Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Oct 13;95(21):e0131021.
doi: 10.1128/JVI.01310-21. Epub 2021 Aug 11.

Determinants in Nonstructural Protein 4A of Dengue Virus Required for RNA Replication and Replication Organelle Biogenesis

Mirko Cortese  1 , Klaas Mulder  1 , Laurent Chatel-Chaix  1 , Pietro Scaturro  1 , Berati Cerikan  1 , Anna Plaszczyca  1 , Uta Haselmann  1 , Marie Bartenschlager  1 , Christopher J Neufeldt  1 , Ralf Bartenschlager  1   2
Affiliations

Determinants in Nonstructural Protein 4A of Dengue Virus Required for RNA Replication and Replication Organelle Biogenesis

Mirko Cortese et al. J Virol. .

Abstract

Dengue virus (DENV) constitutes one of the most important arboviral pathogens affecting humans. The high prevalence of DENV infections, which cause more than 20,000 deaths annually, and the lack of effective vaccines or direct-acting antiviral drugs make it a global health concern. DENV genome replication occurs in close association with the host endomembrane system, which is remodeled to form the viral replication organelle that originates from endoplasmic reticulum (ER) membranes. To date, the viral and cellular determinants responsible for the biogenesis of DENV replication organelles are still poorly defined. The viral nonstructural protein 4A (NS4A) can remodel membranes and has been shown to associate with numerous host factors in DENV-replicating cells. In the present study, we used reverse and forward genetic screens and identified sites within NS4A required for DENV replication. We also mapped the determinants in NS4A required for interactions with other viral proteins. Moreover, taking advantage of our recently developed polyprotein expression system, we evaluated the role of NS4A in the formation of DENV replication organelles. Together, we report a detailed map of determinants within NS4A required for RNA replication, interaction with other viral proteins, and replication organelle formation. Our results suggest that NS4A might be an attractive target for antiviral therapy. IMPORTANCE DENV is the most prevalent mosquito-borne virus, causing around 390 million infections each year. There are no approved therapies to treat DENV infection, and the only available vaccine shows limited efficacy. The viral nonstructural proteins have emerged as attractive drug targets due to their pivotal role in RNA replication and establishment of virus-induced membranous compartments, designated replication organelles (ROs). The transmembrane protein NS4A, generated by cleavage of the NS4A-2K-4B precursor, contributes to DENV replication by unknown mechanisms. Here, we report a detailed genetic interaction map of NS4A and identify residues required for RNA replication and interaction between NS4A-2K-4B and NS2B-3 as well as NS1. Importantly, by means of an expression-based system, we demonstrate the essential role of NS4A in RO biogenesis and identify determinants in NS4A required for this process. Our data suggest that NS4A is an attractive target for antiviral therapy.

Keywords: dengue virus; flavivirus; genetic mapping; membrane remodeling; nonstructural protein 4A; replication organelles.

PubMed Disclaimer

Figures

FIG 1
FIG 1
Residues of DENV NS4A critical for viral replication identified by alanine-scanning mutagenesis. (A) Schematic representation of NS4A membrane topology according to reference . pTMS, putative transmembrane segment; 2K, 2 kDa peptide. (B) Multiple-sequence alignment of NS4A from several flaviviruses. Color grades indicate the degree of conservation according to the 0-to-10 coloring scheme given on the top right. The secondary structure prediction of each residue in context is indicated on the bottom (H, helix; E, extended). The putative transmembrane segments and 2K peptide are indicated by cylinders below the corresponding amino acid residues. Black arrowheads indicate residues selected for alanine-scanning mutagenesis. (C) Effects of mutations in NS4A on DENV replication. Mutations specified on the bottom were inserted into a DENV-2 (strain 16681) full-length reporter genome (DVs-R2A). In vitro transcripts were electroporated into Huh7 cells that were collected and lysed at different time points to measure renilla luciferase activity, which reflects RNA replication. Replication efficiency is reported as relative light units (RLU) normalized to the 4-h value (fold 4 h) that reflects transfection efficiency. Values represent the mean and SD from three independent experiments. Significance has been calculated with one-way ANOVA with Dunnett’s posttest. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) Supernatants from cells electroporated as in panel C were collected 72 h postelectroporation and used to infect Huh7 cells. Cells were lysed after 48 h, and renilla luciferase activity was measured (48 h postinfection [h.p.i.). Replication efficiency is reported as relative light units (RLU) normalized to the value determined 4 h postelectroporation. For comparison, replication levels of each construct at 72 h postelectroporation are shown (72 h.p.e.). Values represent the mean and SD from at least two independent experiments. (E) Reinfection values from panel D normalized to the respective values determined 72 h postelectroporation. Each dot represents one experiment. Significance has been calculated with one-way ANOVA with Dunnett’s posttest. ns, not significant.
FIG 2
FIG 2
Selection for pseudoreversions rescuing replication deficiency of primary mutations in NS4A. (A) Schematic representation of the selectable subgenomic DENV-2 (strain 16681) replicon (sgDVs-H2A) used to select for pseudoreversions. The hygromycin phosphotransferase gene (HygrB) is preceded by the cis-acting cyclization sequence residing in the capsid-coding region (CS). The sequence coding for the 2A-like peptide from the Thosea asigna virus (TaV) ensures cotranslational processing of the polyprotein to release properly cleaved NS1. The sequence coding for the last transmembrane helix of the envelope protein (gray box) and acting as signal sequence is retained upstream of the NS1-coding sequence to allow for ER membrane passage of NS1. (B) To select for second-site mutations rescuing viral replication, Vero E6 cells were electroporated with in vitro transcripts of sgDVs-H2A containing given primary mutations in NS4A. Electroporated cells were cultured in the presence of hygromycin B, and single-cell clones were selected and expanded. Total RNA was extracted, and coding regions of the DENV replicons were amplified by reverse transcriptase PCR (RT-PCR). Amplicons were subjected to nucleotide sequence analysis in the NS4A-coding region. (C) Number of hygromycin B-resistant cell colonies obtained after selection for each of the given constructs. Gray bars indicate confluent cell monolayers that were obtained with cells transfected with given replicon mutants. (D) List of pseudoreversions in NS4A identified in replicons after hygromycin B selection. The numbering of amino acid residues refers to strain 16681. For each cell clone, the pseudoreversion identified in replicon RNA of said clone is given. Revertant indicates a nucleotide substitution that restores the original amino acid residue.
FIG 3
FIG 3
Effect of second-site mutations on replication of wild-type and NS4A mutants. (A) Vero E6 cells were electroporated with the reporter virus DVs-R2A containing the mutations specified on the bottom of each panel. Cell lysates were collected 24, 48, 72, and 96 h after transfection, and renilla luciferase activity was measured as surrogate for viral replication. Values were normalized to the 4-h time point, reflecting transfection efficiency. Data represent the mean and SD of three independent experiments. Significance has been calculated by Student’s t test using, for comparison, values from the same time point obtained with the primary mutant. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B) NS4A genetic interaction map as determined through reverse and forward genetic screens. Arrows connect primary mutations (squares) and the corresponding second-site compensatory mutations (circles).
FIG 4
FIG 4
Impact of mutations in NS4A on the interaction between the NS4A-2K-4B precursor and the NS2B-3 complex. (A) Experimental approach. Plasmids encoding the HA-tagged NS4A-2K-4B precursor into which NS4A mutations had been inserted were transfected into Huh7/Lunet-T7 cells stably expressing DENV NS2B-3. Cell extracts were prepared 16 h posttransfection and subjected to HA-specific immunoprecipitation. Captured immunocomplexes were analyzed by SDS-PAGE and Western blotting. (B) Western blot analysis of captured protein complexes. A representative experiment of three independent experiments is shown. GAPDH served as loading control of the input. (C) Quantification of coimmunoprecipitation experiments representing the ratio of NS3 and HA-reactive proteins, i.e., uncleaved NS4A-2K-4B-HA precursor and processed NS4B-HA. Values were normalized against the HA-tagged wild-type sample that was set to 1. Data are the mean and SD from three independent experiments. (D) Quantification of the ratio between mature NS4B and NS4A-2K-4B precursor signals. Data are the mean and SD from three independent experiments normalized against the wild-type untagged sample. (E) Restoration of the interaction between NS2B-3- and NS4B-containing proteins (NS4A-2K-4B-HA and NS4B-HA) by second-site compensatory mutations in NS4A. Plasmids expressing the HA-tagged NS4A-2K-4B precursor containing primary and the respective compensatory mutations in NS4A were transfected into Huh7/Lunet-T7 cells stably expressing DENV NS2B-3. Immunoprecipitations were performed as described above. Abundance of proteins specified on the right of each panel was determined by Western blot analysis. A representative experiment of three independent experiments is shown. (F) Quantification of panel E. Values representing the ratio between NS3 and NS4B reactive proteins (uncleaved NS4A-2K-4B-HA precursor and processed NS4B-HA) were normalized against the wild-type sample that was arbitrarily set to 1. Data are the mean and SD from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, as determined by two-tailed t test.
FIG 5
FIG 5
Mutations in NS4A affecting the interaction between the NS4A-2K-4B precursor and NS1. (A) Schematic representation of the experimental approach. Plasmids encoding the NS4A-2K-4B precursor and containing mutations in NS4A were transfected into Huh7/Lunet-T7 cells stably expressing HA-tagged DENV NS1. Cell extracts were prepared 16 h posttransfection and used for HA-specific immunoprecipitation. Captured immunocomplexes were analyzed by SDS-PAGE and Western blotting. (B) Western blot analysis of captured immunocomplexes. A representative experiment of three independent experiments is shown. (C) Quantification of coimmunoprecipitation experiments showing the ratio between HA-NS1 and the NS4A-2K-4B precursor. Values were normalized against the wild-type (WT) sample that was set to 1. Data are the mean and SD from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, as determined by two-tailed t test.
FIG 6
FIG 6
Effect of NS4A mutations on DENV polyprotein processing. (A) Schematic representation of the DENV expression construct (pIRO-D) (17) used in this study. It comprises the T7 RNA polymerase promoter, part of the 5′ nontranslated region (NTR) and the cyclization sequence in the DENV capsid-coding region (CS), the encephalomyocarditis virus internal ribosome entry site (EMCV IRES), the coding region from the signal sequence of NS1 in the 3′ terminal region of E up to the stop codon of the polyprotein, the complete 3′ NTR, the ribozyme of the hepatitis D virus, and the T7 terminator. Mutations specified in the individual panels were inserted into NS4A, and plasmids were transfected into Huh7/Lunet-T7 cells. After 16 h, cells were harvested for immunofluorescence, Western blot analysis, and electron microscopy. (B) Quantification of the transfection efficiency obtained with the different pIRO-D constructs as determined by immunofluorescence using NS4B-specific antibody. Mean and SD of three independent experiments are shown. (C) Polyprotein cleavage and abundance of cleavage products was determined by Western blot analysis using antibodies with specificity given on the left of each panel. Actin was used as loading control. A representative experiment of three independent experiments is shown. (D) Quantification of three experiments, including the one in panel C. Values are the mean and SD of three independent experiments. *, P < 0.05; **, P < 0.01, as determined by two-tailed t test.
FIG 7
FIG 7
Effect of NS4A mutations on the biogenesis of vesicle packets. (A and B) Electron microscopy analysis of Huh7/Lunet-T7 cells transfected with polyprotein constructs specified in Fig. 6A. Transfected cells were fixed and embedded into Epon resin, and thin sections were examined by transmission electron microscopy. For each sample, at least 15 cells were analyzed. Representative examples of vesicle packets or aberrant vesicles are shown in panels A and B, respectively. The experiment has been repeated twice. Scale bar, 200 nm. (C) Left and middle panels show the percentage of cells presenting VPs and aberrant vesicles, respectively. Each dot represents the mean of at least 15 cells analyzed. Two independent experiments were performed. The right panel shows the diameter of aberrant vesicles (nm) as found in cells transfected with the mutants specified in the middle panel. Each dot represents one vesicle. Means and SD are shown. ***, P < 0.001, as determined by two-tailed t test. (D) Graphical summary of the NS4A mutation analysis. Residues in NS4A and their contributions to interaction with NS1 and NS3, as well as their role in protein stability and VP formation, are displayed. Note that mutations affecting residues Y41, N132, and Q133 induced both regular VPs and VPs of aberrant morphology.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Neufeldt CJ, Cortese M, Acosta EG, Bartenschlager R. 2018. Rewiring cellular networks by members of the Flaviviridae family. Nat Rev Microbiol 16:125–142. 10.1038/nrmicro.2017.170. - DOI - PMC - PubMed
    1. Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, Drake JM, Brownstein JS, Hoen AG, Sankoh O, Myers MF, George DB, Jaenisch T, Wint GR, Simmons CP, Scott TW, Farrar JJ, Hay SI. 2013. The global distribution and burden of dengue. Nature 496:504–507. 10.1038/nature12060. - DOI - PMC - PubMed
    1. Kaaijk P, Luytjes W. 2018. Are we prepared for emerging flaviviruses in Europe? Challenges for vaccination. Hum Vaccin Immunother 14:337–344. 10.1080/21645515.2017.1389363. - DOI - PMC - PubMed
    1. Hadinegoro SR, Arredondo-Garcia JL, Capeding MR, Deseda C, Chotpitayasunondh T, Dietze R, Muhammad Ismail HI, Reynales H, Limkittikul K, Rivera-Medina DM, Tran HN, Bouckenooghe A, Chansinghakul D, Cortes M, Fanouillere K, Forrat R, Frago C, Gailhardou S, Jackson N, Noriega F, Plennevaux E, Wartel TA, Zambrano B, Saville M, CYD-TDV Dengue Vaccine Working Group. 2015. Efficacy and long-term safety of a dengue vaccine in regions of endemic disease. N Engl J Med 373:1195–1206. 10.1056/NEJMoa1506223. - DOI - PubMed
    1. Płaszczyca A, Scaturro P, Neufeldt CJ, Cortese M, Cerikan B, Ferla S, Brancale A, Pichlmair A, Bartenschlager R. 2019. 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. PLoS Pathog 15:e1007736. 10.1371/journal.ppat.1007736. - DOI - PMC - PubMed

Publication types

MeSH terms