Cyclophilin A Associates with Enterovirus-71 Virus Capsid and Plays an Essential Role in Viral Infection as an Uncoating Regulator

<div><p>Viruses utilize host factors for their efficient proliferation. By evaluating the inhibitory effects of compounds in our library, we identified inhibitors of cyclophilin A (CypA), a known immunosuppressor with peptidyl-prolyl <i>cis-trans</i> isomerase activity, can significantly attenuate EV71 proliferation. We demonstrated that CypA played an essential role in EV71 entry and that the RNA interference-mediated reduction of endogenous CypA expression led to decreased EV71 multiplication. We further revealed that CypA directly interacted with and modified the conformation of H-I loop of the VP1 protein in EV71 capsid, and thus regulated the uncoating process of EV71 entry step in a pH-dependent manner. Our results aid in the understanding of how host factors influence EV71 life cycle and provide new potential targets for developing antiviral agents against EV71 infection.</p></div

CypA regulated EV71 proliferation.

<p><b>(A)</b> The knockdown of endogenous CypA and CypB proteins. RD cells were infected with shRNA recombinant lentiviruses that were specific for CypA (sh-CypA), CypB (sh-CypB), or with a randomized shRNA (sh-control). At 72 hpi, CypA (top), CypB (middle), and GAPDH, (an internal control) (bottom), were detected in the total cell lysates by immunoblotting analysis. <b>(B)</b> Flow cytometric studies of the RD-sh-control, RD-sh-CypA, and RD-sh-CypB cells that were infected with the EV71-GFP virus at an MOI of 0.5 after 24 h. The frames indicate the virus-infected cells, and the ratio of the infected cells is indicated in the inset. <b>(C)</b> Quantifying the infection of the RD-sh-control, RD-sh-CypA, and RD-sh-CypB cells infected with wt- or S243P-EV71 at an MOI of 1 by qRT-PCR. <b>(D)</b> Quantifying the infection of the RD-sh-control, RD-sh-CypA, and RD-sh-CypB cells infected with wt-EV71 at MOIs of 0.1 and 50 after 12 h and 48 h of infection, respectively. The data were expressed as the percentage of EV71 RNA from the untreated control cells; the mRNA level of GAPDH was used as an internal control. Each data point represents the average of three replicates. The error bars represent the SEM. <b>(E)</b> Detecting the EV71-VP1 protein expression when RD-sh-control, RD-sh-CypA, and RD-sh-CypB cells were infected with wt-EV71 or S243P-EV71 virus at an MOI of 1 after 24 h by immunoblotting analysis. <b>(F)</b> Quantifying the infection of the Huh7.5.1-sh-control and Huh7.5.1-sh-CypA cells that were infected with wt-EV71 at an MOI of 10 by qRT-PCR. <b>(G)</b> Detecting the EV71-VP1 protein expression when Huh7.5.1-sh-control and Huh7.5.1-sh-CypA cells were infected with wt-EV71 at an MOI of 10 by using immunoblotting analysis. <b>(H)</b> Detecting the expression of CypA and CypB in RD cells at different time points following infection with wt-EV71. <b>(I)</b> Detecting the existence of secreted CypA in supernatants from the RD-sh-control and RD-sh-CypA cells infected with wt-EV71. The expression levels of CypA, CypB, EV71-VP1 and GADPH were examined by immunoblotting analysis.</p

CypA functions in the entry step of EV71 infection.

<p><b>(A)</b> The effect of adding compound HL051001P2 (5 µM) at various times during the replicative cycle of EV71. RD cells were infected by wt-EV71 virus at an MOI of 50 and the inhibitor was added at −6, −4, −2, 0, 2, 4, 6 and 8 hpi. The data for the EV71 genome were expressed as the percentage of EV71 RNA from the untreated control cells; the mRNA level of GAPDH was used as an internal control. The data for EV71 viral titers in the supernatant were measured as previously described. <b>(B)</b> The EV71 subgenomic replicon RNA was transfected into RD-sh-control and RD-sh-CypA cells, and the luciferase levels were quantified by measuring the firefly luciferase activity in relative luminescence units at 24 hpi; the firefly luciferase activity of pNL4-3 was used as an internal control. The growth curves of wt-EV71 <b>(C)</b> and S243P-EV71 <b>(D)</b> in RD-sh-control or RD-sh-CypA cells at an MOI of 50. The EV71 infection was quantified by using qRT-PCR to detect the EV71 RNA in the RD-sh-control or RD-sh-CypA cells at different times post infection, and the mRNA level of GAPDH was used as an internal control. The data represent the means of three independent experiments. Error bars represent the SEM. <b>(E)</b> The EV71 VP1 expression was also checked in the EV71-infected RD-sh-control and RD-sh-CypA cells by immunoblotting analysis. <b>(F)</b> Viral titers in the supernatant were measured at different times post infection. <b>(G)</b> The subcellular localization of CypA and EV71. Immunofluorescence analysis was performed on RD-sh-control and RD-sh-CypA cells infected with wt- or S243P-EV71 at an MOI of 100. At 2 hpi, the cells were fixed and stained with anti-CypA (panels a, e and I, green) or anti-EV71 VP1 (panels c, g, and k, red) antibodies, and DAPI was used to visualize the nuclei (panels b, f, and j, blue). Panels a-d, e-h, and i-l show the same cells. The merged images are shown in panels d, h, and l, respectively.</p

The concentration-dependent reduction of EV71 RNA following treatment with compound HL051001P2 and CsA.

<p>Chemical structures of compound HL051001P2 <b>(A)</b> and CsA <b>(B)</b>. After the RD cells were incubated for 6 h with various concentrations of compound HL051001P2 (0.06 to 20 µM for anti-EV71 activity and for cytotoxicity) and CsA (0.06 to 20 µM for anti-EV71 activity and for cytotoxicity), the cells were then infected with EV71 virus at an MOI of 1. <b>(C)</b> The infection of RD cells with EV71 virus in the presence of the compound treatments were quantified by qRT-PCR at 24 hpi, and the data were expressed as the percentage of EV71 RNA from the untreated control cells; the mRNA level of GAPDH was used as an internal control. Each data point represents the average of three replicates. Error bars represent the SEM. <b>(D)</b> To monitor the cytotoxic effects, the viability of the RD cells was determined after compound treatment by using a WST-1 based assay, which was compared with that of the untreated control cells at 24 h post compound treatment. Each data point represents the average of three replicates. The EV71 virus used in these experiments was strain AnHui1.</p

CypA is an uncoating regulator of EV71 entry.

<p><b>(A)</b> and <b>(B)</b> The binding assay of the EV71 virions with the host cells. The binding capacity of the wt- <b>(A)</b> or S243P-EV71 viruses <b>(B)</b>. Two conditions were assessed as follows: 10-fold diluted (1∶10) and 20-fold diluted (1∶20) standardized viral stocks (10⁸ TCID₅₀/ml). The amount of bound virus was measured by using a qRT-PCR assay. Error bars represent the SEM. <b>(C-E)</b> The binding of EV71 virion to reported receptors, without or with CypA treatment, including the immobilized His-tagged SCARB2 <b>(C)</b>, heparin-sepharose column <b>(D)</b>, and Fc-PSGL-1 or control Fc <b>(E)</b>. The amount of bound virus was measured by using a quantitative real-time qRT-PCR assay. Each data point for (C) and (E) represents the average of three replicates and each data point for (D) represents the average of ten replicates. Error bars represent the SEM. <b>(F-I)</b> The virion flotation assay with CsCl density gradient ultracentrifugation. Purified viruses (1×10¹⁰ genome copies) were used in each experiment. The samples were analyzed in a 1.1–1.5 g/ml discontinuous CsCl gradient by ultracentrifugation at 41,000 rpm for 10 h at 4°C in a SIW41Ti rotor. <b>(F)</b> Native 160S virions were treated at 61°C and 68°C under neutral conditions and analyzed by ultracentrifugation. <b>(G)</b> Native 160S virions were incubated with 20 µg of CypA followed by incubation at 37°C for 4 h at pH 5.5 and pH 6.5. <b>(H)</b> Native 160S virions were incubated with 20 µg of CypA or catalytic-defective mutant H126Q CypA followed by incubation at 37°C for 4 h at pH 6.0.</p

CypA interacted with the EV71 virion and the VP1 H-I loop.

<p><b>(A)</b> Inputs that were used in the GST-pulldown assays. <b>(B)</b> The S243P-EV71 virion (upper panel) exhibited a higher, but S243A/P246A-EV71 (bottom panel) showed weakened binding affinity to CypA than the wt-EV71 virion (strain AnHui1). The bands indicating the interactions between GST-CypA with wt-, S243P-, or S243A/P246A-EV71 virions were quantified. Each column represents the average of three replicates. Error bars represent the SEM. <b>(C)</b> The CypA interaction with the EV71 virion (strain AnHui1) was disrupted by CsA in a dose-dependent manner, and the S-to-P substitution rescued this interaction. A GST pulldown assay between the GST-CypA and wt-EV71 or S243P-EV71 virions was performed in the absence (lane 1) or presence (lanes 2–5) of CsA. The CsA concentrations in lanes 2–5 are 20, 4, 0.8, and 0.16 µM, respectively. Similar amounts of wt- and S243P-EV71 viruses and GST-tagged CypA were loaded into the GST-pulldown assay. The bands were quantified and each column represents the average of three replicates. Error bars represent the SEM. <b>(D)</b> CypA was bound to different EV71 virions. SK-EV006 refers to the SK-EV006-LPS1 strain, and BrCr refers to the BrCr strain. <b>(E)</b> The mutation at position 243 increases the interaction of CypA with the H-I loop of EV71 VP1. All experiments were performed at least three times. The sequence of the 1S peptide is GSSKSKYPL, and the sequence of the 1P peptide is GSSKPKYPL. The bands for the wt-EV71 interactions with GST-1S or GST-1P were quantified. Each column represents the average of three replicates. Error bars represent the SEM. <b>(F)</b> A catalytic-defective mutant of CypA called H126Q significantly attenuated the binding of wt-EV71 virions to CypA. The bands for the interactions between wt-EV71 and CypA or the CypA mutant were quantified. Each column represents the average of three replicates. Error bars represent the SEM. The bands were quantified with ImageJ software.</p

An analysis of various H-I loop mutant viruses.

<p><b>(A)</b> A sequencing chromatogram of the mutated H-I loop region. <b>(B)</b> Viral titers in the supernatant were measured at 48 hpi after the transfection of EV71 mutant viral RNA. Each column represents the average of two replicates. Error bars represent the SEM * 0.01(C) or with <b>(D)</b> inhibitor HL051001P2 treatment. The EV71 infection was quantified by using qRT-PCR to detect the EV71 RNA in cells at different times post infection, and the mRNA level of GAPDH was used as an internal control.</p

The active site of CypA in EV71 VP1.

<p><b>(A)</b> A sequence comparison of the H-I loop in VP1 for the different EV71 strains CVA16, CVB3, and PV. The H-I loop is indicated by a blue frame. The drug-resistant site (VP1-S243) and the potential CypA functional site (VP1-P266) in the H-I loop of EV71 VP1 are also indicated. <b>(B)</b> The location of the CypA functional site. Left panel, the electrostatic potential surface of five icosahedral asymmetric units (PDB code: 4AED). Negatively charged surfaces are shown in red, and positively charged areas are shown in blue. The H-I loop region was framed out. The fivefold axis is indicated by a black pentamer. In the middle panel, the molecular surfaces are around the fivefold axis of symmetry. VP1-K242 and -K244, VP1-S243, and VP1-P266 are colored green, red and gold, respectively. Right panel, detailed structure of the H-I loop of EV71 VP1-K242 and -K244, VP1-S243, and VP1-P266. The VP1 molecule is drawn as a transparent cartoon, and the specific residues are shown as colored sticks.</p

Table_1_Determination of the effective dose of dexmedetomidine to achieve loss of consciousness during anesthesia induction.DOCX

BackgroundDexmedetomidine (DEX) is a sedative with greater preservation of cognitive function, reduced respiratory depression, and improved patient arousability. This study was designed to investigate the performance of DEX during anesthesia induction and to establish an effective DEX induction strategy, which could be valuable for multiple clinical conditions.MethodsPatients undergoing abdominal surgery were involved in this dose-finding trial. Dixon's up-and-down sequential method was employed to determine the effective dose of DEX to achieve the state of “loss of consciousness”, and an effective induction strategy was established with continuous infusion of DEX and remifentanil. The effects of DEX on hemodynamics, respiratory state, EEG, and anesthetic depth were monitored and analyzed.ResultsThrough the strategy mentioned, the depth of surgical anesthesia was successfully achieved by DEX-led anesthesia induction. The ED50 and ED95 of the initial infusion rate of DEX were 0.115 and 0.200 μg/kg/min, respectively, and the mean induction time was 18.3 min. The ED50 and ED95 of DEX to achieve the state of “loss of consciousness” were 2.899 (95% CI: 2.703–3.115) and 5.001 (95% CI: 4.544–5.700) μg/kg, respectively. The mean PSI on the loss of consciousness was 42.8 among the patients. During anesthesia induction, the hemodynamics including BP and HR were stable, and the EEG monitor showed decreased α and β powers and increased θ and δ in the frontal and pre-frontal cortices of the brain.ConclusionThis study indicated that continuous infusion of combined DEX and remifentanil could be an effective strategy for anesthesia induction. The EEG during the induction was similar to the physiological sleep process.</p