Background
β-Amyloid (Aβ) is identified as an essential pathological factor involved in Alzheimer's disease (AD) [
1,
2]. Nevertheless, a deeper understanding of the relationship between AD and pathogenic microbial infections is prompting further exploration of the physiological function of Aβ. Aβ, a novel defined antimicrobial peptide (AMP), also reportedly exhibits antiviral action against herpes simplex virus type 1 (HSV-1) and influenza A virus (IAV) [
3‐
6]. The oligomerization of Aβ peptide prevents bacteria from binding to bacterial-surface carbohydrates, which is also the partial mechanism of Aβ in exerting its anti-herpes virus effect [
4,
5,
7]. Meanwhile, Bourgade et al
. suggested that Aβ may be associated with another favorable antiviral mechanism in addition to the carbohydrate-binding-mediated antiviral pathway [
3].
Enterovirus A 71 (EV-A71), a non-enveloped virus with a positive-sense ssRNA genome enclosed within an icosahedron capsid shell, comprises 60 copies of four capsid proteins, VP1, VP2, VP3, and VP4 [
8]. For non-enveloped viruses, the binding of receptors is a critical event in the course of infection. EV-A71 utilizes cell-surface glycoproteins as receptors to infect host cells [
9,
10]. Scavenger receptor class B member 2 (SCARB2), a type III multichannel membrane glycoprotein, is the main receptor for EV-A71 on target cells [
11‐
13]. SCARB2 is pivotal in EV-A71’s attachment and uncoating, facilitating efficient EV-A71 infection [
14].
Besides causing hand, foot and mouth disease (HFMD), EV-A71 also infects the CNS and induces neurology-related severe diseases [
15]. The relationship between EV-A71 and Aβ has not been reported. In the present study, we demonstrated for the first time that EV-A71 infection induced Aβ
1–42 production and accumulation and elucidated the mechanism by which Aβ
1–42 inhibited EV-A71 replication. Our results revealed that Aβ presented antiviral activity against EV-A71 infection, paving the way for further research on the potential role of Aβ peptides in other brain-infecting viruses.
Methods
Oligomeric synthetic peptide preparation and compounds
Aβ peptides 1–42 are a major pathological factor of senile plaques in AD [
16,
17]. Accordingly, we utilized Aβ
1–42 as a test subject to perform our study.
Synthetic Aβ1–42 peptides were obtained from Anaspec (Fremont, CA. USA). Dried peptides were solubilized in 150 mM NaCl prior to experimentation. Aβ1–42 was used at 30 µg/mL concentration for the viral life-cycle experiment and 20 µg/mL for the antiviral activity test or pull-down experiment.
Pirodavir was purchased from Biochempartner (Shanghai, China), and NH4Cl was bought from MedChemExpress (USA). Stock solutions of pirodavir (10 mM) and NH4Cl (100 mM) were prepared in dimethyl sulfoxide (Sigma–Aldrich, Carlsbad, CA, USA), respectively. RBV stock solutions (2 mg/mL) were dissolved in a cell culture medium.
Viral strains and cell lines
Human rhabdomyosarcoma (RD) cells and SH-SY5Y cells were bought from the Cellular Cultivation Center of Peking Union Medical College (PUMC) or Chinese Academy of Sciences (CAS) and cultivated in Dulbecco’s adjusted Eagle intermediary comprising 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin–streptomycin (Invitrogen, Carlsbad, CA, USA). Vero cells were acquired from the American Type Culture Collection (ATCC), and cultured in modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% inactivated FBS (Gibco, Grand Island, NY, USA) and 1% penicillin–streptomycin.
EV-A71 strain H (VR-1432) and Coxsackievirus B3 strain Nancy (VR-30) were purchased from ATCC. CVA16 strain (shzh05-1/GD/CHN/2005) was offered by Dr. Jianwei Wang, Institute of Pathogen Biology, CAS, and PUMC. Entire viral strains were passaged in Vero cells.
Quantification of Aβ1–42 by ELISA
A human Aβ (1–42 aa) Quantikine ELISA Kit (R&D Systems, DAB142) was used to identify human Aβ (aa1–42) in cellular cultivation supernatant under the condition of EV-A71 infection. The experiment was performed per the manufacturer’s instructions.
Cytotoxicity assay
The cytotoxic effects of Aβ1–42 peptides on SH-SY5Y, Vero, and RD cells were evaluated with a Cell-Counting Kit (CCK) (TransGen Biotech, Beijing, China). In a typical procedure, cells were cultured in 96-well plates and indicated concentrations of Aβ1–42 peptides were added for 48 h. Then, 10 µL of CCK solution was added to each cell well. The absorbance of the plates was detected at 450 nm on an Enspire system (PerkinElmer, Waltham, MA, USA) after incubating at 37 °C for 30 min.
Western blot (WB) assay
Total proteins were lysed with an M-PER mammal-protein abstraction reagent containing a halt protease suppressor cocktail (Thermo Fisher Scientific, USA). An equal amount of cell lysate was used in a 10%–12% (w/v) SDS-PAGE gels, electro-transferred onto PVDF membranes (Millipore, USA), and blocked with 5% (w/v) milk at room temperature for 2 h. The films were incubated with the first antibody against EV-A71 VP1 (Abnova, Taipei, China), EV-A71 VP2 (GeneTex, California, USA), EV-A71 3AB (GeneTex, California, USA), SCARB2 (Abcam, UK), Aβ
1–42 (CST, USA), and β-actin (CST, USA) overnight at 4 °C. Finally, an ECL identification kit (GE Healthcare Life Sciences, USA) was used with an appropriate second antibody (CST, USA) for 1 h at room temperature [
18]. Software “Gel-Pro analyzer” was used to analysis of the optical density ratio of the bands.
To detect of Aβ1–42 monomer (4 kDa), the suspensions were mixed with 1 × loading buffer (containing DTT) and boiled for 10 min. We used 20% Tris–Tricine–SDS-PAGE gels prepared per the manufacturer’s instructions (Solarbio, China). The gels were transferred onto PVDF membranes for 1 h under the condition of 200 mA by using a transfer buffer containing 20% methanol, 190 mM glycine, and 25 mM Tris. The membranes were blocked with 5% (w/v) milk at room temperature for 2 h. The films were incubated with the first antibody against Aβ
1–42 (CST, USA) overnight at 4 °C. Finally, an ECL identification kit (GE Healthcare Life Sciences, USA) was used with an appropriate second antibody (CST, USA) for 1 h at room temperature [
18].
Immunofluorescence assay and confocal microscopy
SH-SY5Y and Vero cells were infected with EV-A71 (MOI = 1) for 8 h. Cells were fixed with 4% polyoxymethylene for 30 min before the cultivation in 0.1% Triton X-100 for another 20 min. Cells were then blocked and cultivated with an antibody against VP1 and Aβ1–42. After washing in TBS three times, VP1 protein was visualized using an Alexa Fluor 488-conjugated secondary antibody (Invitrogen), whereas Aβ1–42 protein was visualized with Alexa Fluor 594. Cell nuclei were dyed with DAPI (Beyotime, PRC). Images were captured with an Olympus TH4-200 microscope or PE UltraVIEW VOX.
Real-time reverse transcription-PCR (qRT-PCR)
Overall, RNA was extracted with RNeasy Mini Kit (Qian, USA). The level of EV-A71 VP1 RNA was quantified with One-Step qRT-PCR by using primers VP1-forward (5′-GATATCCCACATTCGGTGA-3′) and VP1-reverse (5′-TAGGACACGCTCCATACTCAAG-3′). The level of CVA16 VP1 RNA was identified using primers VP1-forward (5′-GTTATCCCACCTTCGGAGA-3′) and VP1-reverse (5′-TCGGGCATTGACCATAATCTAG-3′). The level of CVB3 VP1 RNA was amplified with forwarding primers VP1 (5′-TGCTCCGCAGTTAGGATTAGC-3′) and reverse VP1 (5′-ACATGGTGCGAAGAGTCTATTGAG-3′). GAPDH mRNA and β-actin mRNA served as an internal control to standardize the examined mRNAs by using primers GAPDH-forward (5′-GAAGGTGAAGGTCGGAGTC-3′). GAPDH-reverse (5′-GAAGATGGTGATGGGATTTC-3′). The relative fold change of the detected RNA specimens was analyzed by the comparative 2
−ΔΔCT method [
19].
Time-of-addition assay
The virus-replication steps targeted by Aβ1–42 were mapped by identifying the role of sequential supplementation of Aβ1–42 on EV-A71 VP1-level variation. In short, SH-SY5Y cells were subjected to EV-A71 infection (MOI = 10), and Aβ1–42 (30 μg/mL) was supplemented at the time of infection or at a different time post-infection. Entire cells were collected at 8 h post-infection, and VP1 expression was detected by WB assay.
Attachment-inhibition assay
Cell plates were stored at 4 °C for 60 min before starting the assay. SH-SY5Y, Vero, or RD cells were incubated with EV-A71 (MOI = 2.5) and Aβ1–42 (30 µg/mL) at 4 °C for 60 min. Afterwards, cells were washed in cold PBS (pH = 7.4) three times and subjected to a qRT-PCR assay.
Pre-attachment inhibition analysis
The pre-attachment inhibition analysis was designed as two experimental methods to test the various suppression characteristics of Aβ1–42.
For the pre-attachment inhibition assay, EV-A71 virus (MOI = 2.5) was mixed with Aβ1–42 (30 µg/mL) peptides at 4 °C for 1 h and attached to SH-SY5Y or Vero cells in cold 12-well plates at 4 °C for another 1 h. Total cellular RNA was extracted after three washes with cold PBS (pH = 7.4) and analyzed by qRT-PCR.
For the pre attachment inhibition assay of Aβ1–42 peptides, 20 µg/mL Aβ1–42 was incubated with precooled cell plates at 4 °C for 60 min and then washed in cold PBS three times. The processed cells were used in the subsequent pre attachment inhibition assays of the virus.
Virus-penetration inhibition assay
Cells were cultivated with EV-A71 (MOI = 2.5) at 4 °C for 60 min to enable viral attachments, followed by cleaning in cold PBS three times to realize the removal of nonbound viruses. They were subsequently cultivated at 37 °C for 60 min to internalize viruses, and uncoating occurred eventually. After washing the unbound viruses three times, cells were lysed, and the RNA content of EV-A71 was measured by qRT-PCR analysis.
Purification of EV-A71 virions
EV-A71 was propagated in Vero cells at 37 °C for 3 days. The cells were freeze-thawed in three cycles and then centrifuged at 3000 g for 30 min to remove cell debris. EV-A71 was initially concentrated using a 100 kDa centrifugal concentrator (Millipore, Billerica, MA, USA) at 3000 g for 30 min. Then, the virus was sedimented through a 20% density sucrose layer at 4 °C, 14,000 g for 3 h with a Beckman SW41 Ti rotor. Virus fractions at the bottom were harvested and gently resuspended in PBS (pH = 7.4). The purified EV-A71 stock was supplied for transmission electron microscopy (TEM) and pull-down assays [
5,
6].
TEM analysis of virus agglutination
The purified EV-A71 stock was cultivated together with Aβ1–42 peptides in PBS (pH = 7.4) at 4 °C for 1 h, absorbed into formvar carbon-coated copper grids for 60 s, and then stained with 1% (w/v) phosphotungstic acid (pH = 6.8) for another 60 s. The grids were desiccated in air atmosphere and imaged with a Tecnai12 TEM (FEI, Eindhoven, Netherlands) with a CCD camera (EMSIS MRADA g3, Germany).
Pull-down assay
For EV-A71 VP1 pull-down assays, 20 µg/mL Aβ1–42 was incubated with protein A/G magnetic beads at 4 °C overnight before adding IgG or purified EV-A71 after cleaning in cold PBS three times. Subsequently, the mixture was cultivated at 4 °C for 2 h. After cleaning in cold PBS three times, the bound virions were detected by WB assay with VP1 antibody.
For the SCARB2 pull-down assays, 6 µg of SCARB2-Myc plasmid or pcDNA 3.1 + vector was transfected into Vero cells and lysed with protein lysate containing protein phosphatase and protease inhibitor at 24 h post-transfection. Subsequently, the lysis buffer supernatant was mixed with Aβ1–42 immobilized magnetic protein A/G magnetic beads at 4 °C for 2 h. The bounded beads were suspended with a 1 × sample loading buffer and boiled for 10 min. The binding of SCARB2 was detected by WB with anti-SCARB2 antibody.
Statistical analysis
The two groups were contrasted by Student’s T-test, and more groups were contrasted through one-way ANOVA using GraphPad Prism 8.0. Asterisks (*) corresponded with p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).
Discussion
Enterovirus A types, including EV-A71 and CVA16, are primary pathogens related to HFMD and encephalitis, which are typical neural complications from HFMD infection. They are accompanied by complications such as central nervous system (CNS) damage, particularly in East and Southeast Asia [
15,
25,
26]. Aβ has long been recognized as a pathogenetic factor for AD. However, the antimicrobial function of Aβ has been gradually revealed recently. Here, we demonstrated for the first time that Aβ
1–42 exhibited antiviral activity against EV-A71 and CVA16 infections. The anti-EV-A71 effect of Aβ presented in SH-SY5Y cells, and in Vero and RD cells indicates that Aβ
1–42 inhibited EV-A71 replication independent of cell-line specificity. Mechanistically, we revealed that Aβ
1–42 interfered with the attachment and uncoating stage of EV-A71 infection. The effect of Aβ
1–42 on suppressing viral post-attachment was more effective than the attachment in SH-SY5Y cells, whereas an equal inhibition effect was observed in Vero and RD cells. The reason for this apparent difference in nerve cells remains elusive. Aβ exhibited a direct ability to bind microbial surface carbohydrates, primarily mediating its antimicrobe infection capability. The carbohydrate binding of Aβ promoted self-fibrillization, leading to the aggregation of enveloped viruses such as HSV-1, HHV6, and IAV, thereby reducing viral infectivity [
4,
6,
27,
28]. However, we discovered that Aβ
1–42 also induced a slight aggregation of non-envelope virus EV-A71 after co-incubating for 1 h and bound to virus capsid protein VP1 performed by pull-down assay. Meanwhile, either the binding to VP1 or the aggregation of EV-A71 may lead to the impairment of virus attachment (Fig.
4d). Bourgade et al
. suggested that the carbohydrate-binding of Aβ may not be the only pathway to target herpesviruses. Previous research has assumed that Aβ, which has high sequence homology to the HSV-1-gB membrane-proximal region, targets HSV-1 by competing for binding gB's fusion loops, thereby preventing the HSV-1 fusion cell process [
29]. Based on VP1 of EV-A71, which is not a glycoprotein, the binding between VP1 and Aβ may exist in other pathways. Accordingly, we speculated that a noncarbohydrate binding region of Aβ
1–42 may mediate the targeting of EV-A71 capsid protein VP1.
In HSV-1 and IAV, the present research showed that Aβ inhibited virus replication outside the cells but not when peptides were added after virus infection. In contrast to previous reports, we discovered that the antiviral action of Aβ could effectually occur inside the target cells (Figs.
2 and
3c). Unlike enveloped viruses, the cellular entry of non-enveloped viruses primarily relies on the binding to cell surface virus receptors. SCARB2, a membrane glycoprotein, mediates viral entry into the cell and is a common receptor for all enterovirus A virus. Consistent with the glycoprotein-binding function, Aβ could interact with SCARB2 and inhibit EV-A71 attachment and entry (Fig.
5). We further found that Aβ
1–42 inhibited CVA16 rather than CVB3 replication (Fig.
5b), possibly because SCARB2 was not a receptor for CVB3. Hsu et al
. revealed that Aβ could bind to the spike protein S1 sub-unit (S1) of the SARS-CoV-2 and the viral receptor ACE2, enhancing the interaction of the S1 protein with ACE2 and increasing the virus entry along with intracellular IL-6 production [
30]. Figure
5a indicates that pretreating with Aβ
1–42 peptides in various cell lines led to evident reductions in EV-A71 RNA levels. This finding suggested that Aβ exerted a competitive combination with SCARB2 instead of strengthening the binding of EV-A71 to SCARB2. Further investigation is required to confirm the interference of Aβ1–42 on the binding of the virus to SCARB2. Recent research has suggested that the VP1 GH and VP2 EF loops of EV-A71 interact with SCARB2 and further facilitate the low-pH uncoating of the virus in endosomes/lysosomes [
31]. Our results showed that Aβ
1–42 bound to VP1 protein but not VP2 (Additional file
1: Fig. S1), indicating that the viral VP1 GH loop may play a key role in mediating the interaction of VP1 and Aβ
1–42. Our results further showed that Aβ
1–42 interacted with SCARB2 or VP1. However, further studies are needed to confirm whether these two interactions were competitive or synergistic.
Toczylowski K et al. measured Aβ1–42, a traditional AD biomarker, in children with enteroviral meningitis caused by echovirus. The CSF concentration of Aβ1–42 was decreased when compared with the control group without CNS infection. In contrast, other biomarker concentrations are unchanged [
32]. Thus, an enteroviral infection may lead to a specific impact on Aβ1–42 production. Our results showed that Aβ
1–42 generation and accumulation were induced at 12 h after EV-A71 infection in SH-SY5Y cell lines (Fig.
1). The findings of Toczylowski et al
., together with ours, reveal the distinct effect of various enteroviruses infection on Aβ
1–42 production. Nevertheless, whether Aβ production and further aggregation is a protective immune response against the invasion of foreign pathogens such as EV-A71 infection requires further exploration.
Over the past few years, several infection factors have been proposed to be associated with AD development [
33]. Most past research has focused on the relation between Aβ
1–42 and HSV-1. Little is known about enteroviruses that exert a similar inflammatory influence on CNS due to HSV infection [
24,
33]. Thus, more detailed studies on the specific binding of the amino acid region of Aβ
1–42 to capsid VP1 protein or virus receptor SCARB2 should be performed. Apart from SCARB2, PSGL-1 is also a typical glycoprotein receptor of EV-A71. However, PSGL-1 is expressed by macrophages in the intestinal mucosa and lymph-node dendritic cells, and it plays a key role in trafficking inflammation stimulated by infection [
34]. We did not use PSGL-1-expression cell lines in this research and will explore it in a follow-up work if needed. The previous discoveries of Bourgade et al
. have revealed that Aβ
1–42 can not inhibit non-enveloped human adenovirus replication in vitro. Our findings unveiled that Aβ1–42 can sufficiently inhibit non-enveloped EV-A71 effectively, thereby extending the potential function of Aβ in other non-enveloped viruses linked to infectious neurological diseases.
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