strain new guinea c  (ATCC)

Journal: bioRxiv

Article Title: Sec61 translocon inhibitor Flavitransin blocks selectively dengue virus polyprotein insertion into the ER membrane with pan-flavivirus antiviral potency

doi: 10.1101/2025.03.29.646078

Figure Lengend Snippet: A , Schematic representation of a flaviviral polyprotein embedded in the ER membrane with 18 membrane-spanning domains. The different proteolytic cleavage sites are indicated by colored scissors. The viral protease, formed by NS3 and its cofactor NS2B (blue scissors), releases the viral subunits at the cytoplasmic side of the ER membrane. Shaded transmembrane domains (boxes) indicate cleavable SP-like targeting sequences for Sec61 translocation. B , Chemical structure of FT and FT IN . C , Four parameter concentration-response curve of FT or FT IN for virus yield (log 10 reduction in copy number compared to untreated virus control) in DENV2 NGC-infected (MOI 1) Vero cells. Compound treatment started 2 h before infection, and supernatant was collected at 4-5 days post infection for RT-qPCR detection of the viral 3’UTR. Data points show mean values ± SD, n=3. D , Four parameter concentration-response curve of FT or FT IN for viability of Vero cells. Cytotoxic effects were determined at day 5 post treatment by MTS/PMS assay. Data are normalized to untreated cell controls. Graph shows mean values ± SD, n=3. E , Same as in (C) but for Huh7 cells. F , Viral E protein detection in DENV2 NGC (MOI 0.3) infected Huh7 cells, treated with FT or 10 µM FT IN (indicated as ‘IN’) for 48 h. Immunoblot on cell lysate with anti-E or anti-clathrin (loading control) antibody. G , Viral E protein detection in DENV2 NGC (MOI 0.5) infected Huh7 cells, treated with 2 µM FT or 10 µM FT IN for 72h. Flow cytometric histogram plots show mean fluorescence intensity (MFI) values for E protein, acquired from at least 5,000 cells. Mock-infected cells are in black dotted line, virus infected control cells are in green line, FT treated infected cells in red and FT IN infected cells in grey. Representative histogram plots are given for one out of two experiments. H , Time-of-drug-addition assay in which Vero cells were treated with the attachment inhibitor Dextran Sulfate (DS, 4 mg/ml), FT (1 µM) or the viral polymerase inhibitor NITD008 (10 µM) starting before infection (-30’), at the time of infection with DENV2 NGC (0 h) or at several time points after infection (4, 8, 12, 16, 20, 23 h) . Cells were exposed to virus for 2 h and excess of virus was then removed. Compounds were present till end of experiment. At 24 hpi, cells were lysed for viral RNA quantification by RT-qPCR (3’UTR). Graphs represent the viral load as normalized to the viral copy number of the virus control from 3 independent experiments. Values are mean ± SD; n=3.

Article Snippet: The laboratory-adapted DENV2 New Guinea-C strain (NGC; VR-1584) and the prototype ZIKV strain MR766 (MK105975.1; VR-84) were purchased at ATCC.

Techniques: Membrane, Translocation Assay, Concentration Assay, Virus, Control, Infection, Quantitative RT-PCR, Western Blot, Fluorescence

Journal: bioRxiv

Article Title: Sec61 translocon inhibitor Flavitransin blocks selectively dengue virus polyprotein insertion into the ER membrane with pan-flavivirus antiviral potency

doi: 10.1101/2025.03.29.646078

Figure Lengend Snippet: A , Schematic representation of the plasmid constructs expressing DENV2 C 14 -prM-E and E 23 -NS1-V5. N-linked glycosylations are indicated by an asterisk. B, HEK293T cells were transiently transfected with the C 14 -prM-E construct and treated with FT for 18 h. Cells were lysed in NP-40 buffer and analyzed by immunoblotting with an anti-E or an anti-prM antibody. For the cell loading control, an anti-clathrin antibody was used. A representative immunoblot is shown on the left. Bar graphs represent relative prM and E levels from clathrin-corrected samples, normalized to untreated transfected controls. Bars are mean ± SD, n=3. *P< 0.05, **P<0.01, ***P<0.001, ****P<0.0001, compared to untreated transfection control (two-tailed/multiple unpaired t test with Welch’s correction). Note that the processing of prM into pr and M by furin cleavage was not successful in HEK293T cells. C , Same as in (B) but with the E 23 -NS1-V5 construct and with anti-NS1 or anti-V5 antibodies. n.s. = P>0.05.

Article Snippet: The laboratory-adapted DENV2 New Guinea-C strain (NGC; VR-1584) and the prototype ZIKV strain MR766 (MK105975.1; VR-84) were purchased at ATCC.

Techniques: Plasmid Preparation, Construct, Expressing, Transfection, Western Blot, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Sec61 translocon inhibitor Flavitransin blocks selectively dengue virus polyprotein insertion into the ER membrane with pan-flavivirus antiviral potency

doi: 10.1101/2025.03.29.646078

Figure Lengend Snippet: A , Schematic representation of the DENV prM and E transcripts for in vitro translation/translocation assay in which the transcripts are translated in rabbit reticulocyte lysate (RRL) supplemented with ovine rough microsomes (RM) and [ 35 S]-labeled methionine. Only fully translocated proteins in the ER lumen are protected from proteinase K (PK) treatment. Red scissors indicate signal peptidase cleavage sites and the asterisks represent the N-linked glycosylation sites. Cartoon created with BioRender (2025); www.bioRender.com . B , Transcripts encoding DENV2 C 14 -prM 166 were translated and/or translocated in the assay described in (A). Samples were left untreated or were treated with the indicated concentrations of FT or with 15 µM of FT IN . Half of the translated material of each sample was additionally treated with PK. Samples were separated by SDS-PAGE and analyzed by autoradiography. A representative autoradiogram is shown on the left. Graph on the right shows the relative translocation as normalized to the DMSO control from 2 independent experiments. Bars represent mean +/- SD; n=2. ***P<0.001; n.s. = P>0.05 for multiple unpaired t tests with Welch’s correction. Solid arrowhead: unprocessed nascent protein; asterisk: glycosylated (translocated) mature protein. C , Same as in (B) but for the M 21 -E 122 construct. Open arrowhead: SP-cleaved protein that is not being glycosylated. Only a small fraction of these species are translocated into the ER lumen and protected from PK.

Article Snippet: The laboratory-adapted DENV2 New Guinea-C strain (NGC; VR-1584) and the prototype ZIKV strain MR766 (MK105975.1; VR-84) were purchased at ATCC.

Techniques: In Vitro, Translocation Assay, Labeling, Glycoproteomics, SDS Page, Autoradiography, Control, Construct

Journal: bioRxiv

Article Title: Sec61 translocon inhibitor Flavitransin blocks selectively dengue virus polyprotein insertion into the ER membrane with pan-flavivirus antiviral potency

doi: 10.1101/2025.03.29.646078

Figure Lengend Snippet: A , Schematic representation of the modified DENV2 C 14 -prM-E construct with a V5 tag in the pr protein, a FLAG tag in the M protein and a MYC tag in the E protein (tags are represented by colored boxes). Transmembrane domains are indicated by shaded boxes. N-glycosylation sites are indicated by an asterisk. B , CHO-K1 cells were transiently transfected (TF) with the construct from (A) and treated with FT, FT IN (10 µM) and/or the proteasome inhibitor MG132 (200 nM). Compound treatment was started at 6 h post transfection for 18 h and cells were lysed in NP-40 buffer and analyzed by immunoblotting. For the cell loading control, an anti-clathrin antibody was used. The uncleaved precursor (prM-E) is indicated by a black arrow. One representative experiment out of three is shown. C , Cell lysate of control samples from (B) was incubated with Endo-H for removal of N-glycosylation and subjected to immunoblotting like in (B). UG: unglycosylated. D , V5 pull down. Samples from (B) were incubated with anti-V5 beads for pull down of the V5-tagged protein species and immunostained with anti-FLAG and anti-MYC antibodies. Black arrow indicates the uncleaved precursor that is already detectable in the FT sample without MG132. Note that some processed E protein is co-immunoprecipitated with prM as viral envelope protein complexes (MYC detection). In the control sample, an additional species with a higher molecular weight is detected (*), presumably a glycosylated precursor protein (∼ 80kDa) that was not properly cleaved into the prM and E subunits.

Article Snippet: The laboratory-adapted DENV2 New Guinea-C strain (NGC; VR-1584) and the prototype ZIKV strain MR766 (MK105975.1; VR-84) were purchased at ATCC.

Techniques: Modification, Construct, FLAG-tag, Glycoproteomics, Transfection, Western Blot, Control, Incubation, Immunoprecipitation, Molecular Weight

Journal: bioRxiv

Article Title: Sec61 translocon inhibitor Flavitransin blocks selectively dengue virus polyprotein insertion into the ER membrane with pan-flavivirus antiviral potency

doi: 10.1101/2025.03.29.646078

Figure Lengend Snippet: A , Schematic outline of the protocol used to select for FT-desensitized cells. HCT116 cells were seeded at low density in suspension in the presence of FT (25 µM). After 9 days, surviving cells were subcloned, expanded and genotyped. Cartoon created with BioRender (2025); www.bioRender.com . B , Four-parameter concentration-response curves of FT for the proliferation of WT and FT-resistant HCT116 cells. After 72 h of FT incubation, cell proliferation was quantified by measuring resazurin:resorufin conversion. For each cell clone, data are normalized to the DMSO control. Values are mean ± SD; n=3. C, WT and FT-resistant HCT116 cells were infected with DENV2 (MOI 1) and incubated for 5 days in the presence of various FT concentrations. Viral copy number in the supernatant was quantified by RT-qPCR. Graph shows the four-parameter concentration-response curves of FT for viral replication (log 10 reduction in viral load compared to untreated infected controls). Values are mean± SD; n=3. D , WT and FT-resistant HCT116 cells were transiently transfected with a plasmid encoding for DENV2 C 14 -prM 62 -VSV-G protein (same as in ) and treated with FT for 18h. GFP and BFP expression (mean fluorescent intensity) was quantified by flow cytometry and normalized to untreated transfection control. Graph shows the four-parameter concentration-response curves of FT for the tGFP:BFP ratio. Values are mean ± SD; n = 3. See also Suppl. Fig. 8B,C. E , WT and Sec61α A70V mutant HCT116 cells were semi-permeabilized with digitonin and used as RM source in an in vitro translation/translocation assay. Transcripts from a N-Glyc-C 14 -prM 8 -pPL construct were translated and translocated in rabbit reticulocyte lysate with [ 35 S] methionine, FT or FT IN (100 µM), and extracted membranes from semi-permeabilized cells. The pPL construct contains the C14 sequence of DENV that is N-terminally elongated (17 residues) with a N-glycosylation sequon as previously described . Samples were separated by SDS-PAGE and analyzed by autoradiography. The percentage of translocated (glycosylated) protein (relative to unprocessed precursor) is marked below each lane. Translation in absence of membranes produces the unprocessed nascent protein (solid arrowhead). N-glycosylated species are indicated by an asterisk. One out of three representative experiments is shown. See also Suppl. Fig. 8D.

Article Snippet: The laboratory-adapted DENV2 New Guinea-C strain (NGC; VR-1584) and the prototype ZIKV strain MR766 (MK105975.1; VR-84) were purchased at ATCC.

Techniques: Suspension, Concentration Assay, Incubation, Control, Infection, Quantitative RT-PCR, Transfection, Plasmid Preparation, Expressing, Flow Cytometry, Mutagenesis, In Vitro, Translocation Assay, Construct, Sequencing, Glycoproteomics, SDS Page, Autoradiography

Journal: bioRxiv

Article Title: Sec61 translocon inhibitor Flavitransin blocks selectively dengue virus polyprotein insertion into the ER membrane with pan-flavivirus antiviral potency

doi: 10.1101/2025.03.29.646078

Figure Lengend Snippet: A , DENV2 was passaged on Huh7 cells under FT pressure. Compound concentration was gradually increased when viral cytopathic effect on cells was detected. After 75 passages, virus was cultivated in the absence of compounds to generate virus stocks for downstream analysis. At passage 50 and 75, virus was collected and genotyped by nanopore sequencing (see panel C). FT resistance was selected in 2 independent cell cultures. As controls, a culture with FT IN or with control medium was included that underwent the same passaging procedure. Cartoon created with BioRender (2025); www.bioRender.com . B , At end-point, virus stocks were evaluated in Huh7 cells for sensitivity to FT. A virus stock from a low passage (# 3; light blue) was included as reference. Cells were infected with the different virus stocks (MOI ∼ 0.5) and treated with increasing concentrations of FT. After 4-5 days post infection, virus yield in the supernatant was quantified by RT-qPCR detection of the viral 3’UTR. Graph shows the four parameter concentration-response curves of FT for virus release from infected cells. Values (mean ± SD, from two independent experiments; n=2) represent the viral load in FT-treated cells as normalized to the viral copy number of each untreated virus control. C , Summary of the detected AA mutations in the different viral clones as compared to the initial DENV2 control strain (# 3). The numbers in each colored box refer to the residues of the respective viral subunit. The mutations in the medium control (dark blue) and FT IN (grey) are shown at the top; the two FT-resistant viruses (dark and light red) are shown at the bottom. D , Schematic representation of the hydrophobic mutants of the DENV2 C 14 SPs. HEK293T cells were transiently transfected with the VSV-G-tGFP-P2A-BFP constructs and treated with FT for 18 h. GFP and BFP fluorescence (mean fluorescent intensity) was quantified by flow cytometry and relative tGFP:BFP expression ratio (normalized to untreated transfection control) was determined. Graph on the left shows the four parameter concentration response curves of FT for VSV expression for different C14 mutants from three independent experiments. Values are mean ± SD; n=3. Graph at the right shows the relative tGFP:BFP expression ratios at 2 µM FT for additional hydrophobic mutants (A102V/I/L/M/F/Y/W). Values are mean ± SD; n=3.

Article Snippet: The laboratory-adapted DENV2 New Guinea-C strain (NGC; VR-1584) and the prototype ZIKV strain MR766 (MK105975.1; VR-84) were purchased at ATCC.

Techniques: Concentration Assay, Virus, Nanopore Sequencing, Control, Passaging, Infection, Quantitative RT-PCR, Clone Assay, Transfection, Construct, Fluorescence, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Sec61 translocon inhibitor Flavitransin blocks selectively dengue virus polyprotein insertion into the ER membrane with pan-flavivirus antiviral potency

doi: 10.1101/2025.03.29.646078

Figure Lengend Snippet: Structural superposition of snapshots from MD simulation #1 (MD1) of SP and inhibitor bound Sec61 at t= 0 ns (cyan) and at t = 200 ns (orange). Sec61 is shown in surface, SP (DENV2 C14) in cartoon and FT is shown in stick representation, respectively.

Article Snippet: The laboratory-adapted DENV2 New Guinea-C strain (NGC; VR-1584) and the prototype ZIKV strain MR766 (MK105975.1; VR-84) were purchased at ATCC.

Techniques: