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. 2023 Jan 27;15(2):364.
doi: 10.3390/v15020364.

Extracellular Vesicles Are Conveyors of the NS1 Toxin during Dengue Virus and Zika Virus Infection

Daed El Safadi  1 , Grégorie Lebeau  1 , Alisé Lagrave  1 , Julien Mélade  1 , Lauriane Grondin  1 , Sarah Rosanaly  2 , Floran Begue  2 , Mathilde Hoareau  1 , Bryan Veeren  2 , Marjolaine Roche  1 , Jean-Jacques Hoarau  1 , Olivier Meilhac  2 , Patrick Mavingui  1 , Philippe Desprès  1 , Wildriss Viranaïcken  1 , Pascale Krejbich-Trotot  1
Affiliations

Extracellular Vesicles Are Conveyors of the NS1 Toxin during Dengue Virus and Zika Virus Infection

Daed El Safadi et al. Viruses. .

Abstract

Extracellular vesicles (EVs), produced during viral infections, are of emerging interest in understanding infectious processes and host-pathogen interactions. EVs and exosomes in particular have the natural ability to transport nucleic acids, proteins, and other components of cellular or viral origin. Thus, they participate in intercellular communication, immune responses, and infectious and pathophysiological processes. Some viruses are known to hijack the cell production and content of EVs for their benefit. Here, we investigate whether two pathogenic flaviviruses i.e., Zika Virus (ZIKV) and Dengue virus (DENV2) could have an impact on the features of EVs. The analysis of EVs produced by infected cells allowed us to identify that the non-structural protein 1 (NS1), described as a viral toxin, is associated with exosomes. This observation could be confirmed under conditions of overexpression of recombinant NS1 from each flavivirus. Using different isolation methods (i.e., exosome isolation kit, size exclusion chromatography, Polyethylene Glycol enrichment, and ELISA capture), we showed that NS1 was present as a dimer at the surface of excreted exosomes, and that this association could occur in the extracellular compartment. This finding could be of major importance in a physiological context. Indeed, this capacity of NS1 to address EVs and its implication in the pathophysiology during Dengue or Zika diseases should be explored. Furthermore, exosomes that have demonstrated a natural capacity to vectorize NS1 could serve as useful tools for vaccine development.

Keywords: Dengue; NS1; Zika; exosome; flavivirus; viral toxin vectorization.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
ZIKV and DENV2-infected A549 cells produce exosome-like particles associated with NS1 protein. (A) The NS1 protein of both ZIKV and DENV2 is found associated with the exosomal fraction obtained from the cell culture supernatants (CCS) of A549 infected cells. The tetraspanin CD63 detected on the dot blot of exosomal fractions provides insight into the quantity of exosomes extracted. (B) Western blot analysis of the exosome fraction, heated (HT) (+) or not (−), produced by DENV2 infected cells, show the presence of NS1 in dimeric form. A unique band with an apparent mass of ~48 kDa is observed after heating (+). (C) DLS analysis of exosomes produced by A549 cells during ZIKV (green) or DENV2 (blue) infection. On the y axis, the intensity gives the proportions of the different vesicle populations, depending on their size on the x axis. Both DENV2 and ZIKV infections appear to slightly reshape the distribution of vesicles.
Figure 2
Figure 2
The overexpressed ZIKV-NS1FLAG-tag and DENV2-NS1V5-tag co-elute with exosome-like particles. (A) The recombinant ZIKV-NS1FLAG-tag and DENV2-NS1V5-tag, overexpressed in stable HEK cell lines, are present in the CCS, mainly as dimers. (B) The tetraspanins CD81 and CD63 are immunodetected in the exosome fractions of CCS from cells expressing the different secreted recombinant proteins. (C) When detecting the recombinant proteins in the CCS and exosomal fractions, only the NS1 proteins were detected in the exosomal fractions, and a similar signal was obtained whether or not the vesicles had been lysed. (D) SEAP activity was estimated by quantiblue assay. CCS and the flowthrough remaining from the exosome extraction, but not purified exosomes, exhibited SEAP activity. Ordinary one-way ANOVA test was performed using GraphPad Prism **** p < 0.0001, ns (not significant) (E) Western blot analysis of exosome protein extracts shows that ZIKV-NS1FLAG-tag and DENV2-NS1V5-tag are found associated with exosomes in their dimeric form.
Figure 3
Figure 3
ZIKV-NS1FLAG-tag is present in the exosome fraction. (A) Western blot analysis of HEK293-NS1 supernatant following ultrafiltration. Mouse anti-FLAG antibody was used to detect ZIKV-NS1FLAG-tag, while the detection of albumin served as protein loading control. (B) Dot blot analysis of putative exosome fractions. After the ultrafiltration step, size exclusion chromatography was carried out on the >100 kDa fraction. Detection of ZIKV-NS1FLAG-tag was achieved using a mouse anti-FLAG antibody, whereas CD81 was chosen as a marker for exosomes. (C) CCS containing CD63-positive EVs and secreted ZIKV-NS1FLAG-tag (S1) were treated with PEG and centrifuged to obtain a pellet enriched in EVs. CD63 was found in the first pellet (P PEG) and after the second ultracentrifugation step (P2), validating the enrichment of exosomes which were also found associated with ZIKV-NS1FLAG-tag. (D) ELISA capture of the exosomes in the supernatant of HEK293-ZIKV-NS1FLAG-tag versus control. Exosomes were captured by an anti-CD81 antibody coated on Maxisorp® plate. NS1FLAG-tag protein was detected using a mouse anti-FLAG antibody. Ordinary one-way ANOVA test was performed using GraphPad Prism, ** p < 0.001. (E) DLS analysis of the exosomal fractions with the proportion of vesicles by size graph and the corresponding z-average-table (Table 2).
Figure 4
Figure 4
ZIKV and DV2 NS1 recombinant proteins bind to extracellular vesicles. (A) HEK 293 cells expressing ZIKV-NS1Flag--tag protein were treated or not with GW4869 at 10 µM for 16 h in order to inhibit exosome’s biogenesis. The cell culture supernatant (CCS) was collected for exosomes extraction using the Exoeasy kit. A dot blot was performed using anti-Flag and then quantified using imageJ. The flow through (FT) is the remaining part of the first exosome extraction step, and the wash is the second flow through, considered as a negative control. The quantity of NS1 associated with exosomes was reduced when cells were treated with GW4869, thus eliminating the co-elution bias of NS1 protein with exosomes. Unpaired t-test was performed using GraphPad Prism, * p < 0.05. (B) CCS of HEK was collected 48 h post-platting and incubated with ZIKV-NS1HIS-tag or DENV2-NS1HIS-tag for 30 min and 4 h. CCS was then proceeded to PEG exosome precipitation. The negative control (CTL−) corresponds to panserin. The positive control (CTL+) corresponds to 0.2 ng of NS1 solubilized in panserin. Dot blots were performed using anti-His and anti-NS1 antibodies to detect ZIKV and DV2 NS1 proteins, respectively. NS1 of ZIKV and DV2 were found associated with EVs as early as 30 min post-incubation.
Figure 5
Figure 5
Graphical overview of the association of ZIKV and DENV NS1 proteins with exosomes.
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This work was supported by the European Regional development Fund (RUNDENG 20201640-00222937), by the Federation BioST from Reunion Island University (SEVEXDENG project) and by the ZIKAlert project (European Union-Reunion program under grant agreement n° SYNERGY: RE0001902). D.E.S., G.L., F.B. and S.R. have Ph.D. degree scholarships from Reunion University (Ecole doctorale STS) funded by DIRED/2021-1115, DIRED/2021-0161, DIRED/20210155 and D2019/21320 from Région Réunion Council, respectively.