key: cord-309275-soffxxqu authors: Li, Shuang; Zhu, Anjing; Ren, Kai; Li, Shilin; Chen, Limin title: DEFA1B inhibits ZIKV replication and retards cell cycle progression through interaction with ORC1 date: 2020-10-17 journal: Life Sci DOI: 10.1016/j.lfs.2020.118564 sha: doc_id: 309275 cord_uid: soffxxqu AIMS: Zika virus (ZIKV) infection causes a public health concern because of its potential association with the development of microcephaly. During viral infections, the host innate immune response is mounted quickly to produce some endogenous functional molecules to limit virus replication and spread. Exosomes contain molecules from their cell of origin following virus infection and can enter recipient cells for intercellular communication. Here, we aim to clarify whether ZIKV-induced exosomes can regulate viral pathogenicity by transferring specific RNAs. MAIN METHODS: In this study, exosomes were isolated from the supernatants of A549 cells with or without ZIKV infection. Human transcriptome array (HTA) was performed to analyze the profiling of RNAs wrapped in exosomes. Then qPCR, western blotting and ELISA were used to determine ZIKV replication. CCK-8 and flow cytometry were used to test the cell proliferation and cell cycles. Co-culture assay was used to analyze the effect of exosomes on the cell cycles of recipient cells. KEY FINDINGS: Through human transcriptome array (HTA) we found the defensin alpha 1B (DEFA1B) expression was significantly increased within exosomes isolated from ZIKV infected A549 cells. Additionally, we found that the extracellular DEFA1B exerts significant anti-ZIKV activity, mainly before ZIKV entering host cells. Interestingly, up-regulated DEFA1B retards the cell cycle of host cells. Further studies demonstrated that DEFA1B interacted with the origin recognition complex 1 (ORC1) which is required to initiate DNA replication during the cell cycle and increased DEFA1B expression decreased the ORC1 level in the cell nuclei. Accordingly, DEFA1B-containing exosomes can be internalized by the recipient cells to retard their cell cycles. SIGNIFICANCE: Together, our results demonstrated that the anti-ZIKV activity of DEFA1B can be mediated by exosomes, and DEFA1B interacts with ORC1 to retard cell cycles. Our study provides a novel concept that DEFA1B not only acts as an antiviral molecule during ZIKV infection but also may correlate with cell proliferation by retarding the progression of cell cycles. Zika virus (ZIKV) is a re-emerging arbovirus that belongs to the Flaviviridae family and is transmitted by infected Aedes mosquitoes. ZIKV is a single positive-stranded RNA virus with a genome size of approximately 10.8 kilobases [1] . ZIKV infection emerged as a public health concern after increasing evidence linking ZIKV infection with the potential development of microcephaly [2] . Although previous study alluded that ZIKV could directly infect human neural progenitor cells (hNPCs) to induce cell death and disturb cell cycle progression to affect human brain development [3] , the detailed underlying molecular mechanism remains elusive. In the battle between pathogens and hosts, the production of endogenous defensins is one of the first lines of protection for the host. Various types of human cells can produce defensins that have broad antimicrobial activity to many pathogens, including bacteria, viruses and fungi [4, 5] . Defensins have been shown to protect against many virus infections, such as human immunodeficiency virus (HIV), influenza A virus (IAV), human adenovirus (HAdV), severe acute respiratory syndrome coronavirus (SARSC), papillomavirus (HPV), respiratory syncytial virus (RSV), and herpes simplex virus (HSV) [6, 7] . Currently, no approved vaccines or therapies are available for the prevention and treatment of ZIKV infection, therefore defensins may be a promising drug target for the treatment of ZIKV infection. The 'origin recognition complex' (ORC), as one of the most important complexes in all eukaryotes, is involved in the initiation of DNA replication and has been linked to various diseases [8] . Although ORC consists of ORC1-6, ORC1 is the biggest subunit that is necessary for the initiation of DNA replication [9] . Notably, several reports linked ORC to numerous human diseases [10, 11] . Of particular interest, mutations in ORC1 have been shown to cause Meier-Gorlin syndrome, which is characterized by microcephaly and primordial dwarfism [12] . Recent work has pointed out that exosomes function as crucial regulators of cellular cross-talk. Exosomes are phospholipid bilayer-bound structures that can transfer packed mRNA, miRNA, and/or proteins into recipient cells to facilitate intercellular communications and to affect gene expression in recipient cells [14, 15] . In addition, emerging evidence indicated that exosomes can mediate the transfer of pathogen-derived antigens and virulence factors [16] . Exosomes play various roles in the pathogenesis of ZIKV. For example, ZIKV can be wrapped into a sort of cargo of the placental exosomes and be transmitted through exosomes into trophoblast cells [17] . Furthermore, exosomes have been shown to mediate ZIKV transmission through SMPD3 neutral Sphingomyelinase in cortical neurons [18] . In order to further decipher the role of exosomes in the pathogenesis of ZIKV infection, we used human transcriptome array (HTA) and bioinformatics analysis to identify candidate molecules in ZIKV-induced exosomes. We identified defensin alpha 1B (DEFA1B) is involved in ZIKV infection and pathogenesis. Our data demonstrated that DEFA1B not only inhibits ZIKV 5'-GCAGAGCAACGGATGGGATA-3' and the reverse primer was 5'-ATGGTGGGAGCAAAACGGAA-3'. Plasmids expressing DEFA1B were constructed with routine molecular cloning techniques. The full length human DEFA1B gene was amplified by polymerase chain reaction (PCR) from total RNA isolated from A549 cells and cloned into pcDNA3. Secondary antibodies goat anti-rabbit, goat anti-mouse and goat anti-rat were also obtained from CST (MA, USA). Bio-rad Image Acquisition and Analysis Software (UVP, Upland, CA, USA) were performed to quantify the band density. Cell proliferation and viability were evaluated using a CCK-8 assay (TransGene Biotech, China). For CCK8, the cells were allowed to grow in a 96-well plate, at the concentration of 5000 cells per well. At each time point, cells were rinsed with 1/10 CCK-8 diluted in DMEM for 1.5h, and the optical density of the cellular homogenate was measured at 450 nm. Each experiment was performed in quintuplicate. The effect of DEFA1B on cell proliferation was evaluated by measuring the distribution of the and Protein A&G Agarose (Santa Cruz Biotechnology) were used in this co-IP assay. The immunoprecipitates were washed five times and separated by 10% SDS-PAGE gel electrophoresis and blotted using anti-DEFA1B and anti-ORC1 (Santa Cruz), respectively. PKH67 Green Fluorescent Cell Linker Kit (Sigma) was used to label exosomes according to the manufacturer's protocol. Briefly, exosomes were re-suspended in 500 μl Diluent C. In addition, 2 μl PKH67 was mixed with 1 ml Diluent C, followed by mixing with the exosomes suspension and incubated for 4 min. To stop the labeling reaction, an equal volume of 1% BSA was added into the mixed solution. Then, the labeled exosomes were ultracentrifuged at 100,000 ×g for 1 h at 4℃), washed with 1 × PBS, and ultracentrifuged again. To detect exosomes uptake into recipient cells, HEK293T cells and SH-SY5Y cells were grown in 24-well plates at a density of 1 × 10 4 cells per well and PKH67-labeled exosomes were diluted in whole-medium solution and was added into each well. Cells were cultured for 6 h at 37 °C. DAPI was used to stain the nucleus, and the cells were observed using fluorescence microscope (Olympus IX71, Germany). Student's t-tests were used to analyze the differences between groups. All experiments were repeated at least three times. The p-value < 0.05 was considered statistically significant (*p < 0.05; **p < 0.01; ***p < 0.001). 125.3nm, and 97% of Exo-ZIKV is 127.2nm ( Figure 1C , 1D, 1E). Western blot revealed that the exosome-specific proteins, CD63, CD81 and TSG101, were enriched in all exosomes samples but not in cell lysates-confirming these vesicles are indeed exosomes. Calnexin, an endoplasmic reticulum protein, was detectable in whole-cell lysates but not in the exosomes, indicating that the exosomes preparations were not contaminated with other vesicles ( Figure 1F ). Together, these results confirmed the isolated vesicles are indeed purified exosomes and the isolation method is reliable. Western blotting of exosomes were performed to confirm the presence of exosomal marker protein, CD63, CD81 and TSG101. Absence the endoplasmic reticulum protein, Calnexinan, in exosomes but was detectable in whole cell lysates (F). and A549 control cells 48hrs after the plasmids transfection, the mRNA level of DEFA1B was significantly up-regulated ( Figure 3A ). ELISA data showed the DEFA1B could be secreted into culture supernatants, and the concentration of DEFA1B was significantly increased compared with the control group ( Figure 3B ). Furthermore, to examine the anti-viral ability of DEFA1B, we divided the cells into two E. J o u r n a l P r e -p r o o f groups: in one group the supernatants were removed and replaced with fresh medium; the other group the supernatants that contain DEFA1B were added. Then, these two groups of cells were infected with ZIKV at MOI=0.5. Our data showed that DEFA1B contained in culture supernatants significantly inhibited ZIKV replication at mRNA level ( Figure 3C ). NS1 (which is the non-structural protein 1 of ZIKV) expression level was also significantly decreased in the group with DEFA1B in culture supernatants ( Figure 3D ). To further analyze the anti-ZIKV effect of extracellular DEFA1B, we added three different concentrations (2418.1±102.9 pg/mL, 1913.9±92.1 pg/mL, 588.7±31.0 pg/mL) of DEFA1B to the supernatants of HEK293T cells ( Figure 3E ) and infected the cells with ZIKV. Compared with no DEFA1B in supernatants, qPCR data showed that ZIKV replication was significantly inhibited in a dose-dependent manner ( Figure 3F ). We also verified the same tendency at the viral protein levels using western blot ( Figure 3G ). Furthermore, we compared the changes of ZIKV copies in medium with or without DEFA1B to further validate the anti-ZIKV effect of extracellular DEFA1B. The qPCR data showed the ZIKV copies were significantly decreased in medium with DEFA1B when compared with those in normal medium (without DEFA1B) ( Figure 3H ). Furthermore, we compared the changes of ZIKV copies in medium with or without DEFA1B to further validate the anti-ZIKV effect of extracellular DEFA1B. The qPCR data showed the ZIKV copies were significantly decreased in medium with DEFA1B when compared with those in normal medium (without DEFA1B) ( Figure 3H ). In order to test whether the intracellular DEFA1B has anti-ZIKV activity, we transfected different doses (0.2ug, 0.5ug and 1ug per well) of pcDNA3.1-DEFA1B-T2A-EGFP plasmids into HEK293T cells. qPCR ( Figure 3I ) and ELISA ( Figure 3J ) data showed the DEFA1B was indeed up-regulated in HEK293T cells after transfection. We then replaced the culture supernatants with fresh medium and infected the cells with ZIKV (MOI=0.5). Both qPCR ( Figure 3K ) and western blot ( Figure 3L ) data showed no significant changes of ZIKV replication between untreated HEK293T cells and cells with up-regulated DEFA1B group. western blot (L) to analysis ZIKV level. During our research we found that up-regulated DEFA1B expression in HEK293T reduced the cell growth rate. So we used CCK8 to investigate the proliferation of HEK293T cells transfected with pcDNA3.1-DEFA1B-T2A-EGFP or with control vector, and we found that compared with untreated or blank vector control group, the proliferation of cells were obviously decreased at day 2 and day 3 after transfected with pcDNA3.1-DEFA1B-T2A-EGFP (Fig 4) . Proteins often do not just function as a single substance but rather as team players in a dynamic network. Growing evidence shows that protein-protein interactions are crucial in many biological processes in living cells. The database STRING (https://string-db.org/) is a pre-computed global resource for the exploration and analysis of these associations. We found that DEFA1B was the hub of ORC1 interaction that is the largest subunit of the ORC ( Fig 5A) ; therefore, we decided to focus further analysis on ORC1. To confirm whether DEFA1B specifically interacts with ORC1, immunoblot analysis of whole cell lysates and anti-DEFA1B affinity immunoprecipitation (IP) derived from 293T cells transfected with pcDNA3.1-DEFA1B-T2A-EGFP or blank vector control plasmid were performed. Co-IP assay clearly demonstrated that DEFA1B interacts with ORC1 ( Fig 5B) . We further analyzed the expression levels of ORC1 both in the nucleus and cytoplasm. Our results demonstrated that following the up-regulated expression of DEFA1B, the levels of ORC1 in whole cell lysis ( Fig 5C) and cytoplasmic protein lysis (Fig 5D) showed no significant changes compared with the control groups. Interestingly, although DEFA1B was not expressed in the nucleus, the level of ORC1 in nucleus was significantly decreased in DEFA1B -up-regulated cells (Fig 5E) . We used flow cytometry to analyze the cellular DNA content to identify the percentage of G1, S and G2/M in the cell cycle. Our data showed 2 days after transfection, the percentage of G2/M in DEFA1B-up-regulated cells was significantly decreased compared with the control groups (Fig 6A-D) . To be able to follow the progression of cell cycle more precisely, we synchronized cultured HEK293T cells with thymidine which is the most commonly used S-phase blocker and its addition to the culture medium depletes nucleotide pools and inhibits new DNA synthesis, causing the slowdown or arrest of S-phase progression [20] . After released into normal medium, cell populations at distinct cell cycle phase were collected at different time points (1-10h) and the overlapped histograms indicated after about 4-5 hours some cells finished S+G2+M stages and returned to G1 phase. So we compared the velocity to finish S+G2+M stages through analyzing the percentage of G1. Our data showed with time passing by the cultured cells became more and more asynchronous. 6 hours following releasing, the percentage of G1 phase was significantly lower in the DEFA1B-up-regulated group, which indicates that DEFA1B retards cell cycle progression (Fig 6E) . The internalization of exosomes is one mechanism of cargo delivery to recipient cells. To examine whether exosomes isolated from ZIKV infected A549 cells can be taken up by HEK293T and J o u r n a l P r e -p r o o f Journal Pre-proof SH-SY5Y cells, Exo-ZIKV were labeled with PKH67 dye (green) and added to cultured HEK293T cells. Data from fluorescence microscope suggested that Exo-ZIKV were internalized into recipient cells after 6 hours co-culture (Fig 7a) . We further determined whether exosomes can transmit the cell cycle retarding effect of DEFA1B to recipient cells, we discriminated the cell cycles of recipient cells using Flow Cytometry. After co-cultured with Exo-ZIKV for 48h, the percentage of G2/M in HEK293T cells was decreased compared with cells co-cultured with Exo-A549 (Fig 7b, 7c) . As expected, Exo-ZIKV was also internalized into SH-SY5Y cells (Fig 7d) and flow cytometry data showed the percentage of G2 of undifferentiated human neuroblastoma SH-SY5Y cells was decreased compared with cells co-cultured with Exo-A549 (Fig 7E, 7F ). Exosomes, as a "carrier" of material and information, play an important role in the interaction between viruses and host cells. Loading functional genes into virus-induced exosomes has been demonstrated to modulate viral spread and immune response. In this study, we aimed to understand the role of exosomes in the pathogenesis of ZIKV infection. We first isolated and characterized the exosomes from ZIKV infected/uninfected A549 cells. We showed the exosomes with typical shape,size and protein markers, indicating the vesciles we isolated are indeed exosomes and the isolation method is reliable. Next, we used HTA to identify the differentially-expressed genes between exosomes isolated from ZIKV infected A549 and A549 control cells and we found the expression level of DEFA1B was significantly increased within Exo-ZIKV compared with Exo-A549. To further study the role of DEFA1B in ZIKV replication, J o u r n a l P r e -p r o o f we up-regulated the expression level of DEFA1B in A549 cells before infected with ZIKV and the viral replication was assessed by RT-PCR and Western Blot. We found extracellular but not the intracellular DEFA1B inhibited ZIKV replication. Surprisingly, we also revealed that DEFA1B could retard the progression of cell cycle. To further explore the underlying mechanism, we identified that DEFA1B interacts with ORC1 to arrest ORC1 entering into the nuclei. In addition, exosomes as carriers can transmit the cell cycle progression retarding effect of DEFA1B into recipient cells, such as SH-SY5Y and 293T cells (Figure 8 ). in contract with the environment [21] . Our data also showed after ZIKV infection, the DEFA1B increased in host cells as well as in EVs, which can protect from proteinase degradation. Our data showed DEFA1B could be secreted into culture supernatants. Defensins can block viral infection through directly acting on virus particles or indirectly interfering with various stages of the viral life cycle. Available data suggest the antiviral activity of defensins occurs predominantly at viral entry steps; however, antiviral effects at other stages of infection have also been reported, particularly affecting viral trafficking within infected cells [22] . For example, the previous study found that α-Defensin-1 not only had a direct effect on HIV-1 virions but also blocked HIV-1 infection at nuclear import and transcription stages [23] . α-defensins in supernatants also inhibit the infectivity of HSV-1 and Respiratory Syncytial Virus (RSV) [24] . Our data showed DEFA1B could be secreted into culture supernatants. In addition, the extracellular DEFA1B exerts the anti-ZIKV effect, and the inhibiting effect mainly before ZIKV enter host cells. In 2015, physicians in Brazil began to report that the number of microcephaly increased among newborns, which was possibly linked to ZIKV infection during the mothers' pregnancy [25, 26, 27, 28] . The new emergence of patients with severe nerval system prompted public health emergency of international concerns to explore the suspected association between ZIKV infection and microcephaly. Mouse models showed that ZIKV could destroy the central nerval system to leave severe pathological changes in mice [29] . ZIKV can target human brain cells and ZIKV may impact their survival and growth by restricting the growth of neurospheres and brain organoids [30] . Previous studies found that Meier-Gorlin syndrome patients with mutations in ORC1 had increased microcephaly and a significantly proportionally smaller head circumference and brain [31, 32, 33] . Some researchers found that a conserved basic amino acid motif of Orc1 (residues 358-371, Orc1-BP) can specific recognition of thymine residues in the DNA replication origin sequence, and decreased Orc1 in the nuclei could impact the cell growth and be developmentally important [34, 35] . In our study we identified that DEFA1B can bind with ORC1 in the cytoplasm and decrease the level of ORC1 in the cell nucleus. Cell-cycle timings vary markedly during embryo or fetus development, with some stages process rapidly and some stages slow down [36] . For example, in early neurogenesis, neuronal stem cells have a markedly truncated G1 phase in which impaired pre-RC assembly could become rate J o u r n a l P r e -p r o o f Journal Pre-proof limiting. As a result of prolonged cell cycle times, even small changes in the number of cell divisions of progenitor and stem cells could have dramatic effects on eventual tissue and organ size [37] . Our data demonstrated that up-regulated DEFA1B expression could significantly retard the progression of cell cycles, and ZIKV induced exosomes internalized into nerve cells (SH-SY5Y cells) and can develivery the detention cell cycle effect. Whether the ZIKV-induced DEFA1B inhibiting cell cycle progression of fetal nerve cells leading to smaller size of brain needs further investigation. During human development, the placental barrier blood-brain barriers contribute to fetus brain protection [38] . Although the ZIKV RNA has been detected in amniotic fluid samples, placental tissues and newborn and fetal brain tissues, how the virus crosses the placental and blood-brain barriers remains unclear [39] . Previous studies indicated that exosomes as small transporters can easily get through placental barrier and blood-brain barrier [40, 41] . Our data showed the ZIKV induced exosomes could be internalized into recipient cells and inhibit the cells' DNA replication to retard cell cycles. In summary, our innate immune can increase DEFA1B expression during ZIKV infection, and this can effectively control ZIKV replication. Meanwhile, DEFA1B can retard cell cycles. Because there are some correlations between retarded cell cycles and neurodevelopment. So whether the phenomenon we found linked to the formation of microcephaly? If at the early stage of fetal development, can ZIKV induced exosomes go through the placental barrier and blood-brain barrier to entry fetal brain? Can the DEFA1B within these exosomes inhibit cell cycles of fetal nerve cells and consequence lead to small size of brain? 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Shuang Li conceived the study, analyzed the data, and wrote the paper; Anjing Zhu, Kai Ren, Shilin Li and Limin Chen provided experimental materials; Limin Chen revised the paper. All authors have read and agreed to the published version of the manuscript. The authors declare no conflicts of interest.