Probing Zika Virus Neutralization Determinants with
Glycoprotein Mutants Bearing Linear Epitope Insertions
Matthew T. Chambers,aMegan C. Schwarz,aMarion Sourisseau,aEssanna S. Gray,aMatthew J. Evansa
aDepartment of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
ABSTRACT Zika virus (ZIKV) glycoproteins are the primary target of the humoral immune response. In this study, we explored the capacity of these glycoproteins to tolerate insertion of linear epitope sequences and the potential of antibodies that bind these epitopes to inhibit infection. We first created a panel of ZIKV mutants with the FLAG epitope inserted in the premembrane (prM) and envelope (E) glyco-protein regions. The insertion locations were based on the results of our recent transposon insertional mutagenesis screen. Although FLAG insertions in prM greatly impaired viral fitness, this sequence was tolerated in numerous surface-exposed E protein sites. We observed that mutants bearing FLAG epitopes in E domains I and II and the E domain I-II hinge region were all neutralized by FLAG antibody; however, the neutralization sensitivity varied highly. We measured the antibody binding effi-ciency and found that this closely matched the pattern of neutralization sensitivity. We determined that E glycosylation did not affect antibody binding to a nearby epitope or its capacity to serve as a neutralization target. Although we could not generate infectious viruses with FLAG epitope insertions in a buried region of E pro-tein domain III, we found that the V5 epitope could be inserted at this site without greatly impacting fitness. Furthermore, this virus was efficiently neutralized by V5 antibodies, highlighting that even buried epitopes can function as neutralization tar-gets. Finally, we analyzed the timing of antibody neutralization activity during cell entry and found that all antibodies blocked a step after cell attachment.
IMPORTANCE Zika virus (ZIKV) infections are associated with severe birth defects and neurological disease. The structure of the mature ZIKV particle reveals a virion surface covered by the envelope glycoprotein, which is the dominant target of the humoral immune response. It is unclear if all regions of the envelope protein surface or even buried epitopes can function as neutralization targets. To test this, we cre-ated a panel of ZIKV mutants with epitope insertions in different regions of the en-velope protein. In characterizing these viruses, we found that the strength of anti-body binding to an epitope is the major determinant of the neutralization potential of an antibody, that even a buried region of the envelope protein can be efficiently targeted, and that the sole potential envelope glycan does not impact nearby epitope antibody binding and neutralization. Furthermore, this work provides impor-tant insights into our understanding of how antibodies neutralize ZIKV.
KEYWORDS Zika virus, antibody, glycoproteins, neutralization, structure
T
he recent emergence of Zika virus (ZIKV) is a public health concern (1, 2). From its first identification in 1947 in a sentinel monkey in Uganda (3) until recently, ZIVK was thought to cause only a self-limiting mild febrile illness. Beginning in 2007, widespread outbreaks occurred in the Yap Islands, French Polynesia, and later Brazil (4–6), and these have been associated with severe birth defects and neurological disease (7, 8). Since that time, additional outbreaks have occurred in the Pacific Islands, Southeast Asia, and the Americas (9–12).Received23 March 2018Accepted2 July
2018
Accepted manuscript posted online5 July
2018
CitationChambers MT, Schwarz MC,
Sourisseau M, Gray ES, Evans MJ. 2018. Probing Zika virus neutralization determinants with glycoprotein mutants bearing linear epitope insertions. J Virol 92:e00505-18.https://doi.org/ 10.1128/JVI.00505-18.
EditorJulie K. Pfeiffer, University of Texas
Southwestern Medical Center
Copyright© 2018 American Society for
Microbiology.All Rights Reserved.
Address correspondence to Matthew J. Evans, matthew.evans@mssm.edu.
crossm
on November 6, 2019 by guest
http://jvi.asm.org/
transited through thetrans-Golgi network, where E proteins undergo rearrangement into antiparallel dimers arranged in a herringbone pattern characteristic of flaviviruses. This rearrangement exposes the prM cleavage site, which is cleaved by a furin-like protease. Upon release of the virion into the more neutral pH of the extracellular environment, the pr protein dissociates from the E protein, resulting in the mature virion (15). The E protein in the mature virion is folded into three domains (Fig. 1) (16, 17). Domain I (DI) is comprised of a central-barrel and a highly flexible loop that bears the sole E protein N-linked glycosylation site, N154. This glycan is highly conserved among most flaviviruses but is absent from some ZIKV isolates, particularly those of the African lineage (18, 19). Domain II (DII) forms an elongated finger-like shape that is the major interaction surface between monomers of an E dimer. The fusion loop is located at the end of DII that is distal to domain I and domain III (DIII). The major feature of DIII is an Ig-like region that is thought to contribute to host cell binding (20, 21).
The ZIKV E protein is the main target of the humoral response. Presumably, an efficient neutralizing antibody would both exhibit strong binding characteristics and block a region of the E protein that mediates critical host cell entry functions (22). The majority of characterized ZIKV antibodies elicited by natural infection in humans target E DI-II and bind conformational and often quaternary epitopes that span across E monomers (23). Comparing the relationship between epitope location and antibody inhibitory capacity is complicated by such epitope complexity and the different binding affinities of naturally occurring antibodies. Furthermore, antibody neutralization of flaviviruses is thought to be as much of a function of epitope accessibility as of antibody affinity (22). Cryo-electron microscopy structures of ZIKV provide a static view of the mature viral particle and reveal surface-exposed residues that are likely to be accessible to antibodies; however, it is unclear if all surface regions are available for antibody binding or if regions that appear to be buried under the E protein surface can be neutralization targets.
In this study, we created a panel of ZIKV mutants each bearing a linear epitope insertion in different regions of the prM and E glycoproteins, which allowed us to use monoclonal antibodies that bind to these linear epitopes to precisely target distinct regions of the ZIKV glycoproteins. We further assessed if E glycosylation at N154, which is not conserved among all ZIKV isolates, impacts the binding and inhibitory potential of antibodies that have nearby binding sites. Finally, we performed synchronized infections to delineate the timing of inhibition for antibodies targeted to different E protein regions during ZIKV cell entry.
RESULTS
Characterization of ZIKV mutants with epitope-tagged glycoproteins. To choose locations for epitope insertion within the ZIKV prM and E glycoproteins, we used the results from our recent transposon mutagenesis screen that identified regions of the ZIKV glycoprotein genes that best tolerated 15-nucleotide transposon insertions (24). We suspected that these transposon-tolerant sites represented regions of genetic flexibility that were more likely to tolerate other epitopes. This screen identified 59 such sites in the E gene and 14 sites in prM that together grouped into seven clusters. We selected 20 sites for insertion of 27 nucleotides encoding a SalI restriction site and a
on November 6, 2019 by guest
http://jvi.asm.org/
FLAG epitope sequence (DYKDDDDK) (Fig. 1B). In the carboxyl terminus of the pr protein, two candidate insertion sites were selected (Fig. 1B, pr). Though the pr protein is removed from mature particles, it is unknown what proportion of the viral population is structurally heterogeneous, including immature and partially immature particles that may retain pr epitopes (25, 26). Two sites within the ectodomain M-loop of the M protein were selected (Fig. 1B, M). As the E protein covers the M-loop in the mature particle, such epitopes would likely be accessible only in partially immature virions or if ZIKV glycoproteins display structural dynamics similar to the breathing-like motions displayed by dengue virus (27). Within the E protein, insertion sites were selected in five distinct regions. Three insertion sites were selected within the glycan loop (Fig. 1B, E DI) in DI. Within DII, three sites that were proximal to the E dimer interface and two sites that were distal to the E dimer interface were selected (Fig. 1B, E DII). In the hinge region between DI and DII, we chose three sites within a highly flexible exposed loop (Fig. 1B, E DI/II). All candidate insertion sites within DIII lay buried beneath the surface FIG 1Structure of mature M-E dimer and epitope insertion sites. (A) Structure of the ZIKV E protein dimer, viewed along the 2-fold axis (left) and rotated 90° (right). E domains I (red), II (yellow), and III (blue) and the transmembrane stem and anchor (E-TM, teal) are colored in a single E monomer. The fusion loop and N154 glycan are colored purple and orange, respectively. The M protein transmembrane (M-TM), stem, and M-loop regions are colored pink, and the M-loop is indicated with a pink line. (B) List of candidate FLAG epitope insertion locations within the ZIKV prM and E glycoproteins. The specific mature protein, glycoprotein region, nucleotide position, and amino acid that precede each insertion are indicated in the left four columns. The remaining three columns show the M and E protein structures, colored as defined above, with the insertion region for each cluster shown in green. An additional side view of the isolated M protein shows the insertion location within the M-loop of both M monomers.
on November 6, 2019 by guest
http://jvi.asm.org/
[image:3.585.40.401.70.460.2]of the mature viral particle (Fig. 1B, E DIII) and thus may also be accessible only on partially immature virions or as a result of viral structural dynamics.
To test the capacity of the mutant genomes to produce infectious virus, we transfected 293T cells with plasmids encoding the FLAG-tagged ZIKV genomes, as well as wild-type and polymerase-deficient [Pol(⫺)] genomes as controls. Supernatants were collected at 4 days posttransfection, and infectious titers were determined by Vero cell limiting dilution assays that were scored by the virus-induced cytopathic effect (CPE). The titers of rescued viruses ranged from being similar to the those of the wild type to being below the limit of detection of this assay, which represented a more than 5-log impairment (Fig. 2A). All viruses with FLAG insertions in pr and M were either dead or highly impaired. While three of these mutants (739-, 778-, and 787-FLAG) produced no detectable virus, the 730-FLAG mutant titers were inconsistent, near the assay limit of detection, and not high enough for downstream experiments. All three mutant viruses with FLAG insertions near the glycan in E DI (1417-, 1426-, and 1444-FLAG) were rescued with titers equal to or within 4-fold of the wild-type titer. Of the insertions along the E DII dimer interface, only the 1597-FLAG mutant produced detectable levels of infectious virus, although this mutant was impaired by 65-fold compared to the wild type. Mutants 1663- and 1675-FLAG with DII insertions distal to the dimer interface
FIG 2Characterization of FLAG-tagged glycoprotein ZIKV mutants. (A) Limiting dilution assays were performed to compare the infectious titers of wild-type and mutant viruses. Infectious viral supernatant from transfected 293T cells was collected at 4 days posttransfection and used to infect Vero cells. Viral titers were calculated from the Vero cell CPE at 6 days postinfection. Values are expressed as the number of TCID50per milliliter. The data are presented as means and standard deviations from three independent experiments, each performed in triplicate. Arrows denote mutants with the highest TCID50value per FLAG insertion region, which were selected for additional characterization. (B) Number of viral RT-PCR clones from infected cells that maintained the FLAG insertion and surrounding sequence. (C) Alignment of the sequences of the RT-PCR products from cells infected with the 2020-FLAG virus. Nucleotides that are not present indicate gaps. (D) Immunoblot analysis of Vero cell lysates collected at 48 h postinfection with wild-type and mutant ZIKV performed with antibodies against ZIKV E protein, FLAG, and actin. The positions of molecular weight standards (in kilodaltons) are indicated to the left, and the predicted full-length proteins are marked with brackets on the right of each blot.
on November 6, 2019 by guest
http://jvi.asm.org/
[image:4.585.43.498.70.401.2]produced infectious virus with titers reduced by 8- and 295-fold, respectively, relative to the wild-type titer. Insertions in the DI-II hinge region reduced viral titers by 215-fold for the 1813-FLAG virus, by 9,260-fold for the 1816-FLAG virus, and to undetectable levels for the 1822-FLAG virus. Most mutants with E DIII FLAG insertions did not produce detectable levels of infectious virus (2023-, 2029-, 2035-, and 2044-FLAG); however, the titers of the 2020-FLAG mutant virus were just above the limit of detection and consistent enough to warrant downstream analysis. Thus, we were able to rescue viruses with FLAG epitopes inserted into five distinct regions of the E protein. We selected mutants with the highest infectious titer from each region for additional characterization (marked by arrows in Fig. 2A).
We next sought to assess the stability of the FLAG insertions in the mutant ZIKV genomes, as flaviviruses can excise deleterious sequences over multiple rounds of replication. All downstream experiments that we intended to perform involved a single cycle of infection with rescued virus; therefore, we sought to ensure that epitope insertions were maintained within this time frame. To do so, we used reverse transcrip-tion (RT) and PCR to amplify E protein-coding regions from RNA harvested from Vero cells infected for 24 h with rescued viruses. We cloned these PCR products into bacterial plasmids and used Sanger sequencing to score the frequency of each FLAG insertion. The results showed that viral genomes with epitope insertions along the viral surface in the E DI, DII, and DI-II hinge regions did not contain any changes to the FLAG epitope or surrounding sequence (Fig. 2B). In contrast, the epitope sequence of the 2020-FLAG mutant was altered in all 10 of the PCR clones that we analyzed (Fig. 2B). Three of these clones completely lost the FLAG epitope sequence but maintained a silent mutation that created a SalI restriction site, and six others had various in-frame deletions of internal sections of the FLAG epitope (Fig. 2C). Due to this instability, we removed the 2020-FLAG mutant from further analysis. To further assess the stability of the FLAG insertion, we investigated FLAG expression by immunoblot analysis of Vero cell lysates collected at 48 h postinfection. Lysates from cells infected with FLAG-tagged mutants but not those from cells infected with wild-type virus had full-length E proteins that were reactive with the M2 anti-FLAG monoclonal antibody (Fig. 2D). Taken together, the sequencing and immunoblotting results showed that the FLAG insertions in DI-II were stable following at least a single cycle of infection and thus could be safely used in experiments within this time frame.
FLAG antibody inhibits FLAG-tagged ZIKV infection.We next sought to deter-mine the impact of the FLAG monoclonal antibody on infections with FLAG-tagged viruses. To do so, we incubated wild-type and FLAG-tagged viruses with 3-fold serial dilutions of FLAG antibody for 30 min prior to infecting Vero cells. Infected cells were quantified by staining for the presence of ZIKV NS3 or E proteins at 24 h postinfection. The wild-type virus was not impacted at the highest concentration of FLAG antibody tested (Fig. 3A). Conversely, each of the FLAG-tagged viruses was sensitive to neutral-ization with the FLAG antibody. However, the FLAG antibody neutralneutral-ization potency varied dramatically between the mutant viruses, with half-maximal inhibitory concen-tration (IC50) values ranging from 3.8 to 296 ng/ml (Fig. 3C). The 1426-FLAG insertion
within the glycan loop was the most sensitive to FLAG antibody neutralization, fol-lowed by the 1663-FLAG DII insertion distal to the E dimer interface with a 4-fold higher IC50. The 1417-FLAG and 1813-FLAG mutants displayed intermediate neutralization
sensitivities at 37.9 and 68.8 ng/ml, respectively, despite 1417-FLAG being separated from the highly sensitive 1426-FLAG by only 3 residues. The 1597-FLAG epitope, located along the E dimer interface, was the least sensitive to neutralization, with an IC50value 77.9-fold greater than that of 1426-FLAG. These data support our hypothesis
that antibodies targeting distinct regions of the ZIKV E protein will exhibit differential impacts on infection, perhaps due to differences in the accessibility of the targeted domain and/or functional relevance to the cell entry process.
We next sought to determine if FLAG insertions altered global E protein antigenicity. To do so, we performed neutralization assays with the ZKA185 antibody, a potent
on November 6, 2019 by guest
http://jvi.asm.org/
neutralizing antibody that is thought to bind a conformational quaternary E protein epitope (23). All FLAG-tagged and wild-type viruses were neutralized by the ZKA185 antibody (Fig. 3B). IC50values ranged from 18.7 to 61 ng/ml, with the wild-type virus
being the most sensitive and 1663-FLAG being the least sensitive to neutralization (Fig. 3C). The IC50values for FLAG-tagged viruses neutralized with ZKA185 were similar to
those for the wild type, except for 1663-FLAG, which had a less than 4-fold decrease in sensitivity. These data suggest that changes in E protein folding as a result of FLAG insertions do not account for the majority of variation observed in FLAG neutralization potency.
E glycosylation does not alter neutralization of a nearby FLAG epitope. To determine if the E protein N154 glycan affects interactions of antibodies with nearby epitopes, we generated glycosylation-deficient wild-type and 1426-FLAG mutants by replacing the threonine of the NDT glycosylation consensus sequence with an isoleu-cine (T156I), which has been previously shown to abrogate glycosylation (28, 29). The infectious titers of the rescued unglycosylated T156I mutant were reduced 13-fold relative to those of the wild-type virus, and the titers of the T156I 1426-FLAG mutant were reduced less than 4-fold relative to those of the parental 1426-FLAG mutant (Fig. 4A). As described above, FLAG-tagged viruses were assessed for FLAG epitope stability and expression by sequencing and immunoblot analysis. Sequencing of virus from infected cells showed that mutation of the glycosylation consensus sequence was maintained in the untagged and 1426-FLAG mutant clones (Fig. 4B). The FLAG insertion sequence was also maintained in 10/11 T156I FLAG-1426 clones, although this se-quence was completely lost from 1 clone, which recreated the parental T156I sese-quence (Fig. 4B and C). Immunoblot analysis of infected Vero cells showed that glycosylation mutants expressed similar E protein levels as the wild-type virus (Fig. 4D). Furthermore, FLAG expression levels were similar between cells infected with the unglycosylated T156I 1426-FLAG and parental 1426-FLAG viruses (Fig. 4D). These data demonstrate that both the T156I mutation and the FLAG insertion within the T156I 1426-FLAG virus were stable over at least a single cycle of infection and thus could be safely used in experiments within this time frame.
FLAG and ZKA185 antibody neutralization assays of wild-type and glycan-deficient viruses were performed as described above. The FLAG antibody did not inhibit infection with either the untagged wild-type or T156I viruses (Fig. 4E). Comparing the FLAG antibody neutralization sensitivities of the parental and unglycosylated 1426-FLAG viruses, we found that removal of the glycan increased the potency of the FLAG antibody only by a factor of 1.5 (Fig. 4G). Removal of the glycan also increased the neutralization potency of the ZKA185 antibody. Although the T156I 1426-FLAG virus was only 2.2-fold
FIG 3Neutralization sensitivity of FLAG-tagged glycoprotein ZIKV mutants. (A and B) Dose-response neutralization curves for wild-type and FLAG-tagged mutant viruses, indicated in the key, in the presence of anti-FLAG (A) and ZKA185 (B) antibodies. Infection percentages were normalized to those for a no-treatment control. Error bars indicate the standard error of the mean (SEM) of each value. (C) Apparent IC50values calculated from dose-response curves for FLAG and ZKA185 antibodies tested against wild-type and FLAG-tagged mutant Zika viruses. Values are derived from at least two independent experiments performed in triplicate. The value of⬎3,000 indicates that 50% inhibition was not observed at the highest concentration of antibody tested.**,P⬍0.01 (one-way ANOVA) compared to the wild type.
on November 6, 2019 by guest
http://jvi.asm.org/
[image:6.585.41.478.70.215.2]more sensitive to the ZKA185 antibody than its parental 1426-FLAG virus, the untagged T156I mutant was 9.4 times more sensitive to this antibody than the wild-type virus (Fig. 4G). These results demonstrated that the N154 glycan did not greatly alter the neutralization potency of a nearby linear epitope but had a slightly larger effect on the sensitivity of a more distant conformational epitope.
Antibody binding correlated with neutralization.We next sought to determine if differences in the strength of FLAG antibody binding were responsible for the variation in the neutralization sensitivity of FLAG-tagged viruses that we observed above. To do so, we performed indirect four-layer enzyme-linked immunosorbent assays (ELISAs) to measure antibody binding to whole virus. Equivalent 50% tissue culture infective dose (TCID50) units for each virus were first captured with the ZKA185 antibody onto assay
plates. The plates were then incubated with serially diluted FLAG antibody or
D1-4G2-FIG 4Characterization of the impact of removing the E protein glycan on viral fitness and antibody neutralization. (A) Limiting dilution assays were performed to compare the infectious titers of wild-type and glycan loop mutant viruses. Infectious viral supernatant from transfected 293T cells was collected at 4 days posttransfection and used to infect Vero cells. Viral titers were calculated from the Vero cell CPE at 6 days postinfection. Values are expressed as the numbers of TCID50per milliliter. The data represent the means and standard deviations from three independent experiments, each performed in triplicate. Pol(⫺), polymerase-deficient virus. (B) Number of viral RT-PCR clones from infected cells that maintained the FLAG insertion and surrounding sequence. (C) Sequence alignment showing the single RT-PCR product from cells infected with the T156I 1426-FLAG virus from which the FLAG epitope was excised. (D) Immunoblot analysis for wild-type and mutant ZIKV was performed with antibodies against ZIKV E protein, FLAG, and cellular actin from Vero cell lysates collected at 48 h postinfection. Band sizes (in kilodaltons) are indicated to the left, and the predicted full-length proteins are marked with brackets on the right of each blot. (E and F) Dose-response neutralization curves for wild-type and unglycosylated mutant viruses (indicated in the key) in the presence of anti-FLAG (E) and ZKA185 (F) antibodies. Infection percentages were normalized to those for a no-treatment control. Error bars indicate the SEM of each value. (G) Apparent IC50values calculated from dose-response curves for FLAG and ZKA185 antibodies tested against the indicated viruses. The value of⬎3,000 indicates that 50% inhibition was not observed at the highest concentration of antibody tested.***,P⬍0.001 (one-way ANOVA) compared to the wild type. Values are derived from at least two independent experiments performed in triplicate.
on November 6, 2019 by guest
http://jvi.asm.org/
[image:7.585.42.494.70.434.2]4-15 (4G2) antibody, a pan-flavivirus antibody that binds a conformational epitope that overlaps the E protein fusion loop (30). The presence of detergent in the antibody binding buffer may enhance exposure of the fusion loop (31), so ELISA results with this antibody cannot definitively show that the conformation of the E protein in mutant particles was precisely maintained relative to that in the wild-type virus. However, at the very least the quantity of virus loaded onto the plates could be compared with 4G2 binding. Finally, the quantity of these antibodies retained after washing was deter-mined with an anti-mouse immunoglobulin horseradish peroxidase (HRP)-conjugated antibody by colorimetric detection. The ZIKV mutations that we generated caused only minor, although sometimes statistically significant, differences in the binding efficiency of the 4G2 antibody relative to that to wild-type virus (Fig. 5A and C). The greatest increase in 4G2 binding efficiency was observed in mutants with FLAG insertions in the glycan loop (1417- and 1426-FLAG), with the half-maximal effective concentration (EC50) values being approximately 3-fold lower than those for wild-type virus. The
1597-FLAG and unglycosylated T156I 1426-FLAG viruses each bound 4G2 approxi-mately 2-fold more efficiently than the wild type. Binding of 4G2 to the 1663- and 1813-FLAG viruses, with insertions distal to the E2 dimer interface and at the DI-II hinge region, respectively, did not differ significantly from that to the wild-type virus. These results at least indicate that similar numbers of viral particles were present in the ELISAs but may not formally prove that the FLAG insertions did not alter the accessibility of fusion loop epitopes, as, again, the detergent present in the antibody binding buffer may increase the exposure of the fusion loop (31).
In this assay, the location of the FLAG insertion greatly impacted FLAG antibody binding efficiency, which varied by up to 52-fold between the FLAG-tagged viruses (Fig. 5B and C). FLAG antibody bound most efficiently to the unglycosylated and glycosy-FIG 5Binding efficiency of antibodies to FLAG-tagged glycoprotein ZIKV mutants. (A and B) ELISA dose-response binding curves for wild-type and FLAG-tagged mutant viruses in the presence of 4G2 (A) and anti-FLAG (B) antibodies. Error bars indicate the SEM of each value. (C) Apparent EC50 values calculated from dose-response curves for 4G2 and FLAG antibodies tested against wild-type and FLAG-tagged mutant Zika viruses.**,P⬍0.01;***,P⬍0.001 (one-way ANOVA) compared to the wild type. Values are derived from at least two independent experiments performed in triplicate. (D) Regression analysis of Zika virus mutants with FLAG-tagged glycoproteins comparing FLAG antibody binding efficiency (EC50) and neutralization sensitivity (IC50) demonstrates a linear relationship. The formula for the line and the goodness of fit (R2) are displayed on the graph. OD, optical density.
on November 6, 2019 by guest
http://jvi.asm.org/
[image:8.585.71.342.72.330.2]lated 1426-FLAG mutants, which had similar EC50values of 47.2 and 61 ng/ml,
respec-tively, again suggesting that the glycan does not have a significant steric effect on nearby epitopes. FLAG antibody binding to the 1663-FLAG epitope in DII, located distal to the E dimer interface, was 2.4-fold less efficient than binding to 1426-FLAG. The 1813-FLAG epitope, in a loop within the highly flexible DI-II hinge region, bound FLAG antibody 7.8-fold less efficiently than the highly efficient 1426-FLAG virus. The 1417-FLAG insertion, only 3 residues from the 1426-1417-FLAG insertion, bound 1417-FLAG antibody 18.2-fold less efficiently. The 1597-FLAG virus had the least efficient FLAG antibody binding, with an EC50value of 3,170 ng/ml, or 52-fold above that of the 1426-FLAG
virus. The maximal occupancy of all FLAG-tagged viruses could not be determined under the tested conditions; however, at the highest tested concentration of FLAG antibody, the 1597-FLAG virus bound fewer antibodies than the other FLAG-tagged viruses (Fig. 5B).
To determine the relationship between FLAG antibody binding efficiency and neutralization potency, we performed a linear regression analysis of the EC50and IC50
values for each of the FLAG-tagged viruses (Fig. 5D). The results revealed a linear relationship between the EC50and IC50, with a goodness-of-fit (R2) value of 0.98. These
results suggest that nearly all variation in FLAG antibody neutralization potency may be explained by variation in antibody binding efficiency.
Envelope domain III tolerates insertion of the V5 epitope. The 2020-FLAG insertion was poorly tolerated and quickly lost from the ZIKV genome, which prevented us from studying the impact of FLAG antibodies directed against a region of the E protein that is predicted to be buried beneath the surface on a mature virion. We suspected that an epitope with fewer charged residues might be tolerated at this location. We tested several epitopes at the 2020 insertion site and found that the 14-amino-acid paramyxovirus-derived V5 epitope (GKPIPNPLLGLDST) was better toler-ated at this site. We were able to rescue the 2020-V5 mutant with infectious titers only 113-fold below those of the wild-type virus (Fig. 6A). To assess the stability of the V5 epitope in the ZIKV genome, we analyzed viral sequences from infected Vero cells, as described above. The results showed that the V5 insertion was stable within the genome and was altered in only 1 of 20 sequenced RT-PCR clones. The loss of the V5 epitope in this single clone occurred by deletion of 39 nucleotides from the epitope and surrounding viral sequence, which nearly restored the length of the sequence to that of the wild-type region (Fig. 6B). Analysis of V5 epitope stability by immunoblot analysis also showed that infected Vero cells maintained V5 expression at 2 days postinfection (Fig. 6C).
Antibody neutralization assays using the V5 and ZKA185 antibodies were performed as described above. The V5 antibody did not inhibit infection with the wild-type virus but potently neutralized the 2020-V5 mutant with an apparent IC50of 2.9 ng/ml (Fig.
6D). Though we did not investigate how the 2020-V5 insertion might have altered local E conformations and exposure, neutralization of this mutant with the DII-targeting ZKA185 antibody (32) was similar to that of the wild-type virus (Fig. 6E), which suggested that the V5 insertion did not substantially alter global E antigenicity. Thus, an epitope inserted in a region of the E protein that is predicted to be buried can efficiently serve as an antibody neutralization target.
FLAG and V5 antibodies neutralize ZIKV at a late stage of entry. Different structural elements of the flavivirus E protein have been implicated to have distinct cell entry functions (33). We investigated whether antibodies targeted to distinct E protein regions would inhibit different stages of entry. To do so, we compared antibody neutralization activity at different stages of viral entry for each of the epitope-tagged mutants. Briefly, we performed synchronized cell infection assays, in which virus was first bound to cells for 3 h at 4°C, at which temperature only attachment events can occur. At time zero, unattached virus was removed from these cultures by washing, and the cells were shifted to 37°C, a temperature that is permissive to postattachment cell entry events, such as endocytosis and fusion. ZIKV entry inhibitors, including FLAG and
on November 6, 2019 by guest
http://jvi.asm.org/
ZKA185 antibodies, were added at various time points (t) relative to the temperature shift, in order to test inhibition of viral attachment (t⫽ ⫺180 min), postattachment (t⫽ 0 min), or postentry events (t⫽240 min). It is important to point out that once an inhibitor was added, it remained in the culture for 24 h. The resulting infection levels were measured by immunostaining and flow cytometry analysis at 2 days postinfection. Bafilomycin A1, an endosomal ATPase inhibitor that prevents endosomal acidification, was added as a positive control for postattachment inhibition.
As expected, bafilomycin A1 inhibited all viruses when added at both pre- and postattachment (Fig. 7, light and medium gray bars) stages. The ZKA185 antibody also inhibited all virus infections when added at both pre- and postattachment cell entry stages. Furthermore, when an epitope was present in any position of the E protein, FLAG (Fig. 7B to F and H) and V5 (Fig. 7I) cognate antibodies inhibited infection when added during either the pre- or postattachment stage of host cell entry. No inhibitors
FIG 6Characterization of the fitness and antibody neutralization sensitivity of a V5-tagged E protein ZIKV mutant. (A) Limiting dilution assays of wild-type and V5-tagged mutant viruses were performed to compare infectious titers. Infectious viral supernatant from transfected 293T cells was collected at 4 days posttransfection and used to infect Vero cells. Viral titers were calculated from the Vero cell CPE at 6 days postinfection. Values are expressed as the number of TCID50per milliliter. The data represent the means and standard deviations from three independent experiments, each performed in triplicate. (B) Sequence alignment showing the single RT-PCR product with an altered epitope region from cells infected with the 2020-V5 virus. (C) Immunoblot analysis for wild-type and mutant ZIKV was performed with antibodies against the ZIKV E protein, V5, and cellular actin from Vero cell lysates collected at 48 h postinfection. Band sizes (in kilodaltons) are indicated to the left, and the predicted full-length proteins are marked with brackets on the right of each blot. (D and E) Dose-response neutralization curves for wild-type and V5-tagged viruses in the presence of anti-V5 (D) and ZKA185 (E) antibodies. Infection percentages were normalized to those for a no-treatment control. Error bars indicate the SEM of each value. (F) Apparent IC50values calculated from dose-response curves for V5 and ZKA185 antibodies tested against wild-type and 2020-V5 viruses. The value of⬎3,000 indicates that 50% inhibition was not observed at the highest concentration of antibody tested. Values are derived from at least two independent experiments performed in triplicate.
on November 6, 2019 by guest
http://jvi.asm.org/
[image:10.585.41.493.65.442.2]were observed to exclusively inhibit the attachment stage of ZIKV cell entry. This does not mean that they did not also inhibit cell binding but, rather, that the maintenance of inhibition postbinding indicated that this was at least a major stage at which they exerted inhibition. In many infections, a small increase in inhibition was observed when the antibodies were added during the binding phase. However, this increased inhibi-tion may simply have been a result of longer incubainhibi-tions allowing more antibody-virus complexes to form and not the specific inhibition of viral attachment. Finally, none of the inhibitors affected infection at a postentry stage, as inhibitory activity was com-pletely lost when they were added at 4 h postattachment (data not shown).
DISCUSSION
In this study, we examined the capacity of the ZIKV glycoproteins to tolerate epitope insertions and tested how antibodies against these sequences impacted infection. We chose sites for FLAG epitope insertion based on the results of our prior transposon mutagenesis screen of the MR766 ZIKV genome (24). We suspected that sites in the ZIKV glycoproteins, where transposon-mediated insertions did not greatly impair viral replication and spread, might represent regions of genomic flexibility that could tolerate different insertions. We found that this was not always the case, as the majority of our FLAG epitope insertion mutants were highly impaired. This was probably due to differences between the sequences of transposon-mediated insertions and the FLAG epitope sequence. Transposon mutagenesis results in the insertion of 15 nucleotides, or 5 amino acids when inside a coding sequence. Furthermore, only 10 of these nucleo-tides are transposon derived, and 5 nucleonucleo-tides are duplicated from the target site. Thus, within this 5-amino-acid insertion, 1 or 2 of the amino acids are duplications of the target site, which may lessen the likelihood of detrimental effects on the target protein function. Conversely, the FLAG insertion, along with an extra 3 nucleotides to incorporate a restriction site, encoded 9 additional amino acid residues, of which 7 are charged. Thus, such an insertion may have more dramatic effects than the transposon-mediated insertion on glycoprotein structure and function. Furthermore, without solv-ing the protein structure of an epitope-tagged virus, we cannot know how the epitope is displayed on the ZIKV glycoproteins. All of the regions that tolerated transposon insertion are on the surface of glycoproteins, perhaps because insertions inside a
FIG 7Analysis of the timing of antibody neutralization activity during ZIKV cell infection. The results for synchronized infections of Vero cells with wild-type (A), FLAG-tagged (B to F), unglycosylated wild-type (G), unglycosylated 1426-FLAG (H), and 2020-V5 (I) viruses are shown. To probe the relative timing of inhibitor activities during the entry process, the antibodies or bafilomycin A1 (BAF), as specified on thexaxes, was added at the indicated times relative to the time that the culture temperature was shifted to 37°C to permit infection. (A to I) For each inhibitor or antibody, results are relative to results when inhibitor was added 240 min after the temperature shift, at which point inhibition was no longer observed for any of the inhibitors. Infection percentages were normalized for each inhibitor to the amount of infection that occurred when that inhibitor was added 240 min after the shift to 37°C.*,P⬍0.05;**,P⬍0.01 (Mann-Whitney test).
on November 6, 2019 by guest
http://jvi.asm.org/
[image:11.585.42.479.72.256.2]involved in viral assembly, stability, and host cell entry, and we suspected that highly impaired FLAG-tagged viruses experienced disruptions in one or more of these func-tions. Viruses with insertions within the E DI glycan loop had the highest fitness of all the tested mutants, and these insertions did not appear to affect the antigenicity of a DII conformational epitope, as tested by ZKA185 neutralization (Fig. 3B and C). It is possible that glycan loop insertions were well tolerated because this sequence is naturally highly variable. Mutants with FLAG insertions within E DII both proximal and distal to the E dimer interface were less fit than mutants with FLAG insertions within DI. As DII forms an extended interaction surface within an E dimer, we suspect that some DII FLAG insertions might have disrupted intradimer interactions that negatively im-pacted viral assembly or stability. The DI-II hinge region plays an important role during viral maturation and again during fusion, as demonstrated by flavivirus antibodies which target this region and which are thought to prevent structural rearrangements required for fusion (34, 35). It is possible that insertion of FLAG epitopes within the DI-II hinge region could also impair viral fitness by altering the flexibility of this hinge and disrupting essential glycoprotein rearrangements.
All DIII FLAG insertion mutations were located within a flexible loop region, and the mutants were highly impaired for the production of infectious virus. It has been previously demonstrated that altering the length of this loop can negatively impact viral assembly, growth, and stability (36, 37), and we suspect that the FLAG epitope severely impacted these functions. Insertions that negatively impact viral fitness can be rapidly lost from flavivirus genomes (38–40), likely through recombination (41, 42). We tested the stability of the epitope insertions during single-cycle infections, and we saw that the insertions were maintained in all but the most impaired mutants. Indeed, the highly attenuated 2020-FLAG virus was the sole mutant that frequently lost most of the FLAG insertion within 24 h of infection. We speculated that insertions with fewer charged residues might be better tolerated in this region. Indeed, insertion of the larger but more hydrophobic V5 tag at the 2020 insertion site was tolerated and the virus was more stable.
FLAG antibody neutralization potency and binding efficiency varied dramatically between the FLAG-tagged mutants. In contrast, neutralization with the ZKA185 anti-body was similar for most mutants (Fig. 3C), demonstrating that folding of this DII quaternary epitope was not disrupted by FLAG insertions. The 1426-FLAG mutant, with a glycan loop insertion, exhibited the greatest FLAG antibody neutralization sensitivity and binding efficiency of all the FLAG-tagged viruses. The 1417-FLAG mutant exhibited 10-fold less potent neutralization and 18-fold less efficient binding with the FLAG antibody than the 1426-FLAG mutant, despite their FLAG epitopes being separated by only 3 amino acids. It is possible that this disparity in FLAG antibody neutralization and binding results from different orientations of the epitope or structural constraints on the epitope produced by the local structure. Such constraints might impact the capacity of the epitope sequence to bind its cognate antibody. Furthermore, even subtle changes to flavivirus E residues have the potential to strongly affect the antigenicity of the E protein by increasing viral breathing rates and the exposure of viral epitopes. While the neutralization of both FLAG-tagged glycan loop mutants with
on November 6, 2019 by guest
http://jvi.asm.org/
ZKA185 antibody was similar, it is possible that other epitopes, including the FLAG epitope, experience differential exposure as a result of viral breathing.
The 1663-FLAG epitope mediated the second most potent FLAG antibody neutral-ization and binding efficiency of the FLAG-tagged viruses. The loop containing this insertion exists in two different environments: on E proteins bookending each raft, the loop extends toward the linker between the bulky DI and DIII of an E protein in another raft, but within rafts the loop extends toward and slightly above DII of an antiparallel E dimer. The efficient binding of the 1663-FLAG mutant with its cognate antibody suggests that this epitope was accessible in both environments. This was the sole insertion mutant that was observed to alter the neutralization sensitivity of the ZKA185 antibody, though the effect was small.
The 1813-FLAG epitope, located in the DI-II hinge region, was 20-fold less sensitive to FLAG antibody neutralization and had 8-fold less efficient binding to this antibody than the highly sensitive 1426-FLAG epitope. DI-II hinge flexibility permits major conformational changes during fusion when the E protein extends away from the viral surface to form a fusogenic trimer (43, 44). The 1813-FLAG epitope within the DI-II hinge region was both accessible and an efficient neutralization target, with a possible mechanism of preventing glycoprotein structural rearrangements required for fusion. The 1597-FLAG insertion near the E DII dimer interface was the least sensitive to FLAG antibody neutralization. The FLAG-1597 epitope lies next to a cluster of contact points between flavivirus E monomers (43), within a cleft formed by antiparallel E monomers (Fig. 1B, 1597-FLAG side view). This area likely experiences significant steric effects from surrounding structures, which might explain why the 1597-FLAG binding efficiency was 50-fold lower than that for the highly efficient 1426-FLAG.
We were disappointed that a FLAG epitope was not tolerated in E DIII, as this was the only region of E protein candidate insertion sites that was fully buried under the surface of a mature ZIKV particle. However, we were able to recover infectious virus bearing a V5 epitope which was highly sensitive to neutralization with V5 antibody, though we could not directly compare neutralization between FLAG and V5 mutants. The infectivity of the 2020-V5 mutant suggests that the insertion does not prevent maturation. Furthermore, neutralization of the 2020-V5 virus with the ZKA185 antibody suggests that the insertion did not disrupt folding of this distal conformational epitope. Together these results suggest that this region likely remains buried in the mutant virus, which fits with previous work by others that demonstrated that altering this loop region can impact viral growth and stability without altering the overall structural morphology of the E protein (37). Though we did not investigate whether the insertion in DIII altered access or recognition of local DIII epitopes, our results indicate that this region can serve as an efficient neutralization target, despite it being classified as a buried epitope. We expect that these results will be of interest to others as numerous ZIKV vaccines are being developed, including DIII subunit vaccines (45), which have the potential to present epitopes from both buried and surface regions of DIII.
Glycoprotein glycosylation patterns and the glycan loop composition vary between flaviviruses (14), as well as among ZIKV isolates (18, 19, 46). Investigations into the functional consequences of N154 glycosylation have implicated roles in viral assembly and infectivity (47), tissue tropism (29), and neuroinvasion (48) but not neurovirulence (47, 48). We observed that the N154 glycan is dispensable duringin vitro Vero cell infections, which agrees with previous investigations ofin vitroinfectivity (28) as well asin vivoobservations that unglycosylated mutants are less infectious in mosquitoes and less pathogenic in immunocompromised mice (47). Based on the location of the N154 glycan, across from the fusion loop of an adjacent E monomer (14, 49), it has previously been hypothesized that this glycan might help stabilize E dimer interactions and shield the fusion loop in flaviviruses (17). We further hypothesized that the N154 glycan would sterically hinder access to nearby epitopes on the fusion loop. However, our results did not support this hypothesis, as removal of the glycan group from the FLAG-1426 mutant did not alter FLAG antibody neutralization or binding. We observed that removal of the glycan from the wild-type virus increased binding of the fusion
on November 6, 2019 by guest
http://jvi.asm.org/
infection. The results showed that antibodies targeting all E protein regions inhibited infection when added after viral attachment. Although it is conceivable that some inhibition was mediated by blocking binding, none of the DI, DII, or DI-II hinge regions appeared to act only during viral attachment. For several antibody-virus pairings, we did observe a slight increase in neutralization when the antibody was added during the binding phase, which could reflect overlapping functions of the glycoprotein regions at multiple stages of the cell entry process. However, it is more likely that the increased neutralization observed at a preattachment stage is due to the additional incubation time and the concomitant increase in antibody-virus interactions.
In this study, we used a panel of ZIKV mutants with epitope insertions in the E DI, DII, DIII, and DI-II hinge regions to explore the capacity of the ZIKV glycoproteins to tolerate insertions and the impact of antibodies targeting these specific regions on ZIKV infection. We showed that FLAG antibody neutralization potency varies by the location of the epitope insertion. Indeed, perhaps the greatest determinant of FLAG neutraliza-tion potency was epitope accessibility, as measured by antibody binding efficiency. One potential outcome of this study was the identification of glycoprotein regions where the binding of an antibody has relatively little or no impact on infection, which may have highlighted decoy antigens that elicit antibody responses that are not able to interfere with infection. Ultimately, the correlation between antibody binding and neutralization that we observed suggests either that these epitopes do not exist or that our insertions did not cover such regions. The former possibility is supported by the observation that although antibodies that neutralize ZIKV despite binding recombinant forms of the glycoprotein have been isolated (23), none that bind but do not neutralize to some degree have yet been isolated.
MATERIALS AND METHODS
Plasmid construction. (i) FLAG-tagged glycoproteins.Cloning was performed using a plasmid bearing a cytomegalovirus promoter-driven MR766 cDNA (50), with cloning sites in the ZIKV glycopro-teins being added by silent mutation. Candidate sites for FLAG insertions were selected on the basis of previous results from a transposon mutagenesis screen (24) that identified sites where transposon insertions were well tolerated (⬎3 standard deviations above the mean background rate). Insertions were generated by use of synthetic gBlocks (Integrated DNA Technologies, Coralville, IA). The 27-nucleotide inserts (GTC GAC TAC AAG GAC GAC GAC GAC AAG) encode a FLAG epitope (VDYKDDDDK) preceded by a SalI restriction site that was included for screening purposes.
(ii) T156I glycan mutation.The T156I mutant was generated as described above, by using synthetic gBlocks to generate a missense point mutation at C1469T, resulting in a T156I mutation that disrupts the NDT glycosylation consensus sequence.
(iii) V5-tagged glycoprotein.Insertions were generated by use of synthetic gBlocks (Integrated DNA Technologies, Coralville, IA). The 42-nucleotide inserts (GGT AAG CCT ATC CCT AAC CCT CTC CTC GGT CTC GAT TCT ACG) encode a V5 epitope (GKPIPNPLLGLDST). Sanger sequencing of all plasmids was performed to confirm the presence of insertions and the absence of additional mutations.
Modeling ZIKV glycoprotein structures.The locations of epitope insertion sites on ZIKV glycopro-teins M and E were modeled using the protein with Protein Data Bank accession number5IRE(14) as a model, and the locations of inserted epitopes were denoted using PyMOL software (51).
Rescue of mutant ZIKV. Viruses were produced by transfecting plasmids into 293T cells using techniques that we previously reported (50). Briefly, prior to transfection, 6-well plates were seeded overnight with 1.2⫻106cells/well. Cells were transfected using 2g plasmid DNA and theTransIT-LT1 transfection reagent (Mirus Bio, Madison WI) per the manufacturer’s recommendations. Each transfection was performed in triplicate. The viral supernatant was collected at 3, 4, and 5 days posttransfection, filtered (pore size, 0.45m) to remove cellular debris, and stored at⫺80°C.
on November 6, 2019 by guest
http://jvi.asm.org/
Titration of infectious virus.Infectious titers of rescued viruses were determined by limiting dilution assay on Vero cells, followed by quantification of infection by determination of the ZIKV-induced cytopathic effect (CPE). One day prior to infection, 96-well plates were seeded at a density of 104 cells/well in 100l Dulbecco modified Eagle medium (DMEM) supplemented with 2% fetal bovine serum (FBS). Immediately prior to infection, viral supernatants were serially diluted in DMEM with 2% FBS, and 100 l of diluted virus was added to each well (8 wells per dilution). CPE was observed at 6 days postinfection, and TCID50units were calculated according to the method of Reed and Muench (52). Data calculations and graphs were prepared using GraphPad Prism (version 7.0d) software (GraphPad Prism Inc., La Jolla, CA).
Sanger sequencing of viral RNA.One day prior to infection, 24-well plates were seeded at a density of 105Vero cells/well in DMEM supplemented with 2% FBS. Vero cells were infected at a low multiplicity of infection (MOI; 0.1 to 0.15) to obtain infections with single-hit kinetics. At 24 h postinfection, Vero cells were washed to remove input virus, and then total RNA was extracted using a PureLink RNA minikit and treated with DNase to reduce the possibility of plasmid contamination. Epitope insertion regions were amplified by RT-PCR, cloned into pGEM-T Easy plasmids (Promega, Madison, WI), and analyzed by Sanger sequencing (Macrogen USA).
Antibodies.The anti-FLAG M2 monoclonal mouse antibody (Sigma-Aldrich, St. Louis, MO) has an affinity for the linear epitope DYKDDDDK. GTX133314 (anti-envelope) is a rabbit polyclonal antibody against the ZIKV E protein (GeneTex, Irvine, CA). Antiactin is a mouse monoclonal antibody against the beta subunit (monoclonal antibody AC-15; Sigma-Aldrich, St. Louis, MO). The V5 antibody (Invitrogen-Thermo Fisher Scientific, Waltham, MA) binds a 14-residue sequence (GKPIPNPLLGLDST) that is derived from a paramyxovirus. ZKA185 is a human monoclonal antibody that targets a quaternary ZIKV E epitope (antibody Ab00835-10.0; Absolute Antibody, Oxford, UK). The mouse monoclonal antibody D1-4G2-4-15 (4G2) and a humanized version (Millipore Sigma, Burlington, MA) are broadly reactive flavivirus antibod-ies that bind to a fusion loop epitope. Rabbit anti-NS3 (NS3) was raised against a peptide consisting of amino acid residues 456 to 469 of the ZIKV MR766 sequence (GenBank accession numberAAV34151). Immunoblot analysis.Immunoblot assays were performed as previously described (53) with primary antibodies against FLAG, actin, E protein, and V5, as well as secondary anti-mouse and anti-rabbit immunoglobulin horseradish peroxidase (HRP)-conjugated antibodies (Thermo Fisher Scientific, Wal-tham, MA). The Immobilon chemiluminescent HRP reagent (EMD Millipore, Billerica, MA) was used for detection.
Neutralization assays.One day prior to infection, 24-well plates were seeded with 1.25⫻105Vero cells per well. Threefold serial dilutions of anti-FLAG and ZKA185 antibodies were incubated with approximately 2.5⫻104TCID50units of each virus (MOI, 0.1) at 37°C for 30 min and then used to infect Vero cells. At 24 h postinfection, cells were collected and analyzed by flow cytometry, as described below, to determine the percentage of infected cells. The percentage of infected cells in each well was normalized relative to the percentage of infected cells in a no-treatment control. All data sets were derived from at least two independent experiments, each performed in triplicate. Analysis of dose-response curves was performed with Prism (version 7.0d) software (dose-dose-response, three parameters) to calculate the apparent IC50for each antibody. IC50values were compared by one-way analysis of variance (ANOVA) and adjusted for multiple comparisons (by Tukey’s test).
Indirect four-layer ELISA to quantify whole virus binding.The ZKA185 capture antibody (100 ng/well) was bound overnight at 4°C on high-binding Microlon 96-well plates (Greiner Bio-One, Monroe, NC). Approximately 1.25⫻105TCID
50units of each virus were added to each well, and whole virus was captured at 37°C for 3 h. Threefold dilutions of 4G2 and FLAG primary antibodies, both starting at 10
g/ml, were bound at 37°C for 1 h in the presence of 0.05% Tween 20 detergent and 2.5% dry milk powder in phosphate-buffered saline (PBS). Goat anti-mouse immunoglobulin HRP-conjugated second-ary antibody was bound at a concentration of 1.3g/ml for 1 h at 37°C. The plates were developed by a colorimetric assay using SigmaFast OPD (o-phenylenediamine dihydrochloride) per the manufacturer’s recommendations, and the absorbance at 490 nm was measured by spectrophotometry. The data represent the mean⫾standard error of the mean (SEM) from at least two independent experiments, each performed in triplicate. Analysis of the dose-response curves was performed with Prism (version 7.0d) software (dose-response, three parameters) to calculate the apparent EC50for each antibody. EC50 values were compared by one-way ANOVA and adjusted for multiple comparisons (by Tukey’s test).
Linear regression analysis. Antibody neutralization IC50 values and binding EC50 values were plotted, and linear regression analysis was performed with Prism (version 7.0d) software (log-log line) to calculate the goodness of fit between the variables.
Synchronized infection assays.A total of 105cells were seeded on poly-L-lysine-coated 24-well tissue culture plates 24 h prior to infection. Cultures were cooled to 4°C at⫺210 min prior to shifting of the temperature to 37°C. At⫺180 min, the medium was replaced with 500l of virus diluted in DMEM supplemented with 2% FBS to achieve MOIs ranging from 0.05 to 0.2 at 4°C. At 0 min, the cultures were washed twice with cold PBS, and then fresh 37°C DMEM with 2% FBS was added. The cultures were then incubated at 37°C. Inhibitors were added at times of⫺180 min, 0 min, or 240 min. At 24 h postinfection, the medium was exchanged to remove the inhibitors. At 2 days postinfection, cells were collected and analyzed by flow cytometry to determine the percentage of infected cells. The percentage of infected cells in each well was normalized relative to the percentage of infected cells in a no-treatment control. The viral MOI was determined by infecting Vero cells with several dilutions of each virus under conditions similar to those in the kinetics assay and then measuring the percentage of infected cells by flow cytometry. ZKA185 antibody was used at a concentration of 0.5g/ml. FLAG antibody (M2) was used at a concentration of 5g/ml for untagged wild-type, T156I, and 1597-FLAG viruses; 2.5g/ml for
on November 6, 2019 by guest
http://jvi.asm.org/
protein insertion sites and Florian Krammer and Emily Gallichotte (University of North Carolina) for helpful discussions.
This work was supported by the following grants: NIDDK F30DK112514 (to M.T.C.), NIAID F31AI126863 (to E.S.G.), NIH R01DK095125 (to M.J.E.), and R21AI133649 (to M.J.E.). M.J.E. holds an Investigators in Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund.
REFERENCES
1. Fauci AS, Morens DM. 2016. Zika virus in the Americas—yet another arbovirus threat. N Engl J Med 374:601– 604.https://doi.org/10.1056/ NEJMp1600297.
2. Mittal R, Nguyen D, Debs LH, Patel AP, Liu G, Jhaveri VM, Mittal SISKJ, Bandstra ES, Younis RT, Chapagain P, Jayaweera DT, Liu XZ. 2017. Zika virus: an emerging global health threat. Front Cell Infect Microbiol 7:486.
https://doi.org/10.3389/fcimb.2017.00486.
3. Dick GW, Kitchen SF, Haddow AJ. 1952. Zika virus. I. Isolations and serological specificity. Trans R Soc Trop Med Hyg 46:509 –520.https:// doi.org/10.1016/0035-9203(52)90042-4.
4. Duffy MR, Chen TH, Hancock WT, Powers AM, Kool JL, Lanciotti RS, Pretrick M, Marfel M, Holzbauer S, Dubray C, Guillaumot L, Griggs A, Bel M, Lambert AJ, Laven J, Kosoy O, Panella A, Biggerstaff BJ, Fischer M, Hayes EB. 2009. Zika virus outbreak on Yap Island, Federated States of Micronesia. N Engl J Med 360:2536 –2543. https://doi.org/10.1056/ NEJMoa0805715.
5. Musso D, Nilles EJ, Cao-Lormeau VM. 2014. Rapid spread of emerging Zika virus in the Pacific area. Clin Microbiol Infect 20:O595–O596.https:// doi.org/10.1111/1469-0691.12707.
6. Campos GS, Bandeira AC, Sardi SI. 2015. Zika virus outbreak, Bahia, Brazil. Emerg Infect Dis 21:1885–1886.https://doi.org/10.3201/eid2110.150847. 7. Broutet N, Krauer F, Riesen M, Khalakdina A, Almiron M, Aldighieri S, Espinal M, Low N, Dye C. 2016. Zika virus as a cause of neurologic disorders. N Engl J Med 374:1506 –1509.https://doi.org/10.1056/NEJMp1602708.
8. Mlakar J, Korva M, Tul N, Popovic M, Poljsak-Prijatelj M, Mraz J, Kolenc M, Resman Rus K, Vesnaver Vipotnik T, Fabjan Vodusek V, Vizjak A, Pizem J, Petrovec M, Avsic Zupanc T. 2016. Zika virus associated with microceph-aly. N Engl J Med 374:951–958.https://doi.org/10.1056/NEJMoa1600651. 9. WHO. 10 March 2017. Situation report. Zika virus, microcephaly,
Guillain-Barré syndrome. WHO, Geneva, Switzerland.
10. Lim SK, Lim JK, Yoon IK. 2017. An update on Zika virus in Asia. Infect Chemother 49:91–100.https://doi.org/10.3947/ic.2017.49.2.91. 11. Faria NR, Azevedo R, Kraemer MUG, Souza R, Cunha MS, Hill SC, Theze J,
Bonsall MB, Bowden TA, Rissanen I, Rocco IM, Nogueira JS, Maeda AY, Vasami F, Macedo FLL, Suzuki A, Rodrigues SG, Cruz ACR, Nunes BT, Medeiros DBA, Rodrigues DSG, Queiroz ALN, da Silva EVP, Henriques DF, da Rosa EST, de Oliveira CS, Martins LC, Vasconcelos HB, Casseb LMN, Simith DB, Messina JP, Abade L, Lourenco J, Alcantara LCJ, de Lima MM, Giovanetti M, Hay SI, de Oliveira RS, Lemos PDS, de Oliveira LF, de Lima CPS, da Silva SP, de Vasconcelos JM, Franco L, Cardoso JF, Vianez-Junior J, Mir D, Bello G, Delatorre E, Khan K, et al. 2016. Zika virus in the Americas: early epidemiological and genetic findings. Science 352: 345–349.https://doi.org/10.1126/science.aaf5036.
12. Ferguson NM, Cucunuba ZM, Dorigatti I, Nedjati-Gilani GL, Donnelly CA, Basanez MG, Nouvellet P, Lessler J. 2016. Epidemiology. Countering the Zika epidemic in Latin America. Science 353:353–354.
13. Lindenbach CLM, Thile BDH-J, Rice CM. 2013. Flaviviridae, p 712–746.In
Knipe DM, Howley PM, Cohen JI, Griffin DE, Lamb RA, Martin MA, Racaniello VR, Roizman B (ed), Fields virology, 6th ed, vol 1. Lippincott Williams & Wilkins, Philadelphia, PA.
14. Sirohi D, Chen Z, Sun L, Klose T, Pierson TC, Rossmann MG, Kuhn RJ. 2016. The 3.8 Å resolution cryo-EM structure of Zika virus. Science 352:467– 470.https://doi.org/10.1126/science.aaf5316.
15. Yu IM, Zhang W, Holdaway HA, Li L, Kostyuchenko VA, Chipman PR, Kuhn RJ, Rossmann MG, Chen J. 2008. Structure of the immature dengue virus at low pH primes proteolytic maturation. Science 319:1834 –1837.
https://doi.org/10.1126/science.1153264.
16. Modis Y, Ogata S, Clements D, Harrison SC. 2004. Structure of the dengue virus envelope protein after membrane fusion. Nature 427: 313–319.https://doi.org/10.1038/nature02165.
17. Rey FA, Heinz FX, Mandl C, Kunz C, Harrison SC. 1995. The envelope glycoprotein from tick-borne encephalitis virus at 2 Å resolution. Nature 375:291–298.https://doi.org/10.1038/375291a0.
18. Haddow AD, Schuh AJ, Yasuda CY, Kasper MR, Heang V, Huy R, Guzman H, Tesh RB, Weaver SC. 2012. Genetic characterization of Zika virus strains: geographic expansion of the Asian lineage. PLoS Negl Trop Dis 6:e1477.https://doi.org/10.1371/journal.pntd.0001477.
19. Nybakken GE, Oliphant T, Johnson S, Burke S, Diamond MS, Fremont DH. 2005. Structural basis of West Nile virus neutralization by a therapeutic antibody. Nature 437:764 –769.https://doi.org/10.1038/nature03956. 20. Thullier P, Demangel C, Bedouelle H, Megret F, Jouan A, Deubel V, Mazie JC,
Lafaye P. 2001. Mapping of a dengue virus neutralizing epitope critical for the infectivity of all serotypes: insight into the neutralization mechanism. J Gen Virol 82:1885–1892.https://doi.org/10.1099/0022-1317-82-8-1885. 21. Chen Y, Maguire T, Hileman RE, Fromm JR, Esko JD, Linhardt RJ, Marks
RM. 1997. Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate. Nat Med 3:866 – 871.https://doi.org/10 .1038/nm0897-866.
22. Rey FA, Stiasny K, Vaney MC, Dellarole M, Heinz FX. 2017. The bright and the dark side of human antibody responses to flaviviruses: lessons for vaccine design. EMBO Rep 19:206 –224.https://doi.org/10.15252/embr .201745302.
23. Stettler K, Beltramello M, Espinosa DA, Graham V, Cassotta A, Bianchi S, Vanzetta F, Minola A, Jaconi S, Mele F, Foglierini M, Pedotti M, Simonelli L, Dowall S, Atkinson B, Percivalle E, Simmons CP, Varani L, Blum J, Baldanti F, Cameroni E, Hewson R, Harris E, Lanzavecchia A, Sallusto F, Corti D. 2016. Specificity, cross-reactivity, and function of antibodies elicited by Zika virus infection. Science 353:823– 826.https://doi.org/10 .1126/science.aaf8505.
24. Fulton BO, Sachs D, Schwarz MC, Palese P, Evans MJ. 2017. Transposon mutagenesis of the Zika virus genome highlights regions essential for RNA replication and restricted for immune evasion. J Virol 91:e00698-17.
https://doi.org/10.1128/JVI.00698-17.
25. Prasad VM, Miller AS, Klose T, Sirohi D, Buda G, Jiang W, Kuhn RJ,
on November 6, 2019 by guest
http://jvi.asm.org/
Rossmann MG. 2017. Structure of the immature Zika virus at 9 Å resolution. Nat Struct Mol Biol 24:184 –186. https://doi.org/10.1038/ nsmb.3352.
26. Pierson TC, Diamond MS. 2012. Degrees of maturity: the complex struc-ture and biology of flaviviruses. Curr Opin Virol 2:168 –175.https://doi .org/10.1016/j.coviro.2012.02.011.
27. Dowd KA, Mukherjee S, Kuhn RJ, Pierson TC. 2014. Combined effects of the structural heterogeneity and dynamics of flaviviruses on anti-body recognition. J Virol 88:11726 –11737.https://doi.org/10.1128/ JVI.01140-14.
28. Goo L, DeMaso CR, Pelc RS, Ledgerwood JE, Graham BS, Kuhn RJ, Pierson TC. 2018. The Zika virus envelope protein glycan loop regulates virion antigenicity. Virology 515:191–202.https://doi.org/10.1016/j.virol.2017 .12.032.
29. Wen D, Li S, Dong F, Zhang Y, Lin Y, Wang J, Zou Z, Zheng A. 2018. N-glycosylation of viral E protein is the determinant for vector midgut invasion by flaviviruses. mBio 9:e00046-18.https://doi.org/10.1128/mBio .00046-18.
30. Crill WD, Chang GJ. 2004. Localization and characterization of flavivirus envelope glycoprotein cross-reactive epitopes. J Virol 78:13975–13986.
https://doi.org/10.1128/JVI.78.24.13975-13986.2004.
31. Heinz FX, Tuma W, Guirakhoo F, Kunz C. 1986. A model study of the use of monoclonal antibodies in capture enzyme immunoassays for antigen quan-tification exploiting the epitope map of tick-borne encephalitis virus. J Biol Stand 14:133–141.https://doi.org/10.1016/0092-1157(86)90032-6. 32. Wang J, Bardelli M, Espinosa DA, Pedotti M, Ng TS, Bianchi S, Simonelli
L, Lim EXY, Foglierini M, Zatta F, Jaconi S, Beltramello M, Cameroni E, Fibriansah G, Shi J, Barca T, Pagani I, Rubio A, Broccoli V, Vicenzi E, Graham V, Pullan S, Dowall S, Hewson R, Jurt S, Zerbe O, Stettler K, Lanzavecchia A, Sallusto F, Cavalli A, Harris E, Lok SM, Varani L, Corti D. 2017. A human bi-specific antibody against Zika virus with high thera-peutic potential. Cell 171:229 –241.e15. https://doi.org/10.1016/j.cell .2017.09.002.
33. Rey FA, Stiasny K, Heinz FX. 2017. Flavivirus structural heterogeneity: implications for cell entry. Curr Opin Virol 24:132–139.https://doi.org/ 10.1016/j.coviro.2017.06.009.
34. Teoh EP, Kukkaro P, Teo EW, Lim AP, Tan TT, Yip A, Schul W, Aung M, Kostyuchenko VA, Leo YS, Chan SH, Smith KG, Chan AH, Zou G, Ooi EE, Kemeny DM, Tan GK, Ng JK, Ng ML, Alonso S, Fisher D, Shi PY, Hanson BJ, Lok SM, MacAry PA. 2012. The structural basis for serotype-specific neutralization of dengue virus by a human antibody. Sci Transl Med 4:139ra83.https://doi.org/10.1126/scitranslmed.3003888.
35. Fibriansah G, Tan JL, Smith SA, de Alwis AR, Ng TS, Kostyuchenko VA, Ibarra KD, Wang J, Harris E, de Silva A, Crowe JE, Jr, Lok SM. 2014. A potent anti-dengue human antibody preferentially recognizes the con-formation of E protein monomers assembled on the virus surface. EMBO Mol Med 6:358 –371.https://doi.org/10.1002/emmm.201303404. 36. Xie X, Yang Y, Muruato AE, Zou J, Shan C, Nunes BT, Medeiros DB,
Vasconcelos PF, Weaver SC, Rossi SL, Shi PY. 2017. Understanding Zika virus stability and developing a chimeric vaccine through functional analysis. mBio 8:e02134-16.https://doi.org/10.1128/mBio.02134-16. 37. Gallichotte EN, Dinnon KH, III, Lim XN, Ng TS, Lim EXY, Menachery VD,
Lok SM, Baric RS. 2017. CD-loop extension in Zika virus envelope protein key for stability and pathogenesis. J Infect Dis 216:1196 –1204.https:// doi.org/10.1093/infdis/jix473.
38. McGee CE, Shustov AV, Tsetsarkin K, Frolov IV, Mason PW, Vanlanding-ham DL, Higgs S. 2010. Infection, dissemination, and transmission of a
West Nile virus green fluorescent protein infectious clone by Culex pipiens quinquefasciatus mosquitoes. Vector Borne Zoonotic Dis 10: 267–274.https://doi.org/10.1089/vbz.2009.0067.
39. Zou G, Xu HY, Qing M, Wang QY, Shi PY. 2011. Development and characterization of a stable luciferase dengue virus for high-throughput screening. Antiviral Res 91:11–19.https://doi.org/10.1016/j.antiviral.2011 .05.001.
40. Schoggins JW, Dorner M, Feulner M, Imanaka N, Murphy MY, Ploss A, Rice CM. 2012. Dengue reporter viruses reveal viral dynamics in inter-feron receptor-deficient mice and sensitivity to interinter-feron effectors in vitro. Proc Natl Acad Sci U S A 109:14610 –14615. https://doi.org/10 .1073/pnas.1212379109.
41. Perez-Losada M, Arenas M, Galan JC, Palero F, Gonzalez-Candelas F. 2015. Recombination in viruses: mechanisms, methods of study, and evolutionary consequences. Infect Genet Evol 30:296 –307.https://doi .org/10.1016/j.meegid.2014.12.022.
42. Sanjuan R, Domingo-Calap P. 2016. Mechanisms of viral mutation. Cell Mol Life Sci 73:4433– 4448.https://doi.org/10.1007/s00018-016-2299-6. 43. Zhang Y, Zhang W, Ogata S, Clements D, Strauss JH, Baker TS, Kuhn RJ,
Rossmann MG. 2004. Conformational changes of the flavivirus E glyco-protein. Structure 12:1607–1618. https://doi.org/10.1016/j.str.2004.06 .019.
44. Hasan SS, Sevvana M, Kuhn RJ, Rossmann MG. 2018. Structural biology of Zika virus and other flaviviruses. Nat Struct Mol Biol 25:13–20.https:// doi.org/10.1038/s41594-017-0010-8.
45. Poland GA, Kennedy RB, Ovsyannikova IG, Palacios R, Ho PL, Kalil J. 2018. Development of vaccines against Zika virus. Lancet Infect Dis 18: e211– e219.https://doi.org/10.1016/S1473-3099(18)30063-X.
46. Heinz FX, Stiasny K. 2017. The antigenic structure of Zika virus and its relation to other flaviviruses: implications for infection and immunopro-phylaxis. Microbiol Mol Biol Rev 81:e00055-16.https://doi.org/10.1128/ MMBR.00055-16.
47. Fontes-Garfias CR, Shan C, Luo H, Muruato AE, Medeiros DBA, Mays E, Xie X, Zou J, Roundy CM, Wakamiya M, Rossi SL, Wang T, Weaver SC, Shi PY. 2017. Functional analysis of glycosylation of Zika virus envelope protein. Cell Rep 21:1180 –1190.https://doi.org/10.1016/j.celrep.2017.10.016. 48. Annamalai AS, Pattnaik A, Sahoo BR, Muthukrishnan E, Natarajan SK,
Steffen D, Vu HLX, Delhon G, Osorio FA, Petro TM, Xiang SH, Pattnaik AK. 2017. Zika virus encoding non-glycosylated envelope protein is attenu-ated and defective in neuroinvasion. J Virol 91:e01348-17.https://doi .org/10.1128/JVI.01348-17.
49. Kostyuchenko VA, Lim EX, Zhang S, Fibriansah G, Ng TS, Ooi JS, Shi J, Lok SM. 2016. Structure of the thermally stable Zika virus. Nature 533: 425– 428.https://doi.org/10.1038/nature17994.
50. Schwarz MC, Sourisseau M, Espino MM, Gray ES, Chambers MT, Tortorella D, Evans MJ. 2016. Rescue of the 1947 Zika virus prototype strain with a cytomegalovirus promoter-driven cDNA clone. mSphere 1(5):e00246-16.
https://doi.org/10.1128/mSphere.00246-16.
51. Schrodinger Inc. 2015. The PyMOL molecular graphics system, version 2.0, v2.0.6. Schrodinger Inc., New York, NY.
52. Reed LJ, Muench H. 1938. A simple method of estimating fifty per cent endpoints. Am J Hyg 27:493– 497.
53. Michta ML, Hopcraft SE, Narbus CM, Kratovac Z, Israelow B, Sourisseau M, Evans MJ. 2010. Species-specific regions of occludin required by hepa-titis C virus for cell entry. J Virol 84:11696 –11708.https://doi.org/10 .1128/JVI.01555-10.
