Journal: PLoS Neglected Tropical Diseases

Article Title: Intron-derived small RNAs for silencing viral RNAs in mosquito cells

doi: 10.1371/journal.pntd.0010548

Figure Lengend Snippet: (A) Schematic of the CHIKV genome and the location of the 268 nt nsP2 target region (CHI) and each of the 10 sites targeted by small RNAs. (B) Schematic of small RNA expressing plasmids. (C) Strategy for testing small RNA mediated suppression of a CHIKV split replication system (CHIKVRep, replication complex nsP1-4 and viral reporter), chimeric firefly luciferase reporter (CHILuc) and synthetic reporter with the target sequence in the 3’ UTR (LucCHI). Small RNAs containing sequence mismatches (nt 2 and 11) or fully complementary sequences were assessed for their ability to target viral sequences in mosquito cell lines. AAA: polyadenylation signal, Hr5-IE1: Autographa californica nuclear polyhedrosis virus homologous region 5 enhancer-immediate-early gene 1 promoter, Luc: luciferase, mB: Drosophila melanogaster pre- miRNA-1 miRNA backbone, nt: nucleotide, ORF: open reading frame, PUb intron: Ae . aegypti polyubiquitin gene intron, SG: CHIKV subgenomic promoter, SV40: simian virus 40 polyadenylation signal, UTR: untranslated regions, ZsY: ZsYellow fluorescent protein.

Article Snippet: The D . melanogaster pre- miRNA1 stem loop sequence validated by Haley et al. [ ] was modified to target a 268 nt conserved region of the nsP2 viral protease coding region of CHIKV (CHI, ).

Techniques: Expressing, Luciferase, Sequencing, Virus

Journal: PLoS ONE

Article Title: In vitro study of Hesperetin and Hesperidin as inhibitors of zika and chikungunya virus proteases

doi: 10.1371/journal.pone.0246319

Figure Lengend Snippet: Based on PAHO and WHO (PAHO/WHO, 2020) . ZIKV infections colored in blue, DENV in orange and CHIKV in green. A: Confirmed ZIKV, DENV and CHIKV cases in the Americas from 2015 to 2019. B: Confirmed ZIKV, DENV and CHIKV cases in Brazil from 2015 to 2019. C: Confirmed ZIKV, DENV and CHIKV cases in Brazil 2016. D: Confirmed ZIKV, DENV and CHIKV cases in Brazil 2017.

Article Snippet: The DNA fragment encoding CHIKV nsP2 pro (residues 466–798) containing the N-terminal cysteine protease domain and the C-terminal SAM-dependent methyltransferase domain (GenBank Protein Accession number AAN05101.1, strain S27-African prototype) was synthesized (BioCat GmbH, Heidelberg, Germany) and implemented in the kanamycin resistant vector pET-24a (+).

Techniques:

Journal: PLoS ONE

Article Title: In vitro study of Hesperetin and Hesperidin as inhibitors of zika and chikungunya virus proteases

doi: 10.1371/journal.pone.0246319

Figure Lengend Snippet: The tested compound concentration was 20 µM. HST inhibit ZIKV NS2B/NS3 pro activity about 40% and CHIKV nsP2 pro about 90%. The inhibition of CHIKV protease activity by HSD was higher than 60%. Contrary, HSD shows not relevant inhibition against ZIKV NS2B/NS3 pro . Data shown are the means ± SD from three independent measurements (n = 3). Asterisks mean that the data differs from the control (0 µM inhibitor) significantly at p < 0.01 (**) and p< 0.001 (***), level according to ANOVA and Tukey’s test.

Article Snippet: The DNA fragment encoding CHIKV nsP2 pro (residues 466–798) containing the N-terminal cysteine protease domain and the C-terminal SAM-dependent methyltransferase domain (GenBank Protein Accession number AAN05101.1, strain S27-African prototype) was synthesized (BioCat GmbH, Heidelberg, Germany) and implemented in the kanamycin resistant vector pET-24a (+).

Techniques: Concentration Assay, Activity Assay, Inhibition, Control

Journal: PLoS ONE

Article Title: In vitro study of Hesperetin and Hesperidin as inhibitors of zika and chikungunya virus proteases

doi: 10.1371/journal.pone.0246319

Figure Lengend Snippet: Chemical structures of HST and HSD (structural formula) and CHIKV nsP2 pro 3D experimental model (PDB code: 3TRK) are shown. CHIKV nsP2 protease domain is presented in pink and the methyltransferase domain in cyan. Normalized activity and inhibition of the virus protease and Lineweaver-Burk plots to determine the inhibition mode is presented: [S] is the substrate concentration; v is the initial reaction rate. The data shown are the means ± SD from three independent measurements (n = 3). A and C: Normalized activity and inhibition of CHIKV nsP2 pro under HST and HSD influence, respectively. B and D: Lineweaver-Burk plot for HST and HSD inhibition of CHIKV nsP2 pro , respectively.

Article Snippet: The DNA fragment encoding CHIKV nsP2 pro (residues 466–798) containing the N-terminal cysteine protease domain and the C-terminal SAM-dependent methyltransferase domain (GenBank Protein Accession number AAN05101.1, strain S27-African prototype) was synthesized (BioCat GmbH, Heidelberg, Germany) and implemented in the kanamycin resistant vector pET-24a (+).

Techniques: Activity Assay, Inhibition, Virus, Concentration Assay

Journal: PLoS ONE

Article Title: In vitro study of Hesperetin and Hesperidin as inhibitors of zika and chikungunya virus proteases

doi: 10.1371/journal.pone.0246319

Figure Lengend Snippet: Results summarize HST and HSD inhibition experiments of ZIKV NS2B/NS3 pro , and CHIKV nsP2 pro .

Article Snippet: The DNA fragment encoding CHIKV nsP2 pro (residues 466–798) containing the N-terminal cysteine protease domain and the C-terminal SAM-dependent methyltransferase domain (GenBank Protein Accession number AAN05101.1, strain S27-African prototype) was synthesized (BioCat GmbH, Heidelberg, Germany) and implemented in the kanamycin resistant vector pET-24a (+).

Techniques: Inhibition

Journal: PLoS ONE

Article Title: In vitro study of Hesperetin and Hesperidin as inhibitors of zika and chikungunya virus proteases

doi: 10.1371/journal.pone.0246319

Figure Lengend Snippet: Results summary of the fluorescence spectroscopy experiments of ZIKV NS2B/NS3 pro and CHIKV nsP2 pro with HST and HSD.

Article Snippet: The DNA fragment encoding CHIKV nsP2 pro (residues 466–798) containing the N-terminal cysteine protease domain and the C-terminal SAM-dependent methyltransferase domain (GenBank Protein Accession number AAN05101.1, strain S27-African prototype) was synthesized (BioCat GmbH, Heidelberg, Germany) and implemented in the kanamycin resistant vector pET-24a (+).

Techniques: Fluorescence, Spectroscopy

Journal: PLoS ONE

Article Title: In vitro study of Hesperetin and Hesperidin as inhibitors of zika and chikungunya virus proteases

doi: 10.1371/journal.pone.0246319

Figure Lengend Snippet: A: Decomposition of the binding energy of CHIKV nsP2 pro -HST complex. Arrows label the protease domain (gray) and methyltransferase domain (green). The residues involved in the interaction between protease and HST are labeled. B: Decomposition of the binding energy of CHIKV nsP2 pro -HSD complex. The residues involved in the interaction between protease and HSD are labeled. C: 3D structure of CHIKV nsP2 pro (PDB entry: 3TRK), the protease domain is colored in gray and methyltransferase domain in green. Amino acids highlighted that are involved in the interaction with HST (yellow) based on MD simulations. The H-bond between Lys89 and HST is highlighted. D: 3D structure of CHIKV nsP2 pro , amino acids highlighted are involved in the interaction with HSD (yellow) based on MD simulations. The H-bonds between Tyr77, Met240, Val75 and Glu48 with HSD are highlighted. E: Structural overlay of the representative CHIKV nsP2 pro -HST complex structures of two independent MD runs (RMSD: 1.259 Å). Run1: nsP2 pro (gray), HST (yellow) and run2: nsP2 (brown), HST (orange). The binding region of HST is highlighted, the involved amino acids and HST position differs between run1 and run2. In run2 Pro47, Leu51, Leu63 and Ph91 are not interacting by hydrophobic interactions with HST. However, Lys89 forms in run2 like in run1 a hydrogen bond to HST, the distances between donor and acceptor differs slightly between MD run1 and run2 (3.4 to 3.5 Å). F: Structural overlay of the representative CHIKV nsP2 pro -HSD complex structures of two independent MD runs (RMSD: 1.733 Å). Run1: nsP2 pro (gray), methyltransferase domain (green), HSD (yellow) and run2: nsP2 (brown), methyltransferase domain (cyan), HST (orange). The binding region of HST is highlighted, the involved amino acids and HST position differs slightly between run1 and run2. The main difference concerns Met240, which forms in run1 a hydrogen bond, in run2 this interaction is not more formed, the distance increased from 3.0 to 5.5 Å. Because of this observation, Met240 will be not further considered. The hydrogen bonds formed by Glu48, Val75 and Tyr77 remain unaffected.

Article Snippet: The DNA fragment encoding CHIKV nsP2 pro (residues 466–798) containing the N-terminal cysteine protease domain and the C-terminal SAM-dependent methyltransferase domain (GenBank Protein Accession number AAN05101.1, strain S27-African prototype) was synthesized (BioCat GmbH, Heidelberg, Germany) and implemented in the kanamycin resistant vector pET-24a (+).

Techniques: Binding Assay, Labeling

Journal: PLoS ONE

Article Title: In vitro study of Hesperetin and Hesperidin as inhibitors of zika and chikungunya virus proteases

doi: 10.1371/journal.pone.0246319

Figure Lengend Snippet: Residues involved in forming H-bonds and hydrophobic contacts between the proteins and the ligands.

Article Snippet: The DNA fragment encoding CHIKV nsP2 pro (residues 466–798) containing the N-terminal cysteine protease domain and the C-terminal SAM-dependent methyltransferase domain (GenBank Protein Accession number AAN05101.1, strain S27-African prototype) was synthesized (BioCat GmbH, Heidelberg, Germany) and implemented in the kanamycin resistant vector pET-24a (+).

Techniques: Residue

Journal: PLoS ONE

Article Title: In vitro study of Hesperetin and Hesperidin as inhibitors of zika and chikungunya virus proteases

doi: 10.1371/journal.pone.0246319

Figure Lengend Snippet: Atoms involved in the H-Bond interaction between flavivirus and alphavirus proteases and ligands.

Article Snippet: The DNA fragment encoding CHIKV nsP2 pro (residues 466–798) containing the N-terminal cysteine protease domain and the C-terminal SAM-dependent methyltransferase domain (GenBank Protein Accession number AAN05101.1, strain S27-African prototype) was synthesized (BioCat GmbH, Heidelberg, Germany) and implemented in the kanamycin resistant vector pET-24a (+).

Techniques: