A novel interaction between dengue virus nonstructural protein 1 and the NS4A-2K-4B precursor is required for viral RNA replication but not for formation of the membranous replication organelle

<div><p>Dengue virus (DENV) has emerged as major human pathogen. Despite the serious socio-economic impact of DENV-associated diseases, antiviral therapy is missing. DENV replicates in the cytoplasm of infected cells and induces a membranous replication organelle, formed by invaginations of the endoplasmic reticulum membrane and designated vesicle packets (VPs). Nonstructural protein 1 (NS1) of DENV is a multifunctional protein. It is secreted from cells to counteract antiviral immune responses, but also critically contributes to the severe clinical manifestations of dengue. In addition, NS1 is indispensable for viral RNA replication, but the underlying molecular mechanism remains elusive. In this study, we employed a combination of genetic, biochemical and imaging approaches to dissect the determinants in NS1 contributing to its various functions in the viral replication cycle. Several important observations were made. First, we identified a cluster of amino acid residues in the exposed region of the <i>β-ladder</i> domain of NS1 that are essential for NS1 secretion. Second, we revealed a novel interaction of NS1 with the NS4A-2K-4B cleavage intermediate, but not with mature NS4A or NS4B. This interaction is required for RNA replication, with two residues within the connector region of the NS1 “<i>Wing</i>” domain being crucial for binding of the NS4A-2K-4B precursor. By using a polyprotein expression system allowing the formation of VPs in the absence of viral RNA replication, we show that the NS1 –NS4A-2K-4B interaction is not required for VP formation, arguing that the association between these two proteins plays a more direct role in the RNA amplification process. Third, through analysis of polyproteins containing deletions in NS1, and employing a <i>trans</i>-complementation assay, we show that both <i>cis</i> and <i>trans</i> acting elements within NS1 contribute to VP formation, with the capability of NS1 mutants to form VPs correlating with their capability to support RNA replication. In conclusion, these results reveal a direct role of NS1 in VP formation that is independent from RNA replication, and argue for a critical function of a previously unrecognized NS4A-2K-NS4B precursor specifically interacting with NS1 and promoting viral RNA replication.</p></div

Exposed residues in the <i>Wing</i> and <i>β-ladder</i> domain of NS1 mediate interaction with DENV structural proteins.

<p>(<b>A</b>) <i>Pull-down of wild-type or mutant NS1 in the context of ΔNS1</i>^<i>TCP</i><i>infection</i>. Naïve VeroE6_NS1^WT (WT) or VeroE6 cells stably expressing different HA-tagged NS1 mutants were infected with 1 MOI of DVR2A^ΔNS1. Two days post-infection, cell monolayers were lysed and subjected to HA pull-down as described in the legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005277#ppat.1005277.g007" target="_blank">Fig 7B</a>. Whole cell lysates (Input) or eluates (IP) were resolved on SDS-PAGE and analyzed by immunoblot using mono-specific antibodies given on the right of each panel. Numbers on the left refer to molecular weight standards expressed in kDa. (N.I., non-infected). (<b>B</b>) <i>Relative Envelope and prM IP efficiency</i>. Relative co-immunoprecipitation efficiency was calculated by densitometry normalizing the signals of Envelope or prM to immunoprecipitated NS1. Values represent mean and standard error of two to three independent experiments. (*** p<0.001; * p<0.05). (<b>C</b>) Cell culture supernatants from (A) were clarified by low-speed centrifugation, resolved on SDS-PAGE and analyzed by immunoblot using given antibodies. All the images are representative of 3 to 4 independent experiments. (<b>D</b>) <i>Localization of residues within the NS1 dimer involved in infectious particle production</i>. The DENV NS1 dimer structure (Protein Data Bank [PDB] accession no. 4O6B) is shown with each monomer represented in dark or light blue. Residues involved in infectious particle production are shown as <i>van der Waals</i> spheres. Mutations abrogating binding to E/prM/C or selectively preventing C binding are highlighted in green and red, respectively. <i>Upper panels</i>: ribbon representation of the NS1 dimer. <i>Lower panels</i>: putative functional NS1 domains involved in ER membrane binding, membrane bending and virion binding are highlighted. Note that these surfaces are exposed to the solvent and that the putative prM/E binding domain is located within the unresolved amino acid stretch of the <i>Wing</i> domain (aa 108–128; dashed lines).</p

3D structure of NS1 and highly conserved residues targeted by site-directed mutagenesis.

<p>(<b>A</b>) <i>3D organization of the DENV NS1 dimer and hexamer</i>. The left panel shows a <i>ribbon</i> representation of the 3D crystal structure of the DENV NS1 dimer (Protein Data Bank [PDB] accession no. 4O6B) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005277#ppat.1005277.ref009" target="_blank">9</a>]. The <i>β-roll</i>, <i>Wing</i> and <i>β-ladder</i> domains are highlighted in blue, yellow and red, respectively. The right panel shows the 3D organization of the NS1 hexamer, with NS1 domains highlighted in the same color-code of the dimer. (<b>B</b>) Alignment of NS1 amino acid sequences of different DENV serotypes and related flaviviruses (JEV, Japanese encephalitis virus; KUNV, Kunjin virus; MVEV, Murray Valley Encephalitis virus; TBEV, Tick-Borne Encephalitis virus; WNV, West Nile virus; YFV, Yellow Fever virus). Residues are shown in different colors according to the <i>ClustalW</i> algorithm. The bottom line shows the amino acid conservation across different NS1 sequences. Black arrowheads and circles indicate residues targeted by site-directed mutagenesis with black circles indicating known cysteine residues engaged in disulfide bonds. The dashed line indicates the region that was not resolved in the 3D X-ray crystal structure.</p

Effect of NS1 Alanine substitutions on DENV replication.

<p>(<b>A</b>) <i>Schematic representation of the DENV reporter virus genome and NS1 protein domains</i>. The full-length luciferase reporter DENV genome (DVR2A) is shown at the top, with the 5’ and 3’ NTRs depicted with their putative secondary structures. Polyprotein cleavage products are separated by vertical lines and labeled as specified in the introduction. A <i>Renilla luciferase</i> coding sequence was inserted in-between the capsid cyclization sequence (CAE) and the <i>Tosea asigna</i> virus 2A cleavage site that ensures proper processing after polyprotein synthesis. NS1 protein domains shown below are indicated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005277#ppat.1005277.g001" target="_blank">Fig 1</a>. Glycosylation sites are shown with green hexagons, while the <i>β-roll</i>, <i>Wing</i> and <i>β-ladder</i> domains are shown in blue, yellow and red, respectively. Two <i>connector</i> sub-domains within the <i>Wing</i> domain are shown in orange. (<b>B</b>) <i>Replication kinetics of DENV NS1 mutants</i>. VeroE6 cells were electroporated with <i>in-vitro</i> transcribed luciferase virus RNAs containing NS1 mutations specified at the bottom. Cells were lysed 4, 24, 48, 72, 96 and 120 h after transfection and luciferase activity in cell lysates was determined. Data were normalized to the 4h-value that reflects transfection efficiency. The background of the assay was determined with the active site NS5 polymerase mutant (GND). Colored lines on the top of each panel refer to the color-coded representation of NS1 protein domains as shown in (A). NS1 mutants severely impaired in viral RNA replication (normalized luciferase activity ≤0) are underlined. For ease of comparison, data obtained with WT and GND are repeated in the left of each panel. Mean values and standard deviations of three independent experiments are shown.</p

NS1 secretion is dispensable for the production of infectious DENV particles.

<p>(<b>A</b>) <i>Schematic representation of the delta-NS1 DENV genome and helper cell lines employed</i>. A deletion comprising 97 codons in NS1 (Δ156–253) was introduced into the full-length DVR2A genome (DVR2A^ΔNS1). VeroE6 helper cells (right panel) containing the stably integrated pWPI expression vector without insert (CTRL), or encoding full-length <i>wild-type</i> NS1 (WT), or NS1 C-terminally fused with an HA epitope (HA) or NS1 C-terminally fused with a KDEL ER retention motif (KDEL), were generated by transduction with lentiviral vectors as described in materials and methods. (<b>B</b>) <i>Intra- and extra-cellular NS1 protein levels of VeroE6 helper cell lines</i>. Cell lysates or clarified supernatants were analyzed by immunoblotting, using the antibodies indicated on the right. The positions of molecular weight markers (kDa) are shown on the left. (<b>C-D</b>) <i>Replication efficiency and released infectivity of the ΔNS1 genome trans-complemented with NS1 variants</i>. (C) <i>In-vitro</i> RNA transcripts of DVR2A^ΔNS1 were electroporated into VeroE6 helper cell lines expressing NS1 variants specified in the top and replication efficiency was determined in cell lysates at 4, 24, 48 and 72 h after transfection. (<b>D</b>) Clarified supernatants containing infectious particles were harvested 72h.p.t. and used to infect each respective naïve VeroE6_helper cells. Luciferase activity in the lysates was measured 48h later (right panel). The background of the assay was determined by infecting näive VeroE6 cells lacking exogenous NS1 (CTRL) and therefore not supporting DVR2A^ΔNS1 replication. The dashed line indicates the limit of detection of the assay.</p

Characterization of selected NS1 mutants reveals specific defects in infectious particle production.

<p><b>(A)</b><i>Replication kinetics of NS1 mutants in the context of a sub-genomic replicon</i>. Schematic representation of the sub-genomic reporter replicon (sgDVR2A) is shown at the top. It is derived from the DV2 full-length genome by insertion of a <i>Renilla luciferase</i> coding sequence in-between the capsid cyclization sequence (CAE) and the <i>Tosea asigna</i> virus 2A cleavage site. The last 24 amino acid residues of the envelope coding region (TM) at the N-terminus of NS1 ensure proper membrane topology of the polyprotein after synthesis. Selected NS1 mutations affecting virus production were inserted into sgDVR2A, and <i>in vitro</i> transcribed RNAs were electroporated into VeroE6 cells. Luciferase activity was measured in the lysates 4, 24, 48 and 72 h later. Values are expressed as fold of the 4h-value which reflects transfection efficiency. The background of the assay is determined with the active site NS5 polymerase mutant (GND). Columns represent mean and standard deviations of three independent experiments. (<b>B</b>) <i>Intra- and extra-cellular infectivity titers of NS1 mutants</i>. The structure of the full-length DENV genome used for this analysis is shown at the top (DV). NS1 mutations specified at the bottom were inserted into DV and in vitro transcripts derived therefrom were transfected into Huh7 cells and used for determination of intra- and extracellular infectivity. Seventy-two hours post electroporation, supernatants and cell lysates were subjected to 3 freeze and thaw cycles and titers were determined by TCID₅₀ assay using Huh7 cells and an envelope protein-specific MAb. Mean values and standard deviations of three independent experiments are shown. (<b>C</b>) Evidence that NS1 mutations affect, in part, release of infectious DENV particles. To determine the relative contribution of defects in virus assembly and virus release, data presented in (B) were used to calculate the ratio of intra- vs. extra-cellular infectivity.</p

Secretion kinetics and ultrastructural characterization of ΔNS1^TCP-infected helper cell lines.

<p>(<b>A</b>–<b>B</b>) <i>Kinetics of DVR2A</i>^<i>ΔNS1</i><i>TCPs secretion from helper cell lines</i>. (A) Schematic representation of the experimental setting. DVR2A^ΔNS1 TCPs produced in VeroE6_NS1^WT cells (ΔNS1-WT^TCP) were used to infect (MOI = 1) control or helper cell lines expressing different forms of NS1. Culture supernatants were collected 24, 48 and 72 h later, and virus titers were determined by Focus forming unit (FFU) assay on VeroE6_NS1^WT cells, using an E-specific mouse monoclonal antibody. Cell lysates and culture supernatants were analyzed by western blot. (B) FFU titers were determined as specified in panel (A). As reference, naïve VeroE6 cells were infected with DVR2A. The dashed line indicates the limit of detection of the assay. The lower panel shows representative images of foci morphologies of each trans-complemented NS1 variant. (<b>C</b>) <i>Expression levels of intra- and extra-cellular NS1 in DVR2A</i>^<i>ΔNS1</i><i>TCP infected cells</i>. NS1 expression and secretion were evaluated by western-blotting using cell lysates (Intra-) or clarified supernatants (Extra-) from (B) and NS1-, NS5- or GAPDH specific antibodies. (<b>D</b>, <b>E</b>) <i>Ultrastructural characterization of cells infected with DVR2A</i>^<i>ΔNS1</i><i>TCPs</i>. VeroE6_NS1^HA cells were infected with 1 MOI of DVR2A^ΔNS1 TCPs. Forty-eight hours later, cells were fixed, processed and analyzed by transmission electron microscopy as described in materials and methods. Representative images of the perinuclear area (D) or plasma membrane (E) of infected cells are shown. Black arrowheads indicate Vesicle packets (VPs), red arrowheads indicate virion bags. Electron-dense virus particles in proximity to or at the plasma membrane are indicated with black arrows. Boxed areas on the left panels are shown at higher magnification on the right. Black or white scale bars represent 500 or 200 μm, respectively.</p

Effect of NS1 alanine substitutions on DENV reporter virus replication.

<p>The replication level of a given mutant relative to the wild-type (WT) is displayed. Replication was determined by measuring luciferase activity 48 h post-transfection and normalizing to the 4 h value that reflects transfection efficiency. Values represent mean ± standard deviation of three independent experiments. NS1 mutants with severe defects in viral RNA replication are underlined, while asterisks (*) indicate mutants with selective defects in virus assembly or release.</p><p>Effect of NS1 alanine substitutions on DENV reporter virus replication.</p

Effect of mutations in NS1 on the production of infectious DENV particles.

<p>(<b>A</b>) <i>Released infectivity from DVR2A NS1 mutant-transfected cells</i>. VeroE6 cells were electroporated with DENV genomes containing NS1 mutations specified at the bottom. Seventy-two hours post-electroporation, supernatants containing infectious virus were used to infect naïve VeroE6 cells and luciferase activity measured 48 h later (black columns). Luciferase activity in the lysates 72 h post-electroporation reflecting replication is also shown (white columns). The background of the assay is determined with the active site NS5 polymerase mutant (GND). Mean values and standard deviations of three independent experiments are shown. Colours at the top of the panels refer to NS1 protein domains as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005277#ppat.1005277.g002" target="_blank">Fig 2A</a>. (<b>B</b>) <i>Efficiency of virus production relative to replication fitness</i>. To calculate specific defects in infectious particle production, data presented in (A) are shown as ratio of released infectivity/viral replication, and expressed as fold of wild-type (WT). Note that only NS1 mutants with viral RNA replication above input values were considered (cf. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005277#ppat.1005277.g002" target="_blank">Fig 2B</a>; replication > Log₁₀0).</p

NS1 interacts with the structural proteins.

<p>(<b>A</b>) <i>Capsid</i>, <i>prM and Envelope proteins co-immunoprecipitate with NS1</i>. Naïve VeroE6 (CTRL), VeroE6_NS1^WT (WT) or VeroE6_NS1^HA (HA) cells were infected with 1 MOI of DVR2A^ΔNS1 TCPs. Forty-height hours post-infection, cell lysates clarified by centrifugation were used for immunoprecipitation with HA-affinity agarose beads and eluates (IP) or whole cell lysates (Input) analyzed by western-blotting using antibodies specified on the right of each panel. Numbers on the left refer to molecular weight standards expressed in kDa; black arrowhead on the right indicates the ΔNS1 protein expressed by the DVR2A^ΔNS1 genome. A representative experiment of four independent repetitions is shown. (<b>B</b>) <i>Interaction between NS1 and prM/E in DENV-2 wild-type virus-infected cells</i>. VeroE6 cells were mock infected or infected with DENV-2 at an MOI of 1. Forty-eight hours later clarified cell lysates were used for immunoprecipitation using a NS1-specific rabbit polyclonal antiserum or the corresponding pre-immune (PIS) antiserum and protein A-Sepharose beads. After extensive washing, eluted protein complexes were analyzed by western-blotting using polyclonal anti-NS1 and anti-prM or mouse monoclonal anti-E or anti-C specific antibodies as specified on the right of each panel. DENV proteins are indicated with arrowheads, asterisks refer to the immunoglobulin heavy chain.</p