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 Aβ1–42 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), Aβ1–42 (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 Aβ1–42 (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 (****).