Dengue virus NS5 degrades ERC1 during infection to antagonize NF-kB activation

Abstract

Dengue virus (DENV) is the most important human virus transmitted by mosquitos. Dengue pathogenesis is characterized by a large induction of proinflammatory cytokines. This cytokine induction varies among the four DENV serotypes (DENV1 to 4) and poses a challenge for live DENV vaccine design. Here, we identify a viral mechanism to limit NF-κB activation and cytokine secretion by the DENV protein NS5. Using proteomics, we found that NS5 binds and degrades the host protein ERC1 to antagonize NF-κB activation, limit proinflammatory cytokine secretion, and reduce cell migration. We found that ERC1 degradation involves unique properties of the methyltransferase domain of NS5 that are not conserved among the four DENV serotypes. By obtaining chimeric DENV2 and DENV4 viruses, we map the residues in NS5 for ERC1 degradation, and generate recombinant DENVs exchanging serotype properties by single amino acid substitutions. This work uncovers a function of the viral protein NS5 to limit cytokine production, critical to dengue pathogenesis. Importantly, the information provided about the serotype-specific mechanism for counteracting the antiviral response can be applied to improve live attenuated vaccines.

Keywords: NS5 viral protein activation; dengue virus; dengue virus pathogenesis; evasion of innate antiviral responses; host–virus interactions.

Fig. 1.

The host ERC1 protein has an antiviral activity and is degraded during DENV2 infection. (A) ERC1 has an antiviral function. Luciferase activity showing the replication of DENV2 reporter (RepDENV) 36 hpi in A549 cells transfected 48 h previously with siRNAs directed to ERC1 (blue) or directed to two antiviral proteins: STAT2 and CD2BP2, as indicated. Two additional controls were included, a nonrelated siRNA (NR) and a positive siRNA control directed to Renilla luciferase (Luc). WB assays indicate the levels of silenced proteins. Statistical significance is shown with the following notation: *P < 0.05. (B) ERC1 silencing reduces DENV replication. Viral replication is shown by qRT PCR (mean ± SD) at 8 and 24 hpi (MOI 1) in A549 cells previously transfected with a nonrelated siRNA (NR), or a siRNA directed to ERC1. WB shows the levels of ERC1 in each condition. Statistical significance is shown with the following notation: *P < 0.05. (C) ERC1 levels are reduced in DENV2-infected human cells. Immunofluorescence (IF) assays against ERC1 (red) and the viral protein NS3 (green, to label infection), are shown in human A549 and Huh7 cells either mock infected or infected with DENV2 (MOI 3). Objective: 40× (D) ERC1 protein is degraded upon DENV2 infection at MOI of five in different cell lines. WB analyses showing the levels of ERC1 as a function of time postinfection, as indicated in each panel. Cytoplasmic extracts from infected A549 (human), BHK (hamster), Huh7 (human), DCs (monocyte-derived human DCs) are shown. (E) Schematic description of the multiple functions of ERC1. (I) ERC1 is involved in NF-kB activation by Toll-like and cytokine receptors (TNF-α, IL1) or by DNA damage. ERC1 interacts with the IKKγ/NEMO regulatory subunit of IκB kinase (IKK) complex and enhances IKK activation. This activation induces IκBα phosphorylation, followed by ubiquitination and proteasome degradation of NF-kB inhibitory protein IκBα. NF-kB complex is then translocated to the nucleus and acts as a transcription factor that induces genes related to immune responses, such as proinflammatory cytokines (TNF-α, IL6). (II) ERC1 is involved in secretion. It interacts with Rab6-dependent vesicles acting as a docking and/or fusion protein in the cell cortex, where it tags the site of arrival of Rab6- vesicles for secretion. (III) ERC1 is involved in cell migration. The host protein forms a functional complex with LL5 proteins LL5a and LL5b and liprin-α1 that drives cell motility.

Fig. 2.

ERC1 enhances NF-kB-dependent cytokine production and is necessary for cell motility in human cells (A) ERC1 overexpression activates NF-kB reporter activity. The luciferase activity of the NF-kB reporter was measured in cells expressing or not ERC1. On the top, schematic representation of the experiment. 293T cells were cotransfected with 10 ng of the NF-kB reporter plasmid (pFLucNFkB Rep), 4 ng Renilla luciferase expression plasmid (pRLuc) as control to standardize transfection and either, 26 ng ERC1 plasmid (pERC1) or 26 ng of an empty control plasmid (pControl). Relative luciferase activity (FLuc/RLuc) is shown at 16 h posttransfection. (B) ERC1 is involved in proinflammatory cytokine expression and secretion. On the top, schematic representation of the protocol used and WB showing the efficiency of ERC1 silencing. At the bottom, cytokine expression (mRNA of TNF-α and IL6) relative to GAPDH is shown measured by RT-qPCR, and secreted IL6 protein measured by ELISA, in ERC1 silenced or control A549 human cells treated with 5 ng/mL TNF-α during 2, 4, and 6 h. (C) TNF-α, IL6, and IFN-β mRNA expression and protein secretion after 16 and 24 h of 10 μg/mL poly I:C treatment in ERC1 silenced or control A549 cells. On the Top panel, schematic representation of the protocol used and WB showing the efficiency of ERC1 silencing. (D) ERC1 role on A549 cell motility. On the top, schematic representation of the protocol used and WB showing the efficiency of ERC1 silencing. Wound-healing assays are shown in ERC1 silenced or control A549 cells. Representative images of scratched monolayer at 0 and 24 h are shown for each condition. Objective: 5×. The wound closure was quantified by measuring the distance covered by the migrated leading edge using Zen 2.3 lite software. Statistical significance is shown in the different panels with the following notations: ****P < 0.0001; *P < 0.05.

Fig. 3.

Mechanism of ERC1 degradation during DENV infection. (A) NS5 protein is sufficient for ERC1 degradation. IF and WB assays showing ERC1 levels in cells expressing NS5-GFP or NS3-GFP control, as indicated in each case. IF assays were carried out 24 h posttransfection of A549 cells with either NS5 or NS3 expression plasmids. Objective: 40×. For WB analysis, 293T cells were transfected during 24 h with NS5-GFP plasmid and sorted by FACS to separate GFP-positive (NS5 +) from GFP-negative (NS5 −) cells. (B) The methyltransferase domain (MTase) of NS5 is responsible for ERC1 degradation. IF and WB assays showing ERC1 levels in cells expressing RdRp or MTase domains of NS5. For IF, A549 cells were transfected with 250 ng of each plasmid. Images were acquired with the objective 63× 24 h posttransfection. On the right, WB showing endogenous ERC1 levels in 293T cells expressing different amounts of the viral proteins or control plasmid (pCL), as indicated in each case. (C) ERC1 degradation is proteasome-dependent. WB showing expression of ERC1 or the viral NS5 protein in DENV2-infected A549 cells with an MOI of 3 during 12 h and subsequently treated with 20 uM MG132 or DMSO as control during 2, 6, and 10 h. (D) UBR4 E3 ubiquitin ligase mediates ERC1 degradation. The levels of ERC1 and viral proteins are shown in UBR4-silenced A549 cells infected or not with DENV2. Samples at 24 h postinfection with an MOI of 5 are shown. On the Right panel, levels of UBR4 mRNA relative to GAPDH measured by qPCR are included. (E) DENV2 T2A and G3A mutant viruses are replication competent. On the right, DENV NS5 sequence alignment of the first N-terminal eleven amino acids of the four serotypes. Two N-terminal NS5 amino acid substitutions were introduced independently in the DENV2 infectious clone: T2A or G3A. The amino acid change is shown in red. On the left, IF assays showing that T2A and G3A viruses replicate in A549 cells 24 h post infection with an MOI of 10. The viral NS3 protein is shown in green and DAPI in blue. Objective: 20×. (F) T2A or G3A substitutions in NS5 prevent ERC1 degradation. WB showing ERC1 at 24 h postinfection with DENV2 WT, T2A, or G3A. The viral proteins NS5 and NS3 as well as GAPDH are also shown, as indicated in each case. (G) DENV2 WT degrades mouse ERC1 protein. Quantification of ERC1-positive and -negative melanoma mouse cells (B16-F10) infected during 24 h with an MOI of 10 of WT, T2A, or G3A viruses is shown. The percentage of ERC1-positive cells was calculated for each viral infection.

Fig. 4.

DENV serotypes differentially degrade ERC1 (A) ERC1 protein is not degraded during DENV4 infection. IF showing ERC1 or the viral protein NS3 in cells infected with an MOI of 5 with DENV1, DENV2, DENV3, or DENV4, as indicated. Objective: 40× (B) DENV4 infection differentially degrades STAT2 and ERC1. WB assay showing ERC1, STAT2, NS5, and GAPDH control in extracts from cells infected during 24 h with an MOI of 5 with each of the four DENV serotypes, as indicated. (C) STAT2 and ERC1 degradation by the four DENV serotypes. Schematic representation of the ability of NS5 from different serotypes to degrade STAT2 and ERC1. (D) DENV4 NS5 lacks the ability to degrade ERC1. Levels of ERC1 in cytoplasmic extracts of 293T sorted cells expressing or not GFP from cells transfected during 24 h with 15 μg DENV2-NS5-GFP, DENV4-NS5-GFP, or GFP plasmids. Cytoplasmic extracts from equal amounts of cells were loaded in each lane. Levels of ERC1, GFP, and GAPDH are shown. (E) The MTase domain of DENV4 NS5 lacks the ability to degrade ERC1. Levels of ERC1 in cytoplasmic extracts of 293T sorted cells expressing or not GFP from cells 24 h posttransfection with 15 μg DENV2-MTase-GFP, DENV4- MTase-GFP, or GFP plasmids. Cytoplasmic extracts from equal amounts of cells were loaded in each lane. Levels of ERC1 and GAPDH are shown. (F) Schematic representation of the differential degradation of ERC1 by the MTase of DENV2 and DENV4.

Fig. 5.

Mapping the molecular determinants in NS5 for ERC1 degradation. (A) Sequence alignment of MTases from DENV1, 2, 3, and 4. Amino acid sequences of the four DENV serotypes are shown. R1, R2, and R3 in green boxes show solvent-exposed regions of the MTase domain with significant sequence differences between DENV4 and the other three serotypes (in gray). Residue numbers corresponding to the R1, R2, and R3 are shown in each case with a representation of the protein secondary structure below in red. Below: MTase chimeras R1, R2, and R3 are shown based on DENV2 protein crystal structure (DENV2 in green and the DENV4 region in red). (B) Chimeric R1 and R2 MTase proteins lack the ability to degrade ERC1. Representative images of A549 cells transfected with 250 ng chimeric MTase-GFP (R1-R3), DENV2 MTase-GFP, DENV4 MTase-GFP, or GFP control plasmids are shown by IF. Detection of GFP and ERC1 is indicated in each case. Objective: 63×. (C) ERC1 protein levels by WB in cytoplasmic extracts of 293T cells expressing the chimeric MTase-GFP is shown. Proteins NS5 and GAPDH for each sample are also shown. (D) Replication and propagation of chimeric DENV2 viruses. Viral RNA from R1 and R2 chimeras were transfected in BHK cells, and viral propagation was evaluated by detecting NS3 protein using IF. DAPI-staining nucleus shows the integrity of the monolayer. Objective: 20×. (E) DENV2 chimeric virus R1 lacks the ability to degrade ERC1. A549 cells were infected during 24 h with DENV2 (MOI:3) and chimeric virus R1 (MOI:20). IF showing NS5 in green indicating DENV2-infected cells and ERC1 in red showing degradation of the host protein in infected cells. Objective: 63× (F) WB showing ERC1 levels in cytoplasmic extracts of A549 cells infected during 24 h with DENV2 (MOI:5), DENV4 (MOI:5), or R1 chimeric virus (MOI:20). (G) Amino acid sequence alignment of the four DENV serotypes and seven recombinant DENV2 viruses. In red, the amino acid substitution is shown in each recombinant virus. (H) ERC1 degradation during infection with each recombinant DENV. WB of cytoplasmic extracts of A549 cells infected during 24 h with the recombinant viruses as indicated on the top of the gel. For each infection, ERC1 levels are shown and viral replication evaluated by NS3. DENV2 WT and mock infections were used as controls. (I) G21D and T2A recombinant DENV2 viruses are unable to degrade ERC1. IF showing that infection with an MOI of 5 of DENV2 WT degrades ERC1, while G21D and T2A do not alter ERC1 staining compared to the mock infection. In red ERC1 and in green NS3. Objective: 63× (J) Infection with DENV2 G21D differentially degrades STAT2 and ERC1. WB of cytoplasmic extracts of A549 cells infected during 24 h with an MOI of 5 of WT, G21D, or T2A DENV2. ERC1, STAT2, NS3, NS5, and GAPDH are indicated. (K) G21D amino acid change in NS5 protein alters ERC1 binding. Immunoprecipitation analyses are shown. A549 cells were infected with DENV2 WT, G21D, or T2A viruses (MOI: 5), as indicated on the top of the gels. Eight hours postinfection cells were treated with 20 μM MG132 for 6 h. Lysates were immunoprecipitated with anti-ERC1 antibody (indicated as IP: ERC1), and WB were performed using anti ERC1 and anti NS5, as shown. Before the IP assay, an aliquot of the whole-cell extract (WCE) was spared as input control of the IP, shown on the left.

Fig. 6.

ERC1 degradation limits cytokine production and cell motility. (A) G21D and WT DENV2 show similar replication kinetics in mosquito C6/36 cells. Viral replication as a function of time is shown in one-step growth curve (MOI: 10). (A, Left) RT qPCR of intracellular viral RNA relative to 18S. (A, Right) Plaque assay of infectious viral particles in the supernatant of infected cells. PFU: plaque-forming units. (B) G21D DENV2 is attenuated in human A549 cells. Viral replication as a function of time is shown in one-step growth curve (MOI: 10). (B, Left) RT qPCR of intracellular viral ARN relative to GAPDH. (B, Right) Plaque assay of infectious viral particles in the supernatant of infected cells. (C) ERC1 degradation upon WT or G21D virus infection. WB analysis of cytoplasmic extracts of A549 cells infected with an MOI of 10 with either WT or G21D DENV2 as a function of time. Mock infections were used as a control of ERC1 levels. (C, Left) WB of cytoplasmic extracts of A549 cells infected with WT DENV2 as a function of time is shown. A sample of G21D at 48 h postinfection is included for comparison. (C, Right) WB of cytoplasmic extracts of A549 cells infected with G21D virus as a function of time is shown. A sample of WT at 48 h postinfection is included for comparison. Viral replication is shown by NS3 protein accumulation. (D) G21D virus replication is enhanced in ERC1 knockdown cells to WT levels. G21D and WT viral replication is shown by qRT PCR (mean ± SD) at 24 hpi (MOI 5) in A549 cells previously transfected with an ERC1 siRNA or a nonrelated siRNA (NR). (E) Expression and secretion of cytokines during infection with an MOI of 1 of WT or G21D DENV2s. (E, Upper) replication curve and cytokine mRNA expression of WT and G21D DENV infections. Viral RNA levels are shown as a function of time after infection and measured by RT qPCR and normalized to GAPDH. IL6, TNF-α, and IFN-β mRNA expression as a function of time of infected cells was measured by RT qPCR and expressed relative to GAPDH, as indicated in each case. (E, Lower) IL6, TNF-α, and IFN-β protein secretion measured by ELISA in the supernatant of infected cells. (F) Increased cell motility by G21D DENV infection. (F, Left) schematic representation of the protocol used. (F, Middle) representative images of the wound-healing assays in A549 cells previously infected with an MOI of 3 with either WT or G21D viruses. Images of the wounds at 0 and 24 h are shown in mock or infected cell monolayers. Objective: 5×. (F, Right) the wound closure was quantified by measuring the distance covered by the migrated leading edge using Zen 2.3 lite software. Additionally, an IF assay to detect NS3 viral protein is shown, which was performed once the wound-healing assay was concluded. Statistical significance is shown with the following notations: ****P < 0.0001; *P < 0.05.