Cells and antibodies

Human embryonic kidney HEK-293T (ATCC, CRL-1573), human carcinoma epithelial lung A549 (Invivogen Inc, Toulouse, France), human microglial clone 3 HCM3 (ATCC, CCL-3304) and monkey kidney VeroE6 (CCL-81, ATCC, Manassas, VA, USA) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) growth medium supplemented with 10% (HEK-293T, A549 and HCM3 cells) or 5% (VeroE6 cells) of heat-inactivated fetal bovine serum (FBS, Dutscher, Brumath, France), and antibiotics (PAN Biotech Dutscher, Brumath, France) at 37°C under a 5% CO₂ atmosphere. HEK-293T, A549 and HCM3 cells were a generous gift of Dr N. Jouvenet (Institut Pasteur, Paris). The mouse anti-pan orthoflavivirus envelope E protein monoclonal antibody (mAb) 4G2 was produced by RD Biotech (Besançon, France). The mouse anti-FLAG antibody was purchased from Abcam (Cambridge, UK). Donkey IgG anti-mouse IgG Alexa Fluor 488 or Alexa Fluor 594 and donkey IgG anti-rabbit IgG Alexa Fluor 594 secondary antibodies were purchased from Invitrogen (Thermo Fisher Scientific, Illkirch-Graffenstaden, France). Anti-mouse IgG-horseradish peroxidase (HRP)-conjugated secondary antibody was purchased from Abcam (Cambridge, UK). Blue-fluorescent DNA stain 4’,6-diamidino-2-phenylindole (DAPI) was purchased from Euromedex (Souffelweyersheim, France). Lipofectamine 3000 (Thermo Fisher Scientific, Illkirch-Graffenstaden, France) was used for transfection, according to the manufacturer’s instructions.

Design of infectious molecular clones of ZIKV

The infectious molecular clone MR766^MC of ZIKV strain MR766-NIID (Accession n° LC002520) was generated by reverse genetic approach using the ISA method [21]. The same strategy was used to produce an infectious molecular clone GUINEA-18 of viral strain ZIKV-15555. Because the available ZIKV-15555 genome sequence (Accession n°MN025403) is lacking for the 14 first and the 86 last nucleotides of viral genomic RNA, the ends of 5’ and 3’ NCRs of GUINEA-18 were completed with MR766^MC sequences. The subgenomic amplicons Z-1^GUINEA-18, Z-23^GUINEA-18 and Z-4^GUINEA-18 were designed to mimic Z-1^MR766, Z-23^MR766 and Z-4^MR766 amplicons that were generated to produce MR766^MC [21]. Information on viral protein sequences encoded by different amplicons is available in S1 Fig. The amplicon Z-1^GUINEA-18 includes the CMV promoter immediately adjacent to the 5’NCR followed by the residues 1 to 712 of viral polyprotein. The Z-23^GUINEA-18 codes for residues 702 to 2684 of viral polyprotein. The amplicon Z-4^GUINEA-18 codes for residues 2674 to 3423 of viral polyprotein followed by the 3’NCR of viral genome and ended by a hepatitis delta virus ribozyme and then a SV40 poly(A) signal. The Z-1/Z-23 and Z-23/Z4 amplicons match together on at least 40 nucleotides (S1 Fig). Also, the GUINEA-18 and MR766^MC amplicons can match between them in order to generate chimeric viruses between the two infectious molecular clones. The different Z1, Z-23 and Z-4 amplicons were chemically synthetized and inserted into plasmid pUC57 by Genecust (Boynes, France). Plasmid sequences were verified by Sanger method.

Recovery of infectious ZIKV from molecular clones

The Z-1^GUINEA-18, Z-23^GUINEA-18 and Z-4^GUINEA-18 amplicons were amplified by PCR from their respective plasmids using a set of specific primers (S1 Table). The purified PCR products were co-transfected in HEK-293T cells using Lipofectamine 3,000 (ThermoFisher, France). After 4 days, cell supernatants were recovered and used to infected VeroE6 cells in a first round of amplification (P1). After 3 to 5 days, P1 was recovered and amplified on VeroE6 cells for another 2 to 3 days to produce a working virus stock P2. The resulting P2 virus was designed hereafter as GUINEA-2018. Virus stocks P2 grown on VeroE6 cells were used in this study. Virus stock titers in plaque forming unit per ml (PFU.mL^-1) were determined by a standard plaque-forming assay on VeroE6 cells as previously described [26].

Production of chimeric and mutant ZIKV

The design of chimeric and mutant ZIKV is shown in Fig 1. The Z-1^MR766, Z-23^GUINEA-18 and Z-4^GUINEA-18 amplicons were used to produce a chimeric GUINEA-18 virus containing the 5’ region of MR766^MC. The Z-1^MR766, Z-23^MR766 and Z-4^GUINEA-18 amplicons were used to produce a chimeric MR766^MC virus containing the 3’ region of GUINEA-18. The Z-1^MR766, Z-23^GUINEA-18 and Z-4^MR766 amplicons were used to produce a chimeric MR766^MC virus in which NS1 to NS4B proteins followed by the N-terminal region of NS5 protein were replaced by the GUINEA-18 proteins. To produce chimeric MR766^MC viruses containing GUINEA-18 NS1/NS2A or NS1/NS3 proteins, nested PCR was performed on Z-23^GUINEA-18 and Z-23^MR766 amplicons into plasmids using specific primers (S1 Table) that were designed so that the 3’ ends of Z-23^GUINEA-18 amplicon sub-fragments coding for NS1 to NS2A or NS3 proteins match with the 5’ ends of Z-23^MR766 amplicon sub-fragments coding for NS3 or NS2B to N-terminal part of NS5 (Fig 1). Transfection with Z-1^MR766 amplicon, Z-23^GUINEA-18 sub-fragment amplicon, Z-23^MR766 sub-fragment amplicon, and Z-4^MR766 amplicon was required for the production of chimeric MR766^MC viruses. To produce a mutant MR766^MC virus containing GUINEA-18 NS4B protein, site-directed mutagenesis was performed on Z-23^MR766 amplicon inserted into plasmid in order to introduce the seven amino-acid substitutions of GUINEA-18 NS4B protein (S1 Table). Transfection with Z-1^MR766 amplicon, mutant Z-23^MR766 amplicon, and Z-4^MR766 amplicon was carried out for the production of mutant MR766^MC virus with GUINEA-18 NS4B protein. To produce a mutant MR766^MC virus with the 3’NCR of GUINEA-18, site-directed mutagenesis was performed on the Z-4^MR766 amplicon to introduce the eight mutations that differentiate the 3’NCRs of GUINEA-18 and MR766^MC. Transfection with Z-1^MR766, Z-23^MR766, and mutant Z-4^MR766 amplicon was then carried out for the production of mutant MR766^MC virus with 3’NCR of GUINEA-18. Mutagenesis and sequencing of mutant plasmids were performed by Genecust (Boynes, France).

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Fig 1. Description of infectious molecular clones GUINEA-18 and MR766^MC.

Generation of chimeric ZIKV molecular clones are shown along with infectious molecular clones GUINEA-18 and the MR766^MC. In (A), schematic representation of ZIKV GUINEA-18 (red) and MR766^MC (blue) genomes. The ZIKV structural and nonstructural proteins are indicated. NCR: non-coding region. The red and blue stars show the location of the 49 amino-acid substitutions that differentiate the GUINEA-18 and MR766^MC polyproteins, respectively are shown. The three amplicons Z-1, Z-23, and Z-4 covering the GUINEA-18 and MR766^MC genomes are shown. In (B), the chimeric GUINEA-18/MR766^[5’region] virus contains the 5’NCR and structural protein region of MR766^MC inserted into GUINEA-18 backbone. The chimeric MR766/GUINEA-18^[3’region] virus contains the GUINEA-18 NS5 gene followed by the 3’NCR inserted into MR766^MC backbone. Site-directed mutagenesis was performed on 3’NCR MR766 to introduce the 3’ NCR mutations of GUINEA-18 leading to MR766/GUINEA-18^[3’NCR] mutant. The hatched boxes show the locations the overlapping sequences between Z-1/Z-23 and Z-23/Z-4 amplicons. In (C), the chimeric MR766/GUINEA-18^[NS1-NS4B], MR766/GUINEA-18^[NS1-NS3], MR766/GUINEA-18^[NS1-NS2A] and MR766/NS4B^GUINEA-18 viruses contain different GUINEA-18 NS proteins inserted into MR766^MC backbone. The hatched boxes show the overlapping sequences between Z-1/Z-23 and Z-23/Z-4 amplicons. Site-directed mutagenesis was performed on Z-23 amplicons to generate chimeric MR766^MC viruses with GUINEA-18 NS1/NS3, NS1/NS2A or NS4B gene.

https://doi.org/10.1371/journal.pntd.0012066.g001

RT-qPCR

Total RNA was extracted from cells using RNeasy kit (Qiagen) and reverse transcription was performed using random hexamer primers (intracellular viral RNA) and MMLV reverse transcriptase (Life Technologies). Quantitative PCR was performed on a ABI7500 Real-Time PCR System (Applied Biosystems, Life Technologies, Villebon-sur-Yvette, France). Data was normalized using 36B4 gene encoding RPL0 protein as housekeeping gene. For each single-well amplification reaction, a threshold cycle (Ct) was calculated using the ABI7500 program (Applied Biosystems, Life Technologies) in the exponential phase of amplification. Relative changes in gene expression were determined using the 2∂∂Ct method and reported relative to the control. The primers used in this study are listed in S1 Table.

Recombinant NS4B protein

Mammalian codon-optimized genes coding for ZIKV 2K-NS4B (residues 2246 to 2527 from viral polyprotein) from viral strains MR766, ZIKV-15555, or epidemic Brazilian 2015 strain BeH819015 (Accession n° KU365778) were established using H. sapiens codon usage as reference. A glycine-serine spacer followed by a FLAG tag were inserted in-frame at the C-terminus of recombinant NS4B protein. Gene synthesis and cloning into Nhe-I and Not-I restriction sites of the pcDNA3.1-hygro (+) vector plasmid to generate recombinant plasmids pcDNA3/MR766.NS4B, pcDNA3/ZIKV-15555.NS4B, and pcDNA3/BeH819015.NS4B were performed by Genecust (Boynes, France). A deletion mutant coding for ZIKV-15555 NS4B protein without the 2K peptide (residues 2247 to 2269 from viral polyprotein) was cloned into pcDNA3.1-hygro (+) to generate pcDNA3/(Δ2K)-ZIKV-15555.NS4B. The cloning and the sequencing by Sanger method of plasmids were performed by Genecust (Boynes, France). Both A549 and HCM3 cells were transfected with plasmids by using Lipofectamine 3000.

eGFP-G3BP fusion protein cloning

Mammalian codon-optimized gene coding for enhanced green fluorescent protein (eGFP) with the A206K monomeric mutation (Accession n° AAB02572) followed by Ras GTPase-activating protein-binding protein 1 (G3BP1) protein (Accession n° U32519) was established using H. sapiens codon usage as reference. A glycine-serine spacer was inserted in-frame between eGFP and G3BP proteins. A synthetic gene coding for fusion protein eGFP-G3BP1 was delivered by Genecust (Boynes, France). The synthetic gene was inserted into EcoR-I and Not-I restriction sites of the pcDNA3.1- vector plasmid to generate recombinant plasmid pcDNA3/eGFP-G3BP. The cloning and the sequencing by Sanger method of pcDNA3/eGFP-G3BP were performed by Genecust (Boynes, France). Endotoxin-free plasmids were delivered by Genecust (Boynes, France). Endotoxin-free plasmid was delivered by Genecust (Boynes, France). A549 cells were transfected with pcDNA3/eGFP-G3BP using Lipofectamine 3,000.

Confocal immunofluorescence assay

Cells seeded on coverslips were fixed with 3.7% paraformaldehyde (PFA) in PBS at RT for 10 min. For analysis of eGFP-G3BP expression, cells were directly visualized by confocal fluorescence microscopy. For analysis of cells transfected with pcDNA3/eGFP-G3BP and then infected with ZIKV, fixed cells were permeabilized with nonionic detergent Triton X-100 at the final concentration of 0.1% in PBS. Cells were stained with anti-E mAb 4G2 in PBS containing 1% bovine serum albumin (BSA). Goat anti-mouse IgG Alexa Fluor 594 was used as secondary antibody. The capture of the fluorescent signal was carried out with a confocal fluorescence Nikon Eclipse TI2-S-HU microscope equipped with x63 objectives coupled to the imaging software NIS-Element AR (Nikon, Champigny-sur-Marne, France). Image processing based on FIJI/ImageJ software was performed to estimate the size in μm² of eGFP-positive condensates.

Flow cytometry

Cells were fixed with 3.7% PFA in PBS and then permeabilized with 0.15% Triton X-100 in PBS. After incubation of cells with a blocking solution for 10 min, cells were stained with mouse anti-E protein mAb 4G2 or rabbit anti-FLAG antibody as primary antibody for 1h. anti-mouse IgG Alexa Fluor 488 or anti-rabbit IgG Alexa Fluor 488 served as secondary antibody. Immunostained cells were subjected to flow cytometric analysis using FACScan flow cytometer (CytoFLEX, Beckman Coulter, Brea, CA, USA). For each assay, at least 10000 cells were analyzed, and the percentage of positive cells was determined using CytExpert software (version 2.1.0.92, Beckman Coulter, Brea, CA, USA).

Immunoblotting

Cells were lysed with RIPA lysis buffer and clarified cell lysates separated by in-house 12% SDS–PAGE gel. Proteins were transferred onto nitrocellulose NC Protran membrane and after blocking with 5% no-fat dry milk in PBS-Tween for 30 min, the membrane was probed with rabbit anti-FLAG antibody for 1h at room temperature (RT). Anti-rabbit IgG-HRP conjugate was used as secondary antibody. Membranes were then incubated with Pierce ECL Western blotting substrate (Thermo Fisher Scientific) and exposed using Amersham imager 680 (GE Healthcare).

Cytotoxicity assay

Cell damage was evaluated measuring lactate dehydrogenase (LDH) release. Supernatants of infected cells were recovered and subjected to a cytotoxicity assay, performed using CytoTox 96 non-radioactive cytotoxicity assay (Promega, Madison, WI, USA) according to manufacturer instructions. Absorbance of converted dye was measured at 490 nm (Tecan). Results of LDH activity are presented with subtraction of control values.

Statistical analysis

All statistical tests were done using the software GraphPad Prism version 10.1.1. Unpaired t test and ANOVA were used in this study.

Molecular viral clone GUINEA-18 derived from ZIKV-15555

A reverse genetic approach based on the ISA method was successfully used to generate an infectious molecular clone MR766^MC of viral strain MR766-NIID [21,22]. The ISA method was used to generate an infectious molecular clone that represents the genomic RNA of viral strain ZIKV-15555 based on viral sequence that has been deposited under GenBank accession number MN025403. Because both 5’ and 3’ termini of viral genome were lacking, the 5’NCR and 3’NCR sequences were completed with nucleotides 1 to 14 and 10721 to 10807 from MR766-NIID genome, respectively. It should be noted that the viral genetic information retrieved on accession number OL41476 was not suitable for the study.

For the ISA method, the overlapping sub-genomic amplicons Z-1^GUINEA-18, Z-23^GUINEA-18, and Z-4^GUINEA-18 were designed to cover full-length genome sequence of ZIKV-15555 (Fig 1). The three GUINEA-18 amplicons were designed so that they can match with Z-1^MR766, Z-23^MR766, and Z-4^MR766 in order to generate chimeric viruses between GUINEA-18 and MR766^MC (S1 Fig). The amplicons were amplified by PCR from the respective plasmids and the purified PCR products were transfected into HEK-293T cells. The recovered infectious molecular clones MR766^MR and GUINEA-18 were used to infect VeroE6 cells and after two rounds of amplification, P2 virus stocks were used for further studies. Using viral plaque assay on VeroE6 cells as quantitative method of measuring infectious ZIKV (plaque forming units, PFU) (S2 Fig), infectious virus titers of GUINEA-18 and MR766^MC reached up to 7 and 8.5 log PFU.mL^-1 after passage P2, respectively.

Characterization of the infectious molecular clone GUINEA-18

Polyprotein sequence alignment between GUINEA-18 and MR766^MC identified 49 amino acid substitutions (S1 Fig) which are distributed along structural and non-structural proteins (Table 1). Only prM and NS2B proteins were fully conserved between the two viruses.

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Table 1. Amino acid changes between West African ZIKV strains ZIKV-15555 (Accession n° MN025403) and MR766-NIID (Accession n° LC002520).

https://doi.org/10.1371/journal.pntd.0012066.t001

The GUINEA-18 and MR766^MC E proteins differ by six amino-acid substitutions with the cluster of mutations I152T/T156I/H158Y that are part of ZIKV glycan loop (GL) region (Table 1). The GL region of GUINEA-18 but not MR766^MC may be N-glycosylated to residue E-N154 [27]. Immunoblot assay using anti-E mAb 4G2 showed that GUINEA-18 E protein migrated slower than MR766^MC E protein consistent with a glycan potentially linked to GUINEA-18 E-N154 residue (S1 Fig). To analyze the replication of GUINEA-18, VeroE6 cells were infected at a multiplicity of infection (m.o.i) of 0.1 PFU/cell and progeny virus production examined at various times post-infection (p.i.) (Fig 2A). Infection with MR766^MC served as control. In comparison with MR766^MC, virus progeny production of GUINEA-18 was significantly reduced by 3 log at 48 h p.i. Infectious virus titers for GUINEA-18 peaked at 6 log PFU.mL^-1 at 72 h p.i.. At this time point post infection, extensive cell death was observed in VeroE6 cells infected by MR766^MC but not GUINEA-18. The reduced virus production of GUINEA-18 was associated with a significant delay in production level of E protein (Fig 2B). At 48 h p.i., there was a 30-fold reduction in intracellular viral RNA (vRNA) production in VeroE6 cells infected with GUINEA-18 as compared with MR766^MC (Fig 2C). At higher m.o.i of infection, GUINEA-18 virus production was still reduced by 2 log at 48 h p.i. and this was associated with lower levels of E protein compared with MR766^MC (S4 Fig). A lactate dehydrogenase (LDH) assay was used as indicator of cell damage in ZIKV-infected VeroE6 cells. Viability of VeroE6 cells infected with GUINEA-18 was preserved until 72h p.i. whereas MR766^MC caused extensive cell death early on (Fig 2D). Our data indicated that GUINEA-18 was replicating more slowly, and to lower virus production, in VeroE6 cells compared to MR766^MC.

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Fig 2. GUINEA-18 replication in non-human fibroblastic VeroE6 cells.

Green monkey kidney fibroblastic VeroE6 cells were infected with MR766^MC and GUINEA-18 at m.o.i of 0.1 (A to C) or 1 PFU/cell (D). In (A), virus progeny productions at 24, 48 and 72 h p.i. were quantified by a standard plaque-forming assay. In (B), FACS analysis was performed on ZIKV-infected cells using anti-pan orthoflavivirus E mAb 4G2 and the percentage of 4G2-positive cells was determined. In (C), intracellular viral RNA production was determined by RT-qPCR at 48 h p.i. RPLPO36B4 mRNA served as a house-keeping RNA control for normalization of samples. In (D), LDH activity was measured at 24, 48 and 72 h p.i. and expressed as a percentage relative to control. The results are the mean (± SEM) of three independent experiments. Asterisks indicate that the differences between experimental samples at each time point are statistically significant, using the unpaired t test and one-way ANOVA (**** p < 0.0001, *** p < 0.001, ** p < 0.01; n.s.: not significant).

https://doi.org/10.1371/journal.pntd.0012066.g002

The replication properties of GUINEA-18 were first assessed in human cells that have been shown to be permissive to ZIKV infection. Firstly, A549 cells were infected with GUINEA-18 or MR766^MC at an m.o.i. of 1. Analysis of viral growth identified a peak of virus production reaching about of 7.5 log mL^-1 in A549 cells infected for 72 h with GUINEA-18 (Fig 3A). MR766^MC infection for 48 h was required to give a similar infectious virus titer. At this timepoint, GUINEA-18 virus production was one 1 log lower compared to MR766^MC, alongside a 2-fold reduction in E protein expression levels (Fig 3B) and 8-fold reduction in vRNA production (Fig 3C). Cytopathic effects were observed in MR766^MC-infected A549 cells from 48 h p.i. whereas only a weak loss of cell viability was observed with GUINEA-18 at 72 h p.i. (Fig 3D). These results show that A549 cells are less permissiveness to GUINEA-18 than MR76^MC. Reduced replication of GUINEA-18 was also observed in HCM3 cells (S5 Fig).

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Fig 3. GUINEA-18 replication in human epithelial A549 cells.

A549 cells were infected with MR766^MC and GUINEA-18 at m.o.i of 1. In (A), viral progeny production was determined at various times post-infection. In (B), percentages of ZIKV-infected cells at 48 h p.i. were determined by FACS analysis using anti-E mAb 4G2 as primary antibody. In (C), intracellular viral RNA production was determined by RT-qPCR at 48 h p.i. RPLPO36B4 mRNA served as a house-keeping RNA control for normalization of samples. In (D), LDH activity was measured at 24, 48 and 72 h p.i. and expressed as a percentage relative to mock-infected cells (control). The results are the mean (± SEM) of three independent experiments. Asterisks indicate that the differences between experimental samples at each time point are statistically significant, using the unpaired t test and one-way ANOVA (**** p < 0.0001, ** p < 0.01, ** p < 0.05; n.s.: not significant).

https://doi.org/10.1371/journal.pntd.0012066.g003

Next, we evaluated whether the lower replication capacity of GUINEA-18 in A549 cells was associated to a change in activation of antiviral innate immune responses when compared to MR766^MC. ZIKV is inhibited by type I IFNs such as IFN-β and antiviral effector proteins, so called interferon-stimulated genes (ISGs) following recognition of viral nucleic acids and molecular features associated with viral infection of host cells [26,28–31]. The relative expression of ISGs and IFN-β mRNA was assessed by RT-qPCR on total RNA isolated from ZIKV-infected A549 cells at 48 h p.i. (Fig 4). As evidenced by analysis of fourteen representative ISGs, ISG mRNA levels were significantly lower in A549 cells infected by GUINEA-18 compared to MR766^MC. As a control of ISG protein production, ISG15 was weakly detected in A549 cells infected by GUINEA-18 compared with MR766^MC (S6 Fig). Moreover, mRNA levels of IFN-β was also much lower in A549 cells infected by GUINEA-18 in comparison with MR766^MC. Thus, infection of A549 cells with GUINEA-18 induced lower ISG and IFN-β responses than MR766^MC.

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Fig 4. Expression of ISG and IFN-β mRNA in A549 cells infected by ZIKV.

A549 cells were infected with GUINEA-18 or MR766^MC at an m.o.i. of 1. The relative abundance of IFN-β and ISG mRNA was determined at 48 h p.i. by RT-qPCR. RPLPO36B4 mRNA served as a house-keeping RNA control for normalization of samples. Results are expressed as the fold-induction of IFN-β and ISG mRNA in ZIKV-infected cells relative to those in mock-infected cells. The results are the mean (± SEM) of three independent experiments. Asterisks indicate that the differences between experimental samples at each time point are statistically significant, using the unpaired t test (** p < 0.01, * p < 0.05).

https://doi.org/10.1371/journal.pntd.0012066.g004

Role of the 5’ and 3’ regions in GUINEA-18 replication

We next examined whether the reduced replicative abilities of GUINEA-18 depended on virus-specific determinants. A chimeric GUINEA-18 virus in which the Z-1^GUINEA-18 amplicon was swapped for Z-1^MR766 was used to assess the importance of 5’ region in replication properties of GUINEA-18 (Fig 1B). The resulting GUINEA-18/MR766^[5’region] virus includes the 5’NCR mutations G19A and C41T and eight amino-acid substitutions that are distributed between C [2] and E [6] proteins (Fig 1). Of note, mutation E-T156I removes the N-glycosylation site of the E protein, resulting in a non-glycosylated chimeric virus [22,32]. A549 cells were infected with chimeric GUINEA-18/MR766^[5’region] virus or GUINEA-18 at an m.o.i. of 1 (S7 Fig); with infection by MR766^MC as control. At 48 h p.i., there was no significant change in virus production between GUINEA-18/MR766^[5’region] and GUINEA-18 (S7A Fig). There were also similar expression levels of E protein (S7B Fig). These results do not argue in favor of major role of the 5’NCR and structural proteins in the replication properties of GUINEA-18.

We next addressed the role of 3’ region of viral genome in the replicative abilities of GUINEA-18. A chimeric MR766^MC virus was obtained in which Z-4^MR766 amplicon was replaced by Z-4^GUINEA-18 leading to a swapping of both NS5 and 3’NCR sequences between MR766^MC and GUINEA-18 (Fig 1B). There are eleven amino-acid substitutions that are distributed between MTase and RdRp domains of the NS5 protein (Table 1). Eight mutations differentiate the 3’NCRs from GUINEA-18 and MR766^MC (S8A Fig). The most notable mutations in SLII and DB structures might have an effect on viral RNA replication (S8B Fig) [33]. Transfection with Z-1^MR766, Z-23^MR766, and Z-4^GUINEA-18 amplicons resulted in virus production with an infectious titer of 3 log PFU.mL^-1 at passage 2 on VeroE6 cells. Thus, the swapping of Z-4^GUINEA-18 amplicon into MR766^MC restrained viral replication by at least 5 log compared with MR766^MC. To determine whether the inability of chimeric MR766/GUINEA-18^[3’region] virus to replicate depended on the 3’NCR, site-directed mutagenesis was conducted on Z-4^MR766 amplicon to generate a mutant MR766/GUINEA-18^[3’NCR] virus bearing all GUINEA-18 3’NCR mutations (Fig 1 and S8A). The mutant virus was assessed in VeroE6 cells (S9 Fig). At 48 h p.i., there was comparable virus production between MR766/GUINEA-18^[3’NCR] and MR766^MC (S8A Fig). This was associated with a similar percentage of ZIKV-infected cells (S9B Fig). The mutant virus was more cytopathic in VeroE6 cells than the parent (S9C Fig). These results rule out a role for the 3’NCR in the replicative abilities of a chimeric MR766^MC virus with the 3’ region of GUINEA-18. Our data indicated that a chimeric MR766^MC virus with GUINEA-18 NS5 protein is unable to sustain infection of mammalian cells, precluding the study of the largest NS protein in attenuation of GUINEA-18.

The replicative properties of GUINEA-18 depend on NS1 to NS4B proteins

To evaluate the importance of NS1 to NS4B proteins in the replication of GUINEA-18, a chimeric MR766/GUINEA^[NS1-NS4B] virus was generated, in which the MR766^MC region coding for NS1 to NS4B proteins was swapped with their counterpart in GUINEA-18 (Fig 1C). The chimeric MR766/GUINEA^[NS1-NS4B] virus was compared with MR766^MC in VeroE6 cells infected for 48 h at an m.o.i. of 0.1 (Fig 5). Infection with GUINEA-18 served as control. Analysis of viral growth showed that insertion of GUINEA-18 NS1 to NS4B into MR766^MC strongly reduced virus production (Fig 5A). Virus production was similar between MR766/GUINEA-18^[NS1-NS4B] and GUINEA-18 (Fig 5A), and the two viruses were comparable for the E protein expression level (Fig 5B) and intracellular vRNA production in VeroE6 cells (Fig 5C). Late in infection, chimeric MR766/GUINEA-18^[NS1-NS4B] virus preserved cell viability, as observed for GUINEA-18 (Fig 5D). Our data thus indicated a key role for NS1 to NS4B proteins in the replication properties of GUINEA-18.

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Fig 5. MR766/GUINEA-18^[NS1-NS4B] replication in VeroE6 cells.

VeroE6 cells were infected with MR766/GUINEA-18^[NS1-NS4B] chimeric virus or parental viruses at m.o.i of 0.1. In (A), virus progeny production at 24h, 48h, and 72h. In (B), percentage of ZIKV-infected cells at 24h, 48h, and 72h measured by FACS analysis using mAb 4G2. In (C), RT-qPCR was performed on viral RNA (vRNA) extracted from VeroE6 cells infected for 48h with ZIKV. RPLPO36B4 mRNA served as a house-keeping RNA control for normalization of samples. In (D), LDH activity was measured at 72 h p.i. The results are the mean (± SEM) of three independent experiments. Asterisks indicate that the differences between experimental samples at each time point are statistically significant, using the unpaired t test and one-way ANOVA (**** p < 0.0001, *** p < 0.001; n.s.: not significant).

https://doi.org/10.1371/journal.pntd.0012066.g005

The replication efficiency of the MR766^MC chimera with GUINEA-18 NS1 to NS4B proteins was next assessed in A549 cells (Fig 6). The behavior of chimeric MR766/GUINEA-18^[NS1-NS4B] virus was indistinguishable from GUINEA-18 in viral replication (Fig 6A–6C) and cytotoxicity (Fig 6D) reinforcing the notion that NS1 to NS4B proteins influence GUINEA-18 replication in mammalian host cells.

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Fig 6. MR766/GUINEA-18^[NS1-NS4B] replication in A549 cells.

A549 cells were infected for 48h with MR766/GUINEA-18^[NS1-NS4B] chimera or parental viruses at m.o.i of 1. In (A), virus progeny production. In (B), RT-qPCR was performed on viral RNA (vRNA) extracted from ZIKV-infected VeroE6 cell. RPLPO36B4 mRNA served as a house-keeping RNA control for normalization of samples. In (C), percentage of ZIKV-infected cells measured by FACS analysis using mAb 4G2. In (D), LDH activity was measured at 48 h p.i. The results are the mean (± SEM) of three independent. Asterisks indicate that the differences between experimental samples at each time point are statistically significant, using the unpaired t test and one-way ANOVA (**** p < 0.0001, ** p < 0.01; * P < 0.05; n.s.: not significant).

https://doi.org/10.1371/journal.pntd.0012066.g006

To better understand the impact of NS1 to NS4B proteins on GUINEA-18 replication properties, we produced two additional chimeric viruses, MR766/GUINEA-18^[NS1-NS3] and MR766/GUINEA-18^[NS1-N2A], containing GUINEA-18 NS1/NS2AB/NS3 or NS1/NS2A in an MR766^MC backbone, respectively (Fig 1C). The amino-acid sequence of NS2B protein is conserved between GUINEA-18 and MR766^MC (S1 Fig). The replication of chimeric viruses was analyzed in VeroE6 cells infected at an m.o.i. of 0.1 and compared with parental viral clones (Fig 7A). Infection with MR766/GUINEA^[NS1-NS4B] chimera served as control. Analysis of virus production revealed that MR766^MC chimeric viruses with GUINEA-18 NS1-NS3 or NS1-NS2A proteins have little or no effect on virus replication (Fig 7A). Infection with chimeric MR766^MC viruses resulted in extensive cell death (Fig 7B). Thus, the swapping of NS1 to NS3 proteins was not sufficient to affect MR766^MC replication and cytotoxicity emphasizing the major role of NS4B in the attenuated phenotype of GUINEA-18 in vitro.

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Fig 7. Effects of GUINEA-18 NS proteins on MR766^MC replication.

VeroE6 cells were infected with MR766/GUINEA-18 chimeric viruses containing GUINEA-18 NS proteins or parental ZIKV at an m.o.i. of 0.1. In (A), virus progeny production at 48h p.i. The dotted line indicates MR766^MC progeny production. In (B), LDH activity was measured at 72 h p.i. The dotted line indicates the rate of LDH release with MR766^MC. The results are the mean (± SEM) of three independent experiments. Statistical analysis relative to MR766^MC values was noted. Asterisks indicate that the differences between experimental samples at each time point are statistically significant, using the unpaired t test (*** p < 0.001, ** p < 0.01, * p < 0.05; n.s.: not significant).

https://doi.org/10.1371/journal.pntd.0012066.g007

The above results suggest that the replicative properties of GUINEA-18 depend on NS4B protein. Among seven amino-acid substitutions that differentiate NS4B of ZIKV-1555 and MR766, four have been identified at positions 11, 20, 24 and 26 (Table 1). The ZIKV-15555 residues R20 and T24 have been also identified in West African ZIKV strains Senegal-Kedougou 2011 and Senegal-Kedougou 2015 (European Nucleotide Archive accession number n°PRJEB39677) that have been isolated from mosquito pools in Casamance region of Senegal in 2011 and 2015, respectively [19]. Residues R20 and T24 were not observed in Asian and Asian-related American viruses. A change from non-polar amino-acid alanine to polar residue threonine at position 24 might influence NS4B conformation. A three-dimensional structure of the N-terminal region of NS4B was performed by modelling on Phyre² [34] allowing de novo peptide structure prediction (S10A Fig). Structural analysis showed that ZIKV-1555 NS4B residues 1 to 16 and 35 to 50 have propensity for forming a helical structure and transmembrane helix, respectively. The NS4B amino-acid substitutions at positions 20, 24, and 26 that differentiate ZIKV-15555 from MR766 have been identified in a non-ordered structure between helix α1 and TM1 [35].

We assessed whether the NS4B mutations that differentiate ZIKV-15555 from MR766 impact protein expression. Recombinant 2K-NS4B (hereafter intitled rNS4B) proteins derived from ZIKV-15555, MR766, and BeH819015 were expressed in A549 cells using from pcDNA3. The rNS4B proteins were C-terminally tagged with FLAG epitope. The ZIKV-15555 rNS4B protein expressed without the 2K peptide served as control. Next, rNS4B protein expression in transfected A549 cells was verified by FACS analysis using anti-FLAG antibody (S11 Fig). Levels of rNS4B protein expression were comparable across proteins expressed in A549 cells (Fig 8). Immunoblot assays using anti-FLAG antibody allowed the detection of MR766 and ZIKV-15555 rNS4B proteins in A549 (Fig 8A) and HCM3 cells (Fig 8B). Migration was comparable with an apparent molecular weight similar to rNS4B mutant without 2K indicating that the N-terminal peptide was correctly processed from the N-terminus of protein. The change in migration profile of BeH819015 NS4B protein compared to ZIKV-15555 and MR766 could be due to specific residues that have been identified in epidemic Asian/American ZIKV strains (S10B Fig). We can conclude that the seven NS4B amino-acid substitutions that differentiate ZIKV-15555 from MR766 have no obvious effect on protein expression.

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Fig 8. Intracellular expression of recombinant ZIKV NS4B protein.

A549 (A) and HCM3 (B) cells were transfected for 24 h with pcDNA3 expressing rNS4B protein from ZIKV-15555 (rNS4B^ZIKV-15555), MR766 (rNS4B^MR766), or BeH819015 (rNS4B^BeH819015). The rNS4B^ZIK-15555 protein mutant lacking the 2K peptide is indicated as (Δ2K)-rNS4B^ZIK-15555. Cell lysates in RIPA buffer were assessed by immunoblot assays with anti-epitope antibody FLAG as the primary antibody. The β-tubulin protein served as a protein-loading control. The positions of rNS4B are indicated.

https://doi.org/10.1371/journal.pntd.0012066.g008

To better understand the role of NS4B in GUINEA-18 replication, the NS4B gene of MR766^MC was mutated to introduce the seven amino-acid substitutions of GUINEA-18 NS4B protein (Fig 1C). Because most of the mutations have been identified in the N-terminal region of NS4B protein (Table 1), we produced a mutant MR766^MC virus with the S11/R20/T24/I26/V180 residues. The chimeric MR766^MC/NS4B^GUINEA-18 and mutant MR766^MC-NS4B viruses were assessed for replication in VeroE6 cells at an m.o.i. of 0.1 (Fig 9). Parental viruses and chimeric MR766/GUINEA-18^[NS1-NS4B] virus served as controls. Analysis of MR766^MC/NS4B^GUINEA-18 chimera revealed that insertion of GUINEA-18 NS4B in MR766^MC caused no reduction in virus production (Fig 9A) and no effect on expression level of E protein (Fig 9B). By contrast, infection with mutant MR766^MC-NS4B virus resulted in low virus production, comparable to that observed with GUINEA-18 or MR766/GUINEA-18^[NS1-NS4B] (Fig 9C). This correlated with weak expression levels of E protein (Fig 9D). Viability of VeroE6 cells infected with MR766^MC-NS4B mutant was preserved at 72 h p.i. (Fig 9E). The effects of GUINEA-18 NS4B mutations on MR766^MC were not observed in A549 cells raising the possibility of a cellular context dependent effect of the N-terminal NS4B residues on virus replication (Fig 9F).

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Fig 9. Effect of GUINEA-18 NS4B on MR766^MC replication.

VeroE6 cells (A-E) and A549 cells (F) were infected with GUINEA-18, MR766^M, MR766^MC mutant containing the GUINEA-18 NS4B residues S11/R20/T24/I26/V180 (MR766^MC-NS4B mutant) or MR766^MC chimeric virus containing the GUINEA-18 NS4B protein (MR766^MC/NS4B^GUINEA-18). MR766/GUINEA-18^[NS1-NS4B] served as a control. In (A-B), VeroE6 cells were infected with MR766^MC/NS4B^GUINEA-18 at m.o.i 0.1. Virus progeny production (A), and percentage of ZIKV-infected cells measured by FACS analysis using mAb 4G2 (B). In (C-E), VeroE6 cells were infected with MR766^MC-NS4B mutant at m.o.i 0.1. Virus progeny production (C), and percentage of ZIKV-infected cells measured by FACS analysis using mAb 4G2 (D) were determined at 48 h p.i. LDH activity was measured at 72 h p.i. (E). In (F), A549 cells were infected with MR766^MC-NS4B mutant at m.o.i 1. The percentages of ZIKV-infected cells measured by FACS analysis using mAb 4G2 were determined at 48 h p.i. The results are the mean (± SEM) of two or three independent experiments. Asterisks indicate that the differences between experimental samples at each time point are statistically significant, using the unpaired t test and one-way ANOVA (**** p < 0.0001, *** p < 0.001, ** p < 0.01; * p < 0.05; n.s.: not significant).

https://doi.org/10.1371/journal.pntd.0012066.g009

GUINEA-18 NS1 to NS4B proteins influence stress granule formation

In response to RNA virus infection, cellular stress granules (SGs) are assembled as cytoplasmic condensates which can sequester viral RNA and proteins thus restricting viral growth [36–38]. A common feature of infection with orthoflaviviruses including ZIKV is inhibition of SG assembly facilitating viral replication and limiting antiviral signaling activation in infected host-cells [39–41]. In particular, ZIKV hijacks the core SG proteins such as RasGAP SH3 domain-binding protein (G3BP) [40]. Other studies supported the idea that ZIKV uses NS proteins to interfere on SG formation [28]. In this regard, we were wondering whether GUINEA-18 NS1 to NS4B proteins might differ from MR766^MC NS proteins in their ability to inhibit SG assembly in infected cells. To investigate this further, A549 cells were infected with chimeric MR766^MC/GUINEA-18^[NS1-NS4B] virus at an m.o.i. of 2. Both MR766^MC and GUINEA-18 were used as controls. Prior to virus infection, A549 cells were transfected with a plasmid expressing eGFP reporter fused in-frame to the N-terminus of G3BP which is considered a SG marker [42,43]. SG formation was examined in ZIKV-infected A549 cells under environmental stress. Sorbitol as a physiological osmotic and oxidative stressor was used to drive GFP-G3BP fusion protein to SGs [44,45]. By confocal fluorescence microscopy, cytoplasmic eGFP-positive condensates with an average surface area of 2.5 μm² were observed in A549 cells incubated with 0.4M sorbitol for 1.5 h (S12 Fig).

The abundance of eGFP-positive condensates was determined in A549 cells infected with ZIKV for 40 h and then stressed with 0.4M sorbitol for 1.5 h (Fig 10). Infection by ZIKV was confirmed by IF assay using anti-E mAb 4G2. Confocal fluorescence microscopy detected eGFP-positive condensates in A549 cells infected by MR766^MC (Fig 10A). Almost fifty 4G2-positive cells were scored for the number of eGFP-positive condensates per cell profile. An average of seven defined granules per cell was observed in A549 cells infected by MR766^MC (Fig 10B). Almost 50% of the eGFP-positive condensates had a surface area ranging from 3 to 9 μm² with an average of approximately 4.1 μm² (Fig 10C). As assessed by quantifying eGFP-positive condensates, we observed that GUINEA-18 actively blocked SG formation under environmental stress (Fig 10B). Almost 80% of granules had surface area lower than 3 μm² (Fig 10C). Thus, GUINEA-18 showed greater efficiency to prevent SG formation in A59 cells stressed with sorbitol, compared to MR766^MC.

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Fig 10. Stress granule formation in ZIKV-infected cells stressed with sorbitol.

A549 cells were transfected with pcDNA3/eGFP-G3BP for 6 h and then infected with ZIKV at an m.o.i. of 2. At 40 h p.i., cells were stressed with 0.4 M sorbitol for 1.5 h. In (A), cells infected with MR766^MC, GUINEA-18, or MR766/GUINEA-18^[NS1-NS4B] chimera were identified using anti-E mAb 4G2. Cells positive for ZIKV E (red) and eGFP-G3BP (green) proteins were examined by confocal microscopy analysis. Nuclei were stained with DAPI (blue). Scale bar, 50 μm. In (B), cells positive for E protein expression (n ~ 50) were scored for the number of eGFP-positive condensates per ZIKV-infected cell. Values from 4 independent experiments are presented. Asterisks indicate that the differences between experimental samples at each time point are statistically significant, using 2-way ANOVA test (**** p < 0.0001). In (C), cells positive for E protein and eGFP-G3BP expression (n ~ 100) were scored for the surface of EGFP-positive condensates as small (< 3 μm²), medium (3–6 μm²) and large (6–9 μm²) defined granules. Large SGs were not detected (n.d.) in A549 cells infected by GUINEA-18. Values from two independent experiments are presented. Asterisks indicate that the differences between experimental samples at each time point are statistically significant, using the unpaired t test (** p < 0.01; * p < 0.05; ns: not significant).

https://doi.org/10.1371/journal.pntd.0012066.g010

Infection with chimeric MR766/GUINEA^[NS1-NS4B] virus resulted in a weak number of eGFP-positive condensates in A549 cells (Fig 10A). The abundance of defined granules was reduced by 50% compared to parental virus (Fig 10B). The surface area of eGFP-positive condensates was approximately 2.5 μm² (Fig 10C). Our data using chimeric MR766/GUINEA^[NS1-NS4B] virus suggest that NS1 to NS4B proteins may play an essential role in inhibition of ZIKV-mediated SG formation. We conclude that GUINEA-18 NS proteins have evolved mechanisms to restrain SG formation, with possible implications for viral propagation.