52 which activity dependents on the cysteine located in the active center. 53 ATG4B has 12 cysteines according to NCBI database ( Figure 3). It has been reported that ATG4B has no activity if the cysteine at site 74 is mutated. 54 The plasmid of ATG4B and its cysteine mutants were transfected in Hela cells. To confirm the effect of these mutants on ATG4B activity, Hela cells were treated by RA or 3-MA and then subjected to immunoblot analysis. The results showed that RA and 3-MA could active or depress autophagy when ATG4B had these cysteine mutated ( Figure 4A,B). It suggested that these cysteine were not essential for ATG4B activity. However, H 2 S had lose its effect on autophagy when the cysteine at site 89 was mutated ( Figure 4C).
These data indicated that cysteine 89 of ATG4B was the target of H 2 S in the regulation of autophagy.  Figure 5D).

| CSE expression increased in early period of HTLV-1 infection
And then, we detected the effect of exogenous H 2 S on Hela cells.
The result showed that exogenous H 2 S had no effect on the expression of endogenous enzymes ( Figure 5E). In summary, the expression of CSE decreased the level of autophagy of HTLV-1 positive cells.

| CSE could inhibit HTLV-1 replication via ATG4B
Next, we wanted to make sure whether CSE had the same effect like  the effect of CSE. We found that RA could reduce the effect of CSE, and 3-MA could enhanced the effect of CSE ( Figure 6C,D). To confirm that ATG4B was associated with the effect of CSE. Hela cells, overexpressing of CSE and ATG4B mutant, were cocultured with MT2 cells. Immunoblot assays showed that the level of HTLV-1 protein was restored partly ( Figure 6E). Taken together, these data suggested that the expression of CSE decreased HTLV-1 protein via the cysteine 89 of ATG4B.

| DISCUSSION
As a gasotransmitter, H 2 S plays an important role in the physiological and pathological process of the organism. [16][17][18][19] Nowadays, the mechanism of H 2 S in the process of virus infection is unclear. It has been reported that H 2 S could play an antiviral role through its antioxidant and anti-inflammatory effects, such as COVID-9, HSV, HIV, and paramyxovirus. [56][57][58][59][60][61] Our study found that both endogenous and exogenous H 2 S could inhibit autophagy caused by HTLV-1 and There is a complex relationships between autophagy and pathogenic microorganisms. 62 Paradoxically, autophagosomes are benefit for HTLV-1 to complete its replication. 45 How could H 2 S regulate autophagosomes accumulation? It has been reported that ATG4B has an important role in LC3B and autophagosomes formation. 34 As a cysteine enzyme, ATG4B is a good target for H 2 S regulation. Our findings indicated that H 2 S interacted with ATG4B. Using a serious of ATG4B cysteine mutants, we found ATG4B cysteine 89 was essential for the effect of H 2 S.
However, ATG4B will almost lost the ability when it has a mutant at the site of cysteine 74. 63 Therefore, we could not confirm whether the cysteine 74 was associated with H 2 S function.
It is reported that the function of ATG4B can be changed via posttranslational modification. For example, its activity is enhanced when the serine at 383 site was phosphorylated 64 ; After phosphorylation of serine at 34 and 316 sites or S-nitrosation of cysteine at 189 and 292 sites, the opposite results will appear [65][66][67] ; In addition, when the level of reactive oxygen species increase, a disulfide bond will be formed between cysteines at site 292 and 361 resulting the level of autophagy increases. 68 Our study suggested that H 2 S could sulfurize cysteine at site 89 and inhibit autophagy. However, we had no direct evidence to confirm the sulfhydration of ATG4B because of technical limitations.