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. 2020 Jul 7;32(1):107859.
doi: 10.1016/j.celrep.2020.107859.

A Non-Replicative Role of the 3' Terminal Sequence of the Dengue Virus Genome in Membranous Replication Organelle Formation

Berati Cerikan  1 , Sarah Goellner  1 , Christopher John Neufeldt  1 , Uta Haselmann  1 , Klaas Mulder  1 , Laurent Chatel-Chaix  1 , Mirko Cortese  1 , Ralf Bartenschlager  2
Affiliations

A Non-Replicative Role of the 3' Terminal Sequence of the Dengue Virus Genome in Membranous Replication Organelle Formation

Berati Cerikan et al. Cell Rep. .

Abstract

Dengue virus (DENV) and Zika virus (ZIKV), members of the Flavivirus genus, rearrange endoplasmic reticulum membranes to induce invaginations known as vesicle packets (VPs), which are the assumed sites for viral RNA replication. Mechanistic information on VP biogenesis has so far been difficult to attain due to the necessity of studying their formation under conditions of viral replication, where perturbations reducing replication will inevitably impact VP formation. Here, we report a replication-independent expression system, designated pIRO (plasmid-induced replication organelle formation) that induces bona fide DENV and ZIKV VPs that are morphologically indistinguishable from those in infected cells. Using this system, we demonstrate that sequences in the 3' terminal RNA region of the DENV, but not the ZIKV genome, contribute to VP formation in a non-replicative manner. These results validate the pIRO system that opens avenues for mechanistically dissecting virus replication from membrane reorganization.

Keywords: flavivirus; membrane invagination; membranous organelle; organelle biogenesis; replication complex; replication organelle; vesicle packet; viral replicase.

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Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Expression of the Minimal DENV Replicase Does Not Suffice to Induce VP Formation (A) Schematic representation of the DENV genome organization (left) and the T7 RNA polymerase-driven expression construct encoding the minimal DENV replicase NS1-5 (pTM/NS1-5; right panel). SL, stem-loop; UAR, upstream of AUG region; CS, cyclization sequence; DB, dumbbell; sHP, short-harpin. (B) Huh7/Lunet-T7 cells were either transfected with pTM/NS1-5 for 20 h or infected with DENV (MOI = 5) for 48 h before being lysed and subjected to western blot analysis (left and right panels, respectively). β-actin was used as loading control. (C) Relative abundance of viral proteins was determined by densitometry of the western blots, and values obtained for NS1, NS4B, or NS5 were normalized to NS3 expression levels. Values represent mean and standard error of three independent experiments. n.s., not significant. (D) Cells were infected with DENV (upper row), transfected with the pTM/NS1-5 construct (middle row), or left untreated (bottom row) and fixed for immunofluorescence analysis after 48 h (infection) or 20 h (transfection and mock). PDI (protein disulfide isomerase) and RTN3 (reticulon 3) signals serve as ER makers. Scale bars: 10 μm. (E) Nuclear localization of NS5 in DENV-infected or pTM/NS1-5-transfected cells. Scale bar: 10 μm. (F) DENV-infected or pTM/NS1-5-transfected cells were fixed after 48 h or 20 h, respectively, and processed for TEM analysis. Lower panels are magnifications of areas highlighted with yellow squares in the upper panels. Yellow arrowheads show individual VPs. Scale bars: 500 and 200 nm in upper and lower panels, respectively. (G) Transfection efficiency as determined by immunofluorescence staining of NS3. Error bars (left graph) represent the SEM of three independent experiments. Scale bar: 100 μm.
Figure 2
Figure 2
Induction of VPs upon Expression of the Minimal DENV Replicase Requires Sequences in the 3′ UTR but Not the 5′ UTR (A) Schematic of the pTM/5′UTR/NS1-5/3′UTR DENV expression plasmid (abbreviated 5′ WT). (B) Schematic representation of 5′ UTR truncations introduced into the construct shown in (A). Numbers below each construct refer to the position in the DENV-2 genome (GenBank accession number NC_001474.2). (C) Immunoblot showing the expression of NS3, NS1, NS5, and NS4B in cells 20 h after transfection with constructs specified on the top (left panel) or 48 h after DENV infection (right panel). β-actin served as sample loading control. (D) Relative abundance of viral proteins was determined by densitometry and levels of NS1, NS4B, and NS5 normalized to NS3 signal. Values represent mean and standard error of three independent experiments. n.s., not significant. (E) Thin-section TEM images of VPs induced upon transfection of indicated constructs. Cells were transfected or infected with DENV for 20 h and 48 h, respectively, before being processed for EM analysis. GND indicates the NS5 polymerase-inactivating mutation. Lower panels are magnified views of areas indicated with yellow squares in the upper panel images. Yellow arrowheads indicate individual VPs. Scale bars: 500 nm and 200 nm (upper and lower panels, respectively). (F) For each condition, whole-cell sections of 20 cells were counted. Means ± SEM from three independent quantifications are given. n.s., not significant (∗p < 0.05). (G) Vesicle diameters were measured manually using Fiji software. Means ± SEM were based on three independent measurements. For each condition, 50 vesicles were counted per experiment. n.s., not significant; ∗∗∗p < 0.001. (H) Detection of NS3 by immunofluorescence microscopy to determine transfection efficiency for the indicated constructs. Scale bar: 100 μm. (I) Transfection efficiencies were quantified according to NS3 staining. Error bars represent the standard error of three independent experiments. n.s., not significant. Note that percentages given in (F) refer to all cells analyzed by TEM, but only ~70% of them had been transfected (I); therefore, not more than 70% of cells can contain VPs.
Figure 3
Figure 3
ZIKV 5′ UTR Is Dispensable for Expression-Based Induction of VPs (A and B) Schematic of the pTM/5′UTR/NS1-5/3′UTR ZIKV expression plasmid (abbreviated as 5′ WT) (A) and the 5′ UTR deletion introduced into this construct (B). The number in the bottom refers to position in the ZIKV H/PF/2013 genome (GenBank accession number KJ776791.2). (C) Immunoblot showing abundance of ZIKV NS3, NS1, NS4A, and NS2B in transfected and infected (MOI = 10) cells (left and right panel, respectively). GAPDH served as sample loading control. (D) Relative abundance of ZIKV proteins was determined by densitometry and levels of NS1, NS2B, and NS4A normalized to NS3 signal. Values represent mean and standard error of three independent experiments. n.s., not significant. (E) Thin-section TEM images showing VPs induced upon transfection of constructs specified on the top. Cells were transfected or infected with ZIKV for 18 h and 24 h, respectively, before being processed for EM analysis. GAA indicates the NS5 polymerase-inactivating mutation. Lower panels are magnified views of areas indicated with yellow squares in the upper panel images. Yellow arrowheads indicate individual VPs. Scale bars: 500 nm and 200 nm (upper and lower panels, respectively). (F) For each condition, VPs contained in whole-cell sections from at least 20 cells were counted. Means ± SEM from three independent quantifications are given. n.s., not significant; ∗∗p < 0.01. (G) Vesicle diameters were measured manually using Fiji software. Means ± SEM were calculated from three independent measurements. For each condition, 50 vesicles were counted per experiment. n.s., not significant. (H) Detection of ZIKV NS4B by immunofluorescence microscopy to determine transfection efficiency for the constructs given on the top of each panel. Scale bar: 100 μm. (I) Transfection efficiencies were quantified by counting NS4B-containing cells and normalization to the total number of cells. Error bars represent the standard error of three independent experiments. n.s., not significant. Note that percentages given in (F) refer to all cells analyzed by TEM, but only a fraction of them had detectable amounts of ZIKV NS4B that were used to determine transfection efficiency (I). The higher percentage of cells with VPs attained with some constructs is most likely due to lower sensitivity of the immunofluorescence, allowing detection only of cells expressing high amounts of NS4B.
Figure 4
Figure 4
Electron Tomography of Expression-Induced DENV VPs (A–B) (A) Huh7/Lunet-T7 cells were fixed 24 h post-transfection with the pIRO-D Δ 5′ SLAB construct, embedded in epoxy resin, and analyzed by electron tomography. A tomographic slice showing pIRO-D-induced VPs within the ER is displayed. The red rectangle indicates the cropped section shown in (B), which is a magnified view of the ER lumen-containing vesicles. Two adjacent vesicles within the swollen ER lumen are shown (yellow arrowhead). Red arrowhead shows the pore-like opening to the cytosol. (C–F) 3D surface model of the VPs next to a lipid droplet (magenta). The red rectangles in (C) and (E) indicate the cropped magnified sections shown in (D) and (F). Yellow arrowhead points to the adjacent vesicles shown in (B). (G) Side view of the 3D model showing the pore-like opening (red arrowhead). Scale bars: 50 nm.
Figure 5
Figure 5
Importance of the 3′ SL for DENV RO Formation (A) Schematic representation of 3′ UTR truncations introduced into the pIRO-D Δ 5′ SLAB construct. Numbers refer to the deleted nucleotide positions of the DENV 2 genome (GenBank accession number NC_001474.2). (B) Analysis of viral protein production in Huh7/Lunet-T7 cells 20 h after transfection with given constructs. β-actin served as sample loading control. (C) Relative abundance of viral proteins was determined by densitometry, and levels of NS1, NS4B, and NS5 were normalized to NS3 signal. Values represent mean and standard error of three independent experiments. n.s., not significant. (D) TEM images of Huh7/Lunet-T7 cells transfected with indicated 3′ UTR mutants or the 3′ WT reference construct. Cells were transfected and, after 20 h, fixed, processed, and embedded in resin for sectioning. Upper panel scale bar: 500 nm. Lower panels are magnifications of yellow squared areas in the upper panel images. Yellow arrowheads indicate individual VPs. Lower panel scale bar: 100 nm. (E) For each condition, VPs contained in whole-cell sections from 20 cells or, in the case of the Δ 3′ SL mutant, 40 cells were counted. Means ± SEM from three independent experiments are shown. n.s., not significant; ∗∗p < 0.01. (F) Vesicle diameters were measured by analyzing 25 vesicles per experimental condition using Fiji software. Means ± SEM from three independent experiments are given. n.s., not significant; ∗∗∗p < 0.001. (G) Detection of NS3 by immunofluorescence microscopy to determine transfection efficiency for the indicated constructs. Nuclear DNA was stained with DAPI. Scale bar: 100 μm. (H) Transfection efficiencies were quantified by counting NS3-containing cells and normalization to the total number of cells. Error bars represent the standard error of three independent experiments. n.s., not significant. Note that percentages given in (E) refer to all cells analyzed by TEM, but only ~60% of them had been transfected (H); therefore, not more than 60% of cells can contain VPs.
Figure 6
Figure 6
Elements in the ZIKV 3′ UTR Contribute to, But Are Not Essential for, VP formation (A) Schematic representation of deletions introduced into the 3′ UTR of the parental pIRO-Z Δ 5′ SLAB construct. Numbers refer to the last nucleotide of the ZIKV genome contained in the expression construct (ZIKVH/PF/2013strain; GenBank accession number KJ776791.2). (B) Huh7/Lunet-T7 cells were transfected with the parental construct (3′ WT), indicated mutants, or mock transfected (Control). After 18 h, cells were lysed and analyzed by western blot. GAPDH served as loading control. (C) Relative ZIKV protein abundance was quantified by densitometry of western blots, and values for NS1, NS2B, or NS4A were normalized to NS3. Values represent mean and standard error of three independent experiments. n.s., not significant. (D) TEM images of Huh7/Lunet-T7 cells 18 h after transfection with indicated 3′ UTR mutants. Cells were fixed, processed, and embedded in resin for sectioning. Lower panels are magnified views of areas indicated with yellow squares in the upper panel images. Yellow arrowheads indicate individual VPs. Scale bars: 500 nm and 200 nm (upper and lower panels, respectively). (E) For each condition, VPs contained in whole-cell sections from 40 cells were counted. Means ± SEM from three independent quantifications are shown. ∗p < 0.05; ∗∗p < 0.01. Quantification of 3′ WT is taken from Figure 3F (Δ 5′ SLAB). (F) Vesicle diameters were measured manually using Fiji software. Means ± SEM from three independent measurements (50 vesicles per experiment) are given. n.s., not significant. Vesicle diameter quantification of 3′ WT is taken from Figure 3F (parental construct pIRO-Z Δ 5′ SLAB containing an unaltered 3′ UTR). (G) Detection of NS4B by immunofluorescence microscopy to determine transfection efficiency for the indicated constructs. Scale bar: 100 μm. (H) Transfection efficiencies were quantified by counting NS4B-positive cells and normalization to the total cell number. Error bars represent the standard error of three independent experiments. n.s., not significant. Note that percentages given in (E) refer to all cells analyzed by TEM, but only ~60% of them had been transfected (H); therefore, not more than 60% of cells can contain VPs.
Figure 7
Figure 7
Critical Role of an Authentic 3′ UTR for VP Formation and Rescue of DENV Organelle Formation by the ZIKV 3′ SL (A) Schematic representation of the 3′ terminal regions of used constructs containing or not containing the HDV ribozyme. Sites of RNA cleavage are indicated with arrows. (B) TEM images of Huh7/Lunet-T7 cells transfected with the given pIRO-D constructs. Cells were transfected and, 20 h later, fixed, processed, and embedded in resin for sectioning. Upper panel scale bar: 500 nm. Lower panels are magnifications of yellow squared areas in the upper panel images. Yellow arrowheads indicate individual VPs. Lower panel scale bar: 200 nm. (C) For each condition, VPs contained in whole-cell sections from 20 cells were counted. Means ± SEM from three independent quantifications are given. ∗∗p < 0.01. n.d., not detectable. (D) Schematic representation of the 3′ terminal regions of the DENV constructs containing only the 3′ SL or the complete 3′ UTR of ZIKV (left and right, respectively). (E) TEM images of Huh7/Lunet-T7 cells transfected with the constructs specified at the top of each panel. Cells were transfected and, 20 h later, fixed, processed, and embedded in resin for sectioning. Upper panel scale bar: 500 nm. Lower panels are magnifications of yellow squared areas in the upper panel images. Yellow arrowheads indicate individual VPs. Lower panel scale bar: 100 nm. (F) For each condition, VPs contained in whole-cell sections from 20 cells were counted. Means ± SEM from three independent quantifications are given. ∗p < 0.1; n.s., not significant; n.d., non-detectable.
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