Neuroblastoma cell-adapted yellow fever virus: mutagenesis of the E protein locus involved in persistent infection and its effects on virus penetration and spread
Leonssia Vlaycheva,
Michael Nickells,
Deborah A. Droll and
Thomas J. Chambers
Department of Molecular Microbiology and Immunology, St Louis University Health Sciences Center, 1402 South Grand Avenue, St Louis, MO 63104, USA
Correspondence
Thomas J. Chambers
chambetj{at}slu.edu
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ABSTRACT
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Persistent infection of mouse neuroblastoma NB41A3 cells with yellow fever 17D virus generates viral variants which exhibit defective cell penetration, poor cell-to-cell spread, small plaque size and reduced growth efficiency, caused by substitution of glycine for aspartic acid or glutamic acid at positions 360 and 362 in the envelope protein. These positions occur within a charge cluster, Asp360-Asp361-Glu362, located in domain III, near its interface with domain I. To characterize further the molecular basis for the variant phenotype, a series of mutant viruses containing substitutions at position 360, 361 and 362, were studied for effects on the cell culture properties typical of the neuroblastoma-adapted variant. Most substitutions at position 360 gave rise to viruses that were very defective in cell penetration, growth efficiency and cell-to-cell spread, whereas substitution with glutamic acid yielded a virus indistinguishable from parental yellow fever 17D. Substitution with lysine was not tolerated and substitution with asparagine resulted in frequent wild-type revertants. A glycine residue was not tolerated at position 361, but substitution at 362 yielded a small plaque virus, similar to the effect of substitution at position 360. These data indicate that the yellow fever virus E protein contains a locus within domain III where a negative-charge cluster is important for optimal function of this domain in virus-cell interactions beyond the stage of virus attachment. Modelling predictions suggest that the mutations alter the local properties of the loop within domain III, and may compromise interactions of this domain with an adjacent region of domain I during conformational changes that occur in the E protein in association with virus entry.
Present address: Merck & Co. Inc., PO Box 4, BLB-22, West Point, PA 19486, USA. 
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INTRODUCTION
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The flavivirus envelope (E) protein plays a multifunctional role during virus replication in susceptible host cells (Heinz, 1986
; Rice, 1996
), and is a critical factor for viral pathogenesis because of its importance for virus infectivity, cellular tropism and host range, and its capacity to elicit virus-specific neutralizing antibodies (Heinz, 1986
; Roehrig, 1997
). An increasing number of molecular determinants of virulence are being identified within the E protein, and they generally map to one of several structural regions on the protein: (i) the fusion peptide (Allison et al., 2001
; Rey et al., 1995
); (ii) the hinge region at the interface of domains I and II (Beasley & Aaskov, 2001
; Cecilia & Gould, 1991
; Hasegawa et al., 1992
; Hurrelbrink & McMinn, 2001
; or (iii), the putative receptor-binding site of the virus in domain III, with studies suggesting that attenuation may result from altered interactions with cellular receptors (Crill & Roehrig, 2001
; Hasegawa et al., 1992
; Hurrelbrink & McMinn, 2001
; Jiang et al., 1993
; Lee & Lobigs, 2000
; Lobigs et al., 1990
; Mandl et al., 2000
). Recent studies of the structure of the flavivirus E protein reveal that the transition of the dimeric form to the fusion-active trimeric form and the subsequent post-fusion form involve conformational changes in both domains II and III (Bressanelli et al., 2004
; Modis et al., 2003
, 2004
). It is quite possible that these transitions are affected by mutations that alter the charge and surface properties of domain III, and that functions other than merely the cell attachment process are perturbed. In a previous study, we identified a molecular determinant (residue 360) within domain III of the E protein of yellow fever (YF) 17D virus where substitution of glycine for aspartic acid resulted in defective cell penetration, poor growth efficiency, decreased cell-to-cell spread and establishment of persistent infection of mouse neuroblastoma cells (Vlaycheva & Chambers, 2002
). This phenomenon was not directly related to the cell-binding properties of the virus, suggesting a role of domain III in cell entry beyond simply binding to the cell surface. To investigate further the importance of this determinant for cell culture properties of the virus, we constructed a series of viruses harbouring mutations at residues 360362 of the E protein, and characterized the effects of these mutations on cell penetration and spread. The results suggest that conservation of a charged locus at positions 360362 is a determinant of function of the yellow fever virus (YFV) E protein in relationship to these properties.
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METHODS
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Cells and viruses.
Vero cells (originally obtained from J. Jennings, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, USA), were grown at 37 °C in alpha minimal essential medium supplemented with 10 % fetal bovine serum (FBS) and antibiotics [penicillin/streptomycin (Gibco-BRL)]. YF5.2iv virus was derived from an infectious clone of the YF 17D virus (Rice et al., 1989
). NB41A3 cells (NB cells) and C6/36 cells were used as described previously (Vlaycheva & Chambers, 2002
). The neuroblastoma cell-adapted variant (NB15a) has been described previously (Vlaycheva & Chambers, 2002
). The YFS/232 vaccine strain was obtained from J. Porterfield (University of Oxford, Oxford, UK). The YF 17D-SW strain is a laboratory-passaged derivative of the 17D-204 strain originally described by C. Rice (Rice et al., 1985
). Virus titres were determined by plaque assays on Vero cells at 37 °C. Viruses were serially diluted in alpha-MEM containing 10 % FBS, and used to infect confluent Vero cell monolayers. After infection for 1 h virus was removed, and the monolayers were overlaid with 1 % (w/v) SeaKem ME agarose (BioWhittaker) in alpha-MEM supplemented with 5 % FBS. Plates were incubated for 58 days at 37 °C, and plaques visualized by staining with 1·5 % crystal violet in 20 % ethanol, after fixation in 7 % formalin for 24 h.
Construction of virus mutants.
A two-plasmid system for generation of infectious YF 17D virus (Rice et al., 1989
) was used to construct YFV harbouring engineered mutations in the E protein. To facilitate construction of the mutant viruses, a fragment of pYF5.2iv from nt 1643 to 2980 was incorporated into the Zero Blunt TOPO vector (Invitrogen), and subjected to mutagenesis using a QuikChange site-directed mutagenesis kit (Stratagene). Oligonucleotide primers (Gibco-BRL) used to create the mutations were as follows. Underlined codons represent the introduced mutations. Restriction sites designed to facilitate screening of recombinant plasmids are shown. Ala360, 5'-CCATCGCCTCAACGAATGCAGATGAAGTGCTGATTG-3' (new BsmI); Leu360, 5'-ATCGCCTCAACCAATCTAGATGAAGTGCTGATT-3' (new XbaI); Phe360, 5'-CCATCGCCTCAACCAATTCGATGAAGTGCTGATTG-3' (eliminates XbaI); Ser360, 5'-CCCATCGCCTCAACGAATTCTGATGAAGTGCTGATTG-3' (new EcoRI); Asn360, 5'-CCATCGCCTCAACGAATAATGATGAAGTGCTGATTG-3' (eliminates EcoRI); Lys360, 5'-CATCGCCTCAACCAATAAAGATGAAGTGCTGATTG-3' (eliminates XbaI); Gly361, 5'-CGCCTCAACCAATGATGGTGAGGTACTGATTGAGGTG-3' (eliminates ScaI); Gly362, 5'-CAACCAATGATGATGGAGTACTGATTGAGGTGAAC-3' (new ScaI).
The presence of the mutations was screened using restriction enzymes and confirmed by nucleotide sequencing across the region subjected to mutagenesis. Full-length YF5.2iv templates containing engineered mutations were obtained by ligation of the AatII/NsiI fragment from pYFM5.2 plasmids containing the mutations, with the AatII/NsiI fragment from pYF5'3'IV, after isolation of the DNA fragments from agarose gels and purification with Wizard DNA purification kits (Promega). The fragments were ligated using T4 DNA ligase (NEB). The assembled templates were used to synthesize infectious RNA transcripts using SP6 RNA polymerase (NEB) in the presence of 5' cap analogue as originally described (Rice et al., 1989
). Approximately 300 ng of RNA was transfected into Vero cells in the presence of 20 µg Lipofectin (Gibco-BRL) in PBS, followed by incubation at 37 °C in alpha-MEM plus 5 % FBS. Virus was harvested from the cell culture at time of onset of cytopathic effects (between 5 and 15 days post-transfection, depending on the mutant) and the yield was titrated by plaque assay on Vero cells. Mutant viruses selected for further investigation were plaque-purified twice on Vero cells and amplified once to produce working virus stocks. The region between nt 1643 and 2503 was sequenced to verify the presence of the correct mutation in these virus preparations.
Growth curves.
Monolayers of Vero cells were infected with viruses at an m.o.i. of 0·0003 p.f.u. per cell at 37 °C for 1 h. The cells were maintained in alpha-MEM plus 3 % FBS. Media were harvested and replaced after 1517 h, and at 24 h intervals thereafter. Virus titres were determined by plaque assay on Vero cells. For growth curve experiments, all samples were run in triplicate.
Fluorescent focus assay.
Infectious-centre assay was performed as described previously (Vlaycheva & Chambers, 2002
). Confluent monolayers of Vero cells were infected with viruses for 1 h at 37 °C at multiplicities of 50 and 5 p.f.u. per well. Virus was removed, monolayers were overlaid with 1 % agarose (BMA) in alpha-MEM plus 3 % FBS, and incubated at 37 °C. At intervals of 24 h samples were processed for immunofluorescence. Cells were fixed in 4 % paraformaldehyde (90 min at room temperature) and agarose was removed. Cells were permeabilized with 100 % methanol (6 min at 20 °C), incubated with primary antiserum [anti-YF hyperimmune ascitic fluid (ATCC) diluted 1 : 500 in PBS plus 1 % FBS] for 1 h at 37 °C, washed three times with PBS, and then incubated with secondary antibody [affinity-purified fluorescein-conjugated goat anti-rabbit IgG antibody (ICN), 1 : 50 in PBS plus 1 % FBS] for 30 min at 37 °C. Cells were examined using a Nikon TE-300 fluorescent microscope equipped with a standard FITC fluorescence cube and a SPOT camera (Diagnostic Instruments).
Cell penetration assay.
Penetration rate was determined by a method described previously (Vlaycheva & Chambers, 2002
). Monolayers of Vero cells in 60 mm culture plates in quadruplicates were adsorbed at 37 °C with viruses using inputs of 50100 p.f.u. per plate in three separate experiments. At various intervals up to 60 min, unbound virus was removed, duplicate samples were treated with acid glycine buffer (0·1 M glycine and 0·1 M sodium chloride, pH 3·0), for 3 min to inactivate uninternalized virus, whereas two other samples (non-acid-treated controls) were washed with PBS. All samples were then washed once with PBS, overlaid with agarose and incubated at 37 °C to allow plaque formation. Percentage of penetrated virus for each time point was calculated as the ratio of the number of plaques on the acid-treated cells to the number of plaques on the non-acid-treated cells for each time point multiplied by 100. We previously demonstrated that acid treatment for 2 min was sufficient to inactivate >95 % of input viruses in solution (Vlaycheva & Chambers, 2002
).
Molecular modelling.
A homology-based model for the YF 17D E protein, using the tick-borne encephalitis (TBE) virus E protein as a template, was derived as described previously (Nickells & Chambers, 2003
). Substitutions at positions 360362 were threaded into this model and the predicted energy parameters and hydrogen bond formations for various rotamer conformations were evaluated to draw conclusions about the likely effects of mutations on the local structure of domain III. The figures were created using the modelling platform of Swiss-modeller and portrayed using the accompanying graphics programs.
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RESULTS
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Passage of YF 17D viruses on neuroblastoma cells
We previously reported that serial passage of mouse neuroblastoma NB41A3 cells infected with YF5.2iv virus (YF 17D molecular clone), resulted in the emergence of a viral variant (NB15a), which persistently infected these cells and exhibited defects in cell penetration, cell-to-cell spread and growth efficiency in all cell lines tested (Vlaycheva & Chambers, 2002
). To determine whether this phenomenon was reproducible and to investigate whether other mutants with defects in cell entry might be generated from such cultures, we serially passaged several other YF 17D virus stocks on NB41A3 cells and monitored the cultures for persistent infection by plaque assay of the media. The results are shown in Table 1
. YF5.2iv clone 6 was the virus preparation used to derive the original NB15a variant. YF5.2iv clone 7 was a duplicate preparation of virus from the YF molecular clone from an independent transcription/transfection experiment. YF 17D-SW is a laboratory stock of YF 17D-204 (Rice et al., 1985
), which had been subjected to multiple passages in SW-13 cells. YFS/232 is a YF 17D commercial vaccine stock. Only the YF5.2iv clones 6 and 7 were able to generate persistent infection of the neuroblastoma cells, based on recovery of plaque-forming virus of any plaque size from the media of the cultures after multiple serial passages. Cultures infected with YF 17D-SW and YFS/232 yielded no persistent infection based on this criterion. The NB18 and NBC7 viruses were derived following 18 and 20 passages of the NB41A3 cell cultures infected with clone 6 or 7 of YF5.2iv, respectively. The NB18 and NBC7 viruses were plaque-purified twice on Vero cells, and amplified once in NB cells. NB18a and NB18b represent two different plaque picks from the same persistently infected culture. Four RT-PCR-derived clones of these two isolates were sequenced over the region encoding the prM and E proteins. All four clones contained a substitution of glycine for glutamic acid at position 362 in the E protein. Two clones of NB18b had an additional substitution of alanine for valine at position 71 in the prM region. Sequencing of the same region of five clones from another variant, NBC7, isolated from cells infected with YF5.2iv clone 7 revealed the presence of a glycine residue at position 360 in the envelope region, which is identical to the mutation in the original NB15a variant virus (Vlaycheva & Chambers, 2002
). There were no other substitutions present in the E proteins of these clones. Nucleotide sequencing of the NBC7 virus was also carried out in regions encompassing amino acid residue 173 of the NS2A protein and residue 582 of the NS3 protein, where substitutions in the NB15a virus had also been originally detected (Vlaycheva & Chambers, 2002
). In the case of the NBC7 virus, these positions retained the sequence of parental YF5.2iv virus. These data suggest that establishment of persistent infection of NB41A3 cells by YF 17D virus may be affected by differences in the genetic background of this vaccine strain. Also, mutations at residue 360 or 362 appear sufficient for persistence, rather than mutations within NS2A and NS3, although we cannot exclude the possibility that mutations in the other portions of the non-structural region of the NBC7 variant also contribute to its phenotype, since we did not completely sequence its genome.
Engineered mutants at residues 360362
Based on the observation that glycine at position 360 dramatically altered the cell culture properties of YF5.2iv virus, and that repeated passages of YFV consistently yielded small plaque viruses with mutations at either position 360 or 362, we tested a series of mutations at these positions for their effects on plaque formation. The objective was to gain some insight into the molecular basis for the defective cell culture properties of the NB15a, NB18a and NBC7 viruses, and in particular to determine whether this locus exhibits a strict requirement for a negative-charge cluster. This was done by engineering substitutions at position 360 with several amino acid residues other than aspartic acid, and with glycine for aspartic acid and glutamic acid at positions 361362, respectively. Positions outside of this cluster (e.g. residues 358 and 359) were not targeted for mutagenesis based on lack of evidence that these residues are related to the phenomenon of persistent infection of NB cells (Table 1
). Substitutions at position 360 were chosen so as to include a range of different amino acid structures, such as aliphatic hydrocarbon side chains of increasing size and hydrophobicity (alanine, leucine and phenylalanine), non-hydrophobic chemically different side chains (serine and asparagine), and side chains bearing similar or opposite charges (glutamic acid, which like aspartic acid is negatively charged above pH 3, and lysine, which is positively charged at physiological pH). Table 2
summarizes results of virus recovery and plaque formation for the mutations engineered at these positions. Replacement of residue 360 with glutamic acid yielded a virus that closely resembled the parental YF5.2iv virus, suggesting that a negatively charged amino acid is preferred at this position. Replacement of aspartic acid at position 360 with any one of several uncharged polar or hydrophobic amino acids (serine, alanine, leucine and phenylalanine), resulted in recovery of very small plaque viruses after intervals of 7 to 8 days following transfection (Fig. 1
). The plaque sizes of these mutants was distinguishably smaller than that of the original NB15a virus, and there was generally a low rate of reversion based on plaque sizes of viruses in the transfection harvests. Replacement of residue 360 with asparagine also gave a very small plaque virus, but the reversion rate was high, with large plaques visible in the transfection harvest (Fig. 1
). Replacement of residue 360 with lysine resulted in virus with a plaque size similar to that of the parental YF5.2iv virus. However, this virus was recovered after prolonged incubation of the transfected cells (13 to 14 days), and nucleotide sequencing revealed that reversion to the parental sequence at position 360 had in fact taken place. Replacement of residue 361 (aspartic acid) with glycine did not yield any infectious virus. Replacement of residue 362 (glutamic acid) with glycine resulted in recovery of a very small plaque virus, consistent with the fact that the neuroblastoma-passaged NBC7 variant was found to contain a glycine residue at this position (Table 1
). Taken together, these results suggest that the locus 360362 in the E protein is sensitive to amino acid substitutions, which affect plaque size. A negative charge at 360 and possibly at residues 361 and 362, appears to be critical for preservation of the wild-type plaque phenotype and other cell culture properties (see below). Four mutant viruses (Leu360, Ser360, Phe360 and Asn360) were therefore selected for further characterization. These viruses were plaque-purified and the correct mutations were confirmed to be present by nucleotide sequencing.

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Fig. 1. Plaque morphologies of the engineered E protein mutants. P30 plates of Vero cells were infected with plaque-purified stock viruses as described in the Methods, and plaque assays were stained with crystal violet after incubation for 8 days. Asp (aspartic acid at residue 360) represents the parental YF5.2iv virus.
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Growth efficiency
Neuroblastoma cell-adapted YFV (NB15a) and the corresponding engineered Gly360 virus exhibit defective growth on cell lines of different host origin when compared with parental YF5.2iv (Vlaycheva & Chambers, 2002
). To determine whether mutations engineered at position 360 would also affect the growth properties of YFV in cell culture, plaque-purified mutant viruses were tested for growth efficiency in Vero cells after infection at multiplicity of 0·0003 p.f.u. per cell. A very low multiplicity was chosen to exaggerate the requirement for cell-to-cell spread for virus production. The resulting growth curves revealed that virus production in the case of the mutant viruses Leu360, Ser360, Phe360 and Asn360 was significantly delayed compared with YF5.2iv (Fig. 2a and b
), with lower virus yields over the entire time interval tested in these experiments. Peak titres were not measured in these particular experiments, however the maximal titre of YF5.2iv (approx. 67 logs p.f.u. ml1) is typical of what is recovered from this cell line. Plaque sizes of the mutant viruses remained small at the later time points in these growth assays, suggesting that reversion to viruses with more efficient growth capacity was not occurring. In contrast to these mutants, the Glu360 virus grew equally well as the parental virus in Vero cells, reaching a peak titre of 7 log p.f.u. ml1 at 96 h post-infection (Fig. 2c
). Furthermore, the growth efficiency of Glu360 was also confirmed to be similar to parental virus in mosquito (C6/36) cells (Fig. 2d
), where peak titres of 6·25 log p.f.u. ml1 were reached by 3 to 4 days post-infection. These results indicate that the small plaque phenotypes of the mutants Leu360, Ser360, Phe360 and Asn360 correlate with reduced virus production in cell culture. However, the Glu360 mutant was not distinguishable from parental virus and no host restriction of virus production was apparent, based on use of two cell lines of different host origin.

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Fig. 2. Growth kinetics of parental and engineered viruses with mutations at position 360. (a), (b) and (c) Vero cells; (d) C6/36 cells. Cell monolayers were infected as described in the Methods, and grown at 37 °C (Vero) or 28 °C (C6/36), respectively. Virus was harvested at serial time points after infection, and the media were replaced. Virus yields were determined by plaque assay on Vero cells. YF5.2iv indicates the parental virus. Data represent mean of triplicate samples for each time point. Error bars for standard deviations are shown (at some time points these are too small to be visible).
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Fluorescent focus assay
To determine if the small plaque size of the mutants was in fact due to reduced spread through the cell monolayer rather than merely a reduced capacity for cytopathic effects as detected by plaque assay, the sizes of infectious centres were assessed by immunofluorescence (Fig. 3
). The fluorescent focus assay, aided by a digital image analysis, is a sensitive method to measure and compare infectious centres of different viruses. Using this assay, for example, the neuroblastoma cell-adapted NB15a YFV variant exhibits a very small focus size that does not enlarge significantly compared with that of the YF5.2iv virus (Vlaycheva & Chambers, 2002
). For the engineered mutants in the current study, serial images taken at 3, 5 and 7 days after infection revealed reduced sizes of infectious centres compared with parental virus, with little increase in their sizes over the time interval examined. The serine mutant appeared to induce somewhat larger foci than the other mutants suggesting it has a less severe defect in cell-to-cell spread. These results suggest that similar to the original neuroblastoma cell-adapted NB15a virus, these mutant viruses were severely restricted in their ability to spread from cell-to-cell when tested under the conditions of this assay.

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Fig. 3. Fluorescent focus assay. Vero cells were infected with parental or mutant viruses as described in the Methods. Serial samples were processed for indirect immunofluorescence at days 3, 5 and 7 after infection. Images were taken at x100 (days 3 and 5) and x40 (day 7). Dotted lines indicates the edge of the focus produced by the parental YF5.2iv virus.
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Cell penetration assay
Based on previous studies with the NB15a virus, where defective growth and virus spread were associated with reduced ability to penetrate cells compared with parental virus, we hypothesized that the mutant viruses Leu360, Ser360, Phe360 and Asn360 would also be defective in this property. To compare the rates and efficiencies of cell penetration by the mutants, monolayers of Vero cell were infected at low multiplicities, and the percentage of the original inocula, which resulted in infectious centres as a function of time, was measured. Acid pH treatment of the virus adsorbed to the monolayers was used to distinguish bound versus internalized virus, as described in the Methods. The rate of penetration was calculated as the ratio of the mean number of plaques in the wells treated with acid pH buffer to the mean number of plaques in the non-treated control wells, expressed as a percentage. We previously determined that under the conditions of this assay, the extent of adsorption of the small numbers of p.f.u. employed (50100 p.f.u. per 60 mm plate) was almost complete within 10 min of incubation, which reduces the significance of this variable with respect to interpreting differences in penetration efficiency (Vlaycheva & Chambers, 2002
). Results are shown in Fig. 4
. For the parental YF5.2iv virus, efficiency of penetration was approximately 20 % at 10 min, rose to 60 % by 30 min and reached 94 % by 60 min. For the mutants Asn360 and Phe360, efficiencies of penetration were 20 % or less at 10 min, but did not reach levels higher than 20 % by 60 min. The mutant Leu360 exhibited a similar low level of penetration at 10 min, but reached 40 % by 30 min and 55 % by 60 min. In contrast, the Ser360 mutant was distinguished by exhibiting the highest efficiency of penetration of all the viruses at 10 min (42 %), but peak efficiency was only 58 % at 60 min. These results indicate that each of the mutant viruses exhibited a defect in their maximum penetration efficiency compared with parental YF5.2iv virus. However, this defect was severe for the Asn360 and Phe360 mutants, suggesting that they have a profound disturbance in the cell entry process. Variability in the performance of the penetration assay at the low levels of penetration observed for these mutants probably accounts for the lack of an apparent difference of penetration efficiency over the time interval from 1060 min for these two mutants.

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Fig. 4. Comparison of penetration rate and efficiency of the parental YF5.2iv virus and engineered mutants. Penetration assay was performed using plaque-purified viruses on Vero cells as described in the Methods. Data represent combined results of four independent experiments [except for Phe360 (two experiments)]. In each experiment, both acid pH-treated and control samples for each virus at each time point were performed in duplicate. Error bars represent standard error of the mean for the composite results (not available for Phe360).
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DISCUSSION
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Domain III of the flavivirus E protein is believed to provide a binding site for cellular receptors, but little is known about the molecular interactions that occur during the process of viral attachment to the cell surface and subsequent entry. Evidence has accumulated to suggest that specific receptor-binding determinants map to the lateral surface of domain III, based on studies of monoclonal antibody binding, neutralization-escape mutants, host-range mutants, directed mutagenesis and soluble domain III fragments (Bhardwaj et al., 2001
; Crill & Roehrig, 2001
; Hasegawa et al., 1992
; Holzmann et al., 1997
; Hurrelbrink & McMinn, 2001
; Jiang et al., 1993
; Lee & Lobigs, 2000
; Lobigs et al., 1990
; Mandl et al., 2000
). Some mutants characterized in such studies exhibit decreased pathogenesis for mice, presumably in part as a consequence of an altered interaction with host cell receptors. Other functions in which domain III may participate during virus entry have been characterized less. Recent reports on the structure of dengue 2 and TBE viruses and recombinant subviral particles have indicated that the transition of the E protein from the native dimeric form to the fusion-competent trimeric state requires a conformational change in domain II of adjacent E proteins (Ferlenghi et al., 2001
; Kuhn et al., 2002
; Stiasny et al., 2004
; Zhang et al., 2003
). But this may also involve some movement of domain III as the virion acquires a form that accommodate trimeric E protein structures on its surface (Kuhn et al., 2002
). Furthermore, the structures of the post-fusion form of the dengue 2 and TBE virus envelope proteins indicate that a major conformational change occurs, wherein there is movement of domain III across its interface with domain I in the direction of the membrane-engaged fusion peptide (Bressanelli et al., 2004
; Modis et al., 2004
). These findings suggest that molecular determinants that affect the properties of domain III may involve functions additional to the interactions with cellular receptors for which this region is believed to be responsible for.
In previous studies we demonstrated that the neuroblastoma cell-adapted YF 17D variant NB15a, in which a single amino acid substitution in domain III of the E protein (Asp360 to Gly) causes defective cell penetration and poor cell-to-cell spread compared with the parent virus. These characteristics did not appear to result from any major decrease in binding to host cells (Vlaycheva & Chambers, 2002
). This suggested to us that the 360 locus in the E protein may participate in steps of the virus entry process beyond attachment to the cell surface. Such steps include receptor-mediated endocytosis, the low pH-induced structural rearrangement of the E protein in an early endosomal compartment, and the subsequent fusion of the viral and endosomal membranes. To substantiate the significance of the original mutation at position 360 of the E protein identified in the NB15a variant, we derived additional neuroblastoma cell-adapted YF 17D variants, using the original virus stock as well as other virus stocks of the YF 17D strain. A second persistently infected culture from the same starting virus stock used to generate NB15a yielded variants (NB18a and NB18a), with an amino acid substitution different from NB15a, but located in the same charge cluster (glycine for glutamic acid at position 362). A third persistently infected culture from an independent preparation of the YF5.2iv infectious clone yielded a variant (NBC7) with the identical substitution as the original NB15a variant (glycine for aspartic acid at position 360). In contrast, no variants were selected from a YF 17D commercial vaccine stock or an SW-13 cell-adapted stock of the 17D-204 vaccine substrain. These results may reflect differences in genetic composition of the latter two viruses at locus 360362, compared with YF5.2iv viruses which are derived by transfection with RNA transcripts produced in vitro by bacteriophage polymerases. Lack of such variants in the more cell culture adapted strains such as YF 17D-5W and YFS/232 may result in a failure to select for viruses capable of persistently infecting NB cells, and lead to progressive dilution and disappearance of such viruses from serially passaged NB cells. Alternatively, it is possible that genetic differences exist among YF5.2iv viruses and the other 17D strains in regions outside of the E protein, and that these are deleterious for replication and/or persistence of the latter in NB cells. We also cannot at present exclude the possibility that persistence of YF 17D-SW and YFS-232 viruses occurs below the limit of detection by plaque assay. In any case, it is evident that the selection of variants with mutations at positions 360 or 362 is not a non-specific event, but relates to some functional process during virus entry into this cell line and/or the subsequent establishment of persistent infection. We do not know at present if other flaviviruses will exhibit a similar phenomenon with regard to selection of persistent viral variants on NB cells, in conjunction with mutations at the corresponding locus in their E proteins.
To gain more insight into the molecular basis for the defective cell culture properties of the NB15a virus, mutations at residues 360362 in the E protein were tested for their effects on cell penetration, virus spread and growth efficiency. We found that position 360 was very sensitive to substitution with any amino acid other than glutamic acid, generally yielding small or minute plaque viruses which were reduced in size compared with the original neuroblastoma cell-adapted NB15a variant. These mutants resembled the NB15a variant in terms of growth properties, cell-to-cell spread and cell penetration efficiency. In contrast, a substitution of glutamic acid for aspartic acid at position 360 resulted in a virus that was indistinguishable from the parent. This indicates that there is no absolute requirement for aspartic acid, but rather that preservation of the negative charge at this site is important for normal function of the protein. In support of this conclusion is the fact that glutamic acid is found at position 360 in some wild-type YFV strains (e.g. strain 1899/81; Bhardwaj et al., 2001
). Also, either aspartic or glutamic acid occurs at the corresponding position in dengue 1, 2 and 3 viruses, whereas an asparagine residue occurs in the case of dengue 4. Most Japanese encephalitis virus serogroup viruses contain serine or threonine at this position, whereas the non-vector-borne flaviviruses Rio Bravo virus and Apoi virus also contain either glutamic or aspartic acid, respectively, at the corresponding position (Bhardwaj et al., 2001
). Overall, these observations indicate conservation of charged or polar residues at this position among a range of different flaviviruses.
A previous study on antigenic properties of the YFV E protein suggested that the region of domain III inclusive of aa 358365 is involved in a conformational epitope (Jackson & Phillpotts, 1997
), indicating some local structural complexity of residues in this loop. The 360362 locus consists of three consecutive negatively charged amino acids (aspartic-aspartic-glutamic), which form a central part of this solvent-exposed loop connecting the Dx and E
-sheets. Because of the location of the 360362 locus, the defects conferred by substitutions with residues other than aspartic or glutamic acid may result from effects on intramolecular interactions within the E protein that depend on this charge cluster, rather than merely cell receptor interactions, which are proposed to involve loops on the lateral surface of domain III (Rey et al., 1995
). According to the crystal structure of the TBE virus E protein (Rey et al., 1995
), the 360362 locus in the YFV E protein is located on the upper-medial edge of domain III, adjacent to the domain I/III interface. Interestingly, an attenuated neutralization-escape mutant (Gly368 to Arg) of TBE virus which maps to the corresponding position, exhibited a reduced threshold for acid-induced conformational change, suggesting a role of this region in transition of the E protein to a fusion-active form (Holzmann et al., 1997
). Features of this region of the YF 17D E protein, based on a homology model with the TBE virus E protein are shown in Fig. 5
. Under conditions where rotamer positions for Asn359 and Asp360 are energetically favourable, these residues exhibit an outward orientation with respect to the surface of the E protein, and their side chains are predicted to engage in hydrogen bond formation (Fig. 5a
). Glu360 is also predicted to form such a hydrogen bond, as well as a second bond with the backbone nitrogen atom of Asn359 (not shown). In contrast, none of the other substitutions at position 360 result in similar predictions. Glycine (Fig. 5b
), phenylalanine and leucine (not shown) are all unlikely to form a hydrogen bond with Asn359, and leucine or serine force Asn359 to bend towards the polypeptide backbone to form such a bond, in a manner that is not energetically favourable (not shown). This absence of hydrogen bonding is expected to permit some increase in conformational freedom of Asn359 with respect to the solvent environment. This may lead to more flexibility in this loop of domain III, which may be deleterious for its function. Although modelling of the serine mutation predicts that it has deleterious effects on the structure of the 360362 loop, the experimental data (virus production, fluorescent focus formation and penetration efficiency) suggest that it causes a less severe defect in cell entry than the other mutations. Despite the unfavourable energy predictions for serine, its polar side chain may allow partial stabilization of the 360362 loop in a manner similar to that proposed for aspartic or glutamic acid at position 360, possibly by hydrogen bond interaction with the adjacent Asn359 residue.

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Fig. 5. Model of YF E protein based on identity with the TBE virus E protein. Structural features of the domain III-domain I interface depicting mutations at residues 360362 in the Dx-E loop of domain III. Green indicates the peptide backbone of domain III, orange indicates the backbone of domain I. Hydrogen bonds are depicted by dashed white lines; and the peptide chain is coloured by the standard CPK code (white, carbon; red, oxygen; blue, nitrogen). Positions of the substituted side chains are shown for the most favourable predicted energy levels. (a) Parental residues at positions 360362. A predicted hydrogen bond between the side chain oxygen of Asp360 and the side chain nitrogen of Asp359 is shown. (b) Substitution of glycine for Asp360. Note loss of predicted hydrogen bond with Asn359. (c) Substitution of glycine for Asp361. Note loss of the predicted hydrogen bond between side chain oxygen of Asp361 and backbone oxygens of Val145 and Gly146 in the adjacent loop of domain I. (d) Substitution of glycine for Glu362.
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Predicted effects of glycine at positions 361362 are shown in Fig. 5(c) and (d)
. In the case of position 361, glycine abrogates a predicted hydrogen bond normally formed between Asp361 and residues Val145 and Gly146 on an adjacent loop of domain I (Fig. 5a and c
). As shown for dengue 2 and TBE viruses (Bressanelli et al., 2004
; Modis et al., 2004
), interaction between domains I and III appear to be important during the repositioning of domain III along the domain I surface in the post-fusion form of the E protein, with the contact surface of domain III with domain I actually remaining preserved after this movement. Based on this information, it is possible that the deleterious effect of glycine at position 361 in the YFV E protein is caused by disruption of the normal domain III-I interaction that is involved in the major conformational change of domain III. On the other hand, mutations at positions 360 and 362 may be tolerated because an interaction involving Asp361 and residues in domain I is not expected to be affected to the same degree. In the native structure, Glu362 is predicted to occupy a less exposed position than 360 or 361 (Fig. 5a
), and does not participate in any predicted hydrogen bond formations. Substitution with glycine at this position is deleterious, but may act by indirectly disturbing interactions at the domain I/III interface.
In summary, in this study, we provide evidence that residues 360362 of domain III of the YFV E protein constitute a functional determinant involved in virus entry, acting presumably through effects on post-receptor-binding events that may involve the conformational change in domain III associated with activation of membrane fusion activity. Further investigations should be directed towards understanding whether this locus constitutes a broad functional determinant for the E proteins of other flaviviruses, and on what the actual mechanism of this determinant is in regard to the formation of the fusion-competent E protein. Such information may be of value for engineering live-attenuated flaviviruses for potential vaccine use, and for conducting studies of viral neurovirulence.
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ACKNOWLEDGEMENTS
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This work was supported by grants AI43512 (NIAID) and CI00094 (CDC).
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Received 25 May 2004;
accepted 20 October 2004.