Dengue virus-2 infection hinders autophagosome–lysosome fusion and autophagy–lysosomal degradation pathway in human umbilical vein endothelial cells

doi:10.21203/rs.3.rs-1592405/v1

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Dengue virus-2 infection hinders autophagosome–lysosome fusion and autophagy–lysosomal degradation pathway in human umbilical vein endothelial cells

https://doi.org/10.21203/rs.3.rs-1592405/v1

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Autophagy plays a significant role in antiviral responses. Studies have shown that dengue virus-2 (DENV-2) activates autophagy, causing the endothelial cell injury. However, it remains unclear whether this injury is linked to the whole process of autophagy. This paper aimed to investigate the effects of DENV-2 on autophagic vesicle formation, autophagosome–lysosome fusion, and lysosomal degradation pathway in human umbilical vein endothelial cells (HUVECs). It is found that DENV-2 infection caused the formation of autophagic vesicles and increased the LC3 expression. The expression level of p62, STX17, SNAP29, and VAMP8 increased significantly in the late DENV-2 infection. Additionally, a high number of brighter lysosomal red fluorescent spots in the cytoplasm were observed after DENV-2 infection. In conclusion, DENV-2 infection activated autophagy and induced the formation of autophagic vesicles, but hindered the autophagosome–lysosome fusion and damaged the normal lysosomal acidification, thus blocking the autophagic flux.

Dengue virus-2

Autophagic flux

Autophagosome–lysosome fusion

Lysosomal alkalinization

Dengue virus (DENV) is a positive-sense single-stranded RNA virus belonging to the genus Flavivirus of the Flaviviridae, encoding three structural proteins (core protein C, membrane protein M, and envelope protein E) and seven non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). It is transmitted by vectors such as Aedes albopictus and Aedes aegypti, and has become one of the most extensively distributed and morbidity-causing viruses globally (Wu et al., 2021). Approximately 390 million people worldwide are infected with DENV each year, and about 12,500 of them die due to inadequate treatment (Guzman et al., 2010). Moreover, it is endemic to tropical and subtropical regions of the world. In recent years, DENV outbreaks have been more frequent; large-scale DENV infection occurred in Senegal and Brazil in 2018 and 2019. Hainan, China, which had zero cases in nearly 20 years, reported its first outbreak in 2019, and the epidemic lasted for more than one month. In 2020, the first local DENV infection case was found in Italy (Gaye et al., 2021; Adelino et al., 2021; Lazzarini et al., 2020; Du et al., 2021). DENV infections are gradually increasing in size and scope, and the lack of specific therapeutic drugs poses a major threat to the world public health.

Autophagy, one of the intracellular material degradation pathways, comprises three main processes: autophagic vesicle formation, autophagic vesicle–lysosome fusion, and degradation of autophagic substrates within lysosomes. Autophagy, thus, plays an essential role in maintaining the intracellular environmental homeostasis (Godbole et al., 2020). A smooth autophagic flux shows autophagy activation, a normal fusion of mature autophagosomes and lysosomes, as well as normal lysosomal function. The damaged intracellular organelles, proteins, and other substances are degraded within lysosomes, thus enabling the reuse of intracellular substances. However, with a blocked autophagic flux, autophagic substrates cannot be degraded, and the substances fail to be recycled owing to the failure of autophagosome–lysosome fusion or abnormal lysosomal acidification even if autophagosomes are formed. The continuous formation of autophagic vesicles results in a large accumulation of intracellular autophagic substrates, eventually leading to cell death (Zhao et al., 2021). The soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) is essential in the autophagosome–lysosome fusion; the indispensable autophagosome–lysosome fusion protein in the SNARE family of proteins syntaxin 17 (STX17), synaptosome-associated protein 29 (SNAP29), and vesicle-associated membrane protein 8 (VAMP8) are closely linked during fusion (Tian et al., 2020b). First, STX17 is recruited to the mature autophagosome membrane, which is the first key step of fusion. Then, the SNARE domain of STX17 interacts with SNAP29 and VAMP8, forming the STX17–SNAP29–VAMP8 complex to promote the autophagosome–lysosome fusion; knockdown or deletion of one of the three genes in the complex will hinder the autophagosome–lysosome fusion, thereby blocking the autophagic flux (Tian et al., 2020a; Diao et al., 2015). The normal lysosomal function is crucial to the autophagic lysosomal degradation pathway; normally acidified lysosomes are an important guarantee for hydrolases to hydrolyze the substrate encapsulated in autophagy, making the material recycled. Once the acidic environment within the lysosome is altered, the autophagic flux is blocked, causing the damaged organelles, proteins, and pathogens to accumulate within the cell, threatening the cell survival (Laura Rigon 2021; Zheng et al., 2020).

Autophagy plays a dual role during the viral infection, both inhibiting viral replication and promoting viral proliferation in different environments. For example, Herpes simplex virus type 1 (HSV-1) inhibits autophagy to promote its replication; poliovirus, coxsackievirus B3 (CVB3), enterovirus 68 (EVD68) positive-sense RNA viruses undergo RNA replication by anchoring to autophagosome membranes (Khabir et al., 2020; Corona et al., 2018; Choi et al., 2018). Many studies have demonstrated that DENV induces autophagy onset and that the autophagic vesicles provide a favorable site for viral replication. Lee et al. (2008) reported a significant increase in LC3-II expression and autophagy onset when DENV infected Huh7 cells for 36 h. However, autophagy was inhibited when DENV proliferated heavily. The authors did not elucidate the effects on the autophagosome–lysosome fusion and lysosomal degradation pathway. DENV infection damaged vascular endothelium and caused autophagy-related plasma leakage. When autophagy-related gene ATG5 was silenced, the symptoms of plasma leakage induced by recombinant DENV NS1 in mice were relieved (Chen et al., 2016). Therefore, considering the close relationship between DENV infection and autophagy, and the critical role the vascular endothelium plays in the pathogenesis of DENV infection, there are few studies on DENV that take endothelial cells as the research object. Similarly, DENV infection activates autophagy to form autophagic bodies, but there are few studies on the effect of DENV on the autophagic flux. This study takes human umbilical vein endothelial cells (HUVECs) as the research object to explore the effect of DENV-2 on the autophagic pathway. We also want to reveal the mechanism of DENV-2 on the autophagic flux at protein and cell morphology levels to provide a theoretical basis for further study of the pathogenic mechanism of DENV at the autophagic level and the antivirus response of autophagy.

2.1. Cells, viruses, and reagents

C6/36 (preserved in laboratory liquid nitrogen) used to amplify the virus and the experimental model HUVECs (ScienCell, Catalog #8000) were cultured separately in RPMI-1640 or endothelial cell medium (ECM) containing 10% fetal bovine serum (FBS, Gibco, America; ScienCell, America). The DENV-2 NGC strain was preserved in liquid nitrogen in laboratory.

2.2. DENV-2 infection

The HUVECs cells (5 × 10⁴ /well) were grown in six-well plates, incubated with DENV-2 (1x10⁵ TCID50) at 37°C and 5% CO2 for 2 h, shaking every 30 min to homogenize the venom; the supernatant was discarded, washed once with phosphate buffered saline (PBS), and sufficient culture medium was added to continue the incubation.

2.3. Western blotting

Total proteins were extracted from cells using RIPA lysis buffer (Beyotime, China) and were determined using the BCA protein kit. The proteins were electrophoresed by 15% sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidine difluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany). The blots were blocked in 5% skim milk for 2 h and incubated with primary antibodies LC3 (1:1000, CST, America), p62 (1:1000, MBL, Japan), STX17 (1:1000, GeneTex, America), SNAP29 (1:3000, Abcam, U.K), VAMP8 (1:5000, Abcam, U.K), β-actin (1:50,000, Abclonal, China) overnight at 4°C, incubated with secondary antibody for 2 h (BIOPRIMACY, China), and developed using Western Lightning ECL (NCM Biotech, China).

2.4. Lysotracker red staining

Lysotracker red working solution at a final concentration of 75 nM was prepared in advance. Cells were collected from each treatment group, washed twice with precooled PBS, and the residual PBS was completely aspirated. Further, the cells were covered by Lysotracker red working solution and incubated for 1 h at 37°C. Finally, the cells in a fresh medium were observed under an inverted fluorescent microscope.

2.5. Statistical analysis

Graphpad 8.0.1 was used to analyze and graph the experimental data. One-way analysis of variance (ANOVA) was used for comparison between groups, and Student’s t-test was used for comparison between two groups. The two-way ANOVA analysis was used for multi-group measures with normal distribution and chi-square. P < 0.05 indicated that the difference was statistically significant.

3.1. DENV-2 infection promotes cellular autophagic vesicle formation

Currently, transmission electron microscopy (TEM) is the gold standard method for detecting autophagic vesicles. Autophagic vesicle morphology exhibited a bilayer or monolayer membrane structure of undigested organelles or proteins encapsulated within the membrane (Klionsky et al., 2021). According to ultrastructural results, the double-layered membrane-encapsulated undigested organelles were formed in the cytoplasm after DENV-2 infection for 36 h (Fig. 1), indicating that DENV-2 infection induced the formation of autophagic vesicles.

3.2. DENV-2 infection changes autophagy-related proteins in HUVECs

Western blotting was used to detect the expression level of LC3 to elucidate that DENV-2 activation of autophagy induces the formation of autophagic vesicles. LC3, a signature autophagic protein, is converted from LC3-I to LC3-II and localized to the autophagic vesicle membrane after autophagy activation. LC3-I is continuously converted to LC3-II, and increased LC3-II protein levels imply an increase in the number of autophagic vesicles (Klionsky et al., 2016). The results showed that LC3-II in the rapid assessment of physical activity (RAPA)-positive control and DENV-2-infected groups increased continuously from 12 h, and the LC3-II levels were higher than those in the blank group at four time-points of 12 h, 24 h, 36 h and 48 h (P < 0.05), and the difference was not statistically significant at 60 h (P > 0.05).

The ultrastructure of autophagic vesicles and the increased levels of LC3-II protein demonstrated that DENV-2 infection of HUVECs activated autophagy and induced the autophagosome formation.

3.3. DENV-2 infection blocks autophagic flux in HUVECs

The autophagy integrity process mainly includes autophagic vesicle formation, autophagic vesicle–lysosome fusion, and intra-lysosomal autophagic substrate degradation pathways. The smooth autophagic flux is primarily connected with the smooth autophagosome–lysosome fusion and the degradation of autophagic substrates within the lysosome. p62/SQSTM1, as an autophagy-selective substrate, enters the autophagosome through interaction with the LC3 Interacting Region (LIR) structural domain and is subsequently degraded within the lysosome, so the degradation level of p62 reflects the level of clearance of autophagic vesicles and whether the autophagic flux is unobstructed (Yla-Anttila., 2021). Western blotting was used to detect changes in p62 protein expression levels at different times of DENV-2 infection at 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 18 h, 24 h, 30 h, 36 h, and 48 h to investigate the effect of DENV-2 infection on the autophagic flux. The autophagy late inhibitor chloroquine (CQ) was treated for 24 h, 36 h, and 48 h, and the LC3-II protein expression level changed. The results showed that the differences in p62 protein expression levels were not statistically significant (P > 0.05) or not significant compared with the blank group before 30 h of infection, and were higher than the blank group after 30 h (P < 0.05). Totally, the level of p62 started to increase after 4 h of infection and was at a relatively stable level until 24 h, but increased further from 30 h, reached a peak at 36 h, and then decreased. As an autophagic inhibitor, CQ can alter the acidic environment inside the lysosomes and increase the pH of the lysosomes, thus affecting the degradation of autophagosomes and causing an increase in LC3-II (Harhaji-Trajkovic et al., 2012). LC3-II levels were higher in the infected and/or combined CQ-treated groups than in the blank group at all time points of late infection (P < 0.05), while LC3-II levels were similar in the infected and CQ-treated groups. The above results indicated that the impairment of autophagic substrate degradation was not obvious in the early and middle stages of DENV-2 infection, the level of autophagic substrate degradation decreased in the late stage of infection, and p62 accumulated continuously. Furthermore, DENV-2 reduced the autophagic vesicle degradation, decreased the autophagic lysosome synthesis, and blocked the autophagic flux in the late stage of autophagy.

3.4. DENV-2 blocks autophagic vesicle–lysosome fusion in HUVECs

The smooth autophagic flux is a crucial stage of the complete autophagic response. The fusion process of autophagic vesicles and lysosomes is the first step in determining the smoothness of the autophagic flux, with the STX17–SNAP29–VAMP8 complex playing a key role in the autophagic vesicle–lysosome fusion process; reduction of any one protein will block the fusion (Li et al., 2020). To further investigate the process of DENV-2 blocking the late autophagic flux and inhibiting cellular autophagic degradation, Western blotting was used to detect the expression levels of STX17, SNAP29, and VAMP8, the key proteins of fusion at 24 h, 30 h, 36 h, and 48 h of DENV-2 infection. The results indicated that the levels of STX17, SNAP29, and VAMP8 in the infected group showed an increasing trend at first then decreased with time, but compared with the blank group, the levels of STX17 were relatively lower at all time points of infection; at 24 h of infection, the levels of SNAP29 and VAMP8 were higher than those of the blank group, but then they were lower than those of the blank group (P < 0.05). This indicated that DENV-2 infection reduced the levels of key proteins for the late autophagic fusion and blocked the autophagosome–lysosome fusion, thus blocking the autophagic flux.

3.5. DENV-2 affects normal acidification of lysosomes to block autophagic flux

The normal function of lysosomal degradation depends on the acidic environment within the lysosome; with the autophagosome–lysosome fusion, their contents are degraded within the lysosome and thus recycled. When the lysosome is not properly acidified, the autophagic substrate cannot be degraded, causing the blockage of the autophagic flux (Zhang et al., 2021). To further investigate the process of DENV-2 blocking the late autophagic flux, DENV-2-infected HUVECs were stained with Lysotracker red, and changes in intracellular lysosomal pH were observed by an inverted fluorescent microscope. The results showed that the blank group showed more red highlights in the cytoplasm and was bright staining, while the infected group had dull color and significantly fewer red highlights. It indicated that DENV-2 infection impeded the normal acidification of lysosomes, thus blocking the autophagic flux.

Dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) caused by DENV are main fatalities, manifested by endothelial cell injury leading to plasma leakage, which is currently considered to be associated with multiple mechanisms, such as cellular autophagic antiviral response, release of inflammatory factors, and endothelial barrier protein deficiency (Ramirez et al., 2018; Cabezas et al., 2018). Cellular autophagy, an important pathway for maintaining the intracellular environment homeostasis, degrades intracellular harmful protein accumulations, senescent organelles, and harmful pathogenic microorganisms through lysosomes (Xie et al., 2021). Autophagy is important for innate and adaptive immunity against bacteria and viruses, and many studies have demonstrated the interaction between viral infection and various stages of autophagy, especially in the stages of autophagosome–lysosome fusion and lysosome degradation. Hepatitis C virus (HCV) inhibits lysosomes and lysosome fusion by increasing the expression level of Arl8b, blocking autophagy and lysosome degradation. Varicella-zoster virus (VZV) mediates the formation of autophagic vesicles, but the blocked late autophagic flux is associated with the defective autophagosome–lysosome fusion or degradation. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ORF3a protein prevents the autophagosome–lysosome fusion, and the ORF7a protein decreases lysosomal acidity, thereby blocking autophagic degradation (Jones-Jamtgaard et al., 2019; Graybill et al., 2018; Koepke et al., 2021). DENV and autophagy have been found to be closely related, and most current studies suggest that DENV-induced autophagy facilitates viral replication, providing energy for viral replication through the degradation of lipids and proteins, while the formation of autophagic vesicles provides a site for replication (Richards and Jackson, 2013). It was found that DENV infection promoted autophagy in the early stages and may be inhibited in the late stages, blocking the autophagic flux by impeding the autophagosome–lysosome fusion. However, the molecular mechanism is not elucidated and it remains unclear how DENV affects the lysosomal degradation pathway in the subsequent stages of the intact autophagic response (Metz et al., 2015).

In this study, the formation of autophagic vesicles was observed by TEM, along with an increase in the LC3-II expression level, implying that DENV-2 induced the formation of autophagic vesicles; the level of p62 showed no significant change at the early stage of infection and increased significantly after 30 h. The difference in LC3 expression levels between the DNEV2-infected, CQ-treated, and DNEV2 combined with CQ-treated groups was not significant, but higher than the blank group, indicating that infection impaired the autophagic flux at the late stage, and the formation of autophagic vesicles did not depend on the autophagic flux change. It was shown that SNAREs series proteins STX17, SNAP29, and VAMP8 formed a complex to promote the autophagosome–lysosome fusion, and during the fusion process, STX17 first localized on the autophagosomal membrane and then recruited SNAP29 and VAMP8 to form a complex. The absence of one of the three proteins does not affect the formation of autophagosomes but hinders the fusion, thus causing the autophagic flux blockage (Shen et al., 2021; Lorincz and Juhasz, 2020). This study examined the changes in the expression levels of fusion key proteins STX17, SNAP29, and VAMP8 at the late stage of infection. STX17, a primary fusion key protein, showed a low expression level, and although the expression level showed an increasing trend over time, it was always lower than the uninfected group. The proteins, SNAP29 and VAMP8, were higher than the blank group at 24 h of infection, but both were lower than the uninfected group after 24 h. Furthermore, it was confirmed that blocking autophagosome–lysosome fusion and low expression of STX17, SNAP29, and VAMP8 in the late DENV infection were related. Additionally, the blocking fusion may be related to the infection time.

Normal lysosomal acidification is essential for the lysosomal degradation pathway, maintaining normal cell metabolism, growth, and contributing to the clearance of pathogenic microorganisms by autophagy. When the lysosomal function is dysfunctional, even if autophagosomes–lysosomes fuse normally, the substrates within autophagosomes are not degraded and provide more membrane structures for virus replication, allowing viruses localized on autophagosomal membranes to further inhibit the autophagosomal substrate degradation. The studies reporting that DENV-2 impedes the autophagosome–lysosome fusion shows an understudy of autophagic degradation (Metz et al., 2015). This study reported that DENV-2 impeded the autophagosome–lysosome fusion, affected by low expression levels of key fusion proteins. Furthermore, DENV-2 could interfere with the normal lysosomal acidification, thus negatively affecting lysosomal autophagic degradation and blocking the autophagic flux, increasing the intracellular autophagic vesicles in the process.

In summary, it was found that DENV-2 infection activated autophagy in HUVECs at an early stage, induced the autophagosome formation, promoted p62 accumulation at the late stage of infection, decreased the expression levels of STX17, SNAP29, and VAMP8 to hinder the autophagosome–lysosome fusion process, and interfered with the normal lysosomal acidification to inhibit lysosomal autophagic degradation and accelerated cell death. Although this study highlighted the effect of DENV-2 infection on the autophagic process of HUVECs, in-depth studies on the molecular mechanism of how DENV-2 infection interferes with the lysosomal acidification are still needed to provide strong evidence for the autophagic antiviral response.

Funding

This work was supported by the National Natural Science Foundation of China (81860289) and National College Students Innovation and Entrepreneurship Training Program, China (20195200146).

CRediT authorship contribution statement

Ning WuXiaoqin Gou: Conceptualization, and design, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Statistical analysis, Writing – review & editing. Xiaoqin Gou: Study design, Writing – original draft, drafted the manuscript, and performed the experiments. Pan Hu: performed the experiments, formal analysis, and assisted with the statistical analysis. Li Zuo: Conceptualization, Methodology, Validation, Statistical analysis, Resources, Writing – review & editing, Supervision, Project administration, Funding acquisition.

Declaration of competing interests

The authors declare that they have no competing interests to this work.

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You are reading this latest preprint version

Dengue virus-2 infection hinders autophagosome–lysosome fusion and autophagy–lysosomal degradation pathway in human umbilical vein endothelial cells

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Cells, viruses, and reagents

2.2. DENV-2 infection

2.3. Western blotting

2.4. Lysotracker red staining

2.5. Statistical analysis

3. Results

3.1. DENV-2 infection promotes cellular autophagic vesicle formation

3.2. DENV-2 infection changes autophagy-related proteins in HUVECs

3.3. DENV-2 infection blocks autophagic flux in HUVECs

3.4. DENV-2 blocks autophagic vesicle–lysosome fusion in HUVECs

3.5. DENV-2 affects normal acidification of lysosomes to block autophagic flux

4 Discussion

Declarations

References

Status:

Version 1