Attenuation of Murray Valley Encephalitis Virus by Site-DirectedMutagenesis of the Hinge and Putative Receptor-Binding
Regions of the Envelope ProteinROBERT J. HURRELBRINK* AND PETER C. MCMINN
Department of Microbiology, University of Western Australia, Nedlands,Western Australia 6907, Australia
Received 12 March 2001/Accepted 16 May 2001
Molecular determinants of virulence in flaviviruses cluster in two regions on the three-dimensional structureof the envelope (E) protein; the base of domain II, believed to serve as a hinge during pH-dependent confor-mational change in the endosome, and the lateral face of domain III, which contains an integrin-binding motifArg-Gly-Asp (RGD) in mosquito-borne flaviviruses and is believed to form the receptor-binding site of the pro-tein. In an effort to better understand the nature of attenuation caused by mutations in these two regions, afull-length infectious cDNA clone of Murray Valley encephalitis virus prototype strain 1-51 (MVE-1-51) wasemployed to produce a panel of site-directed mutants with substitutions at amino acid positions 277 (E-277;hinge region) or 390 (E-390; RGD motif). Viruses with mutations at E-277 (Ser3Ile, Ser3Asn, Ser3Val, andSer3Pro) showed various levels of in vitro and in vivo attenuation dependent on the level of hydrophobicity ofthe substituted amino acid. Altered hemagglutination activity observed for these viruses suggests that muta-tions in the hinge region may indirectly disrupt the receptor-ligand interaction, possibly by causing prematurerelease of the virion from the endosomal membrane prior to fusion. Similarly, viruses with mutations at E-390(Asp3Asn, Asp3Glu, and Asp3Tyr) were also attenuated in vitro and in vivo; however, the absorption and pen-etration rates of these viruses were similar to those of wild-type virus. This, coupled with the fact that E-390mutant viruses were only moderately inhibited by soluble heparin, suggests that RGD-dependent integrin bind-ing is not essential for entry of MVE and that multiple and/or alternate receptors may be involved in cell entry.
Murray Valley encephalitis virus (MVE) is a member of theFlavivirus genus (family Flaviviridae) and is a small, lipid-en-veloped virus which contains a single-stranded positive-senseRNA genome. The genome is approximately 11 kb in lengthand contains a single open reading frame which is posttrans-lationally cleaved to generate three structural (C, prM, andE) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A,NS4B, and NS5) proteins. Viral genomic RNA also has amethylated cap at its 59 terminus and forms a highly conservedstem-loop structure at its 39 end (61). As for many flaviviruses,MVE causes clinically significant disease in humans and, to-gether with Kunjin (KUN) virus, is responsible for almost allcases of flaviviral encephalitis in mainland Australia (41).
In recent years, infectious cDNA clones have been producedfor a number of flaviviruses, including MVE (31, 39), enablingmanipulation of the genome at the nucleotide level. Suchclones have been used to examine the glycosylation, cleavage,and function of the prM and E (4, 20, 28, 33, 55, 57, 68), NS1(53, 55, 57), NS2B/NS3 (9, 10, 54), and NS5 (34, 35, 36) pro-teins, as well as to generate viruses with deletions in their 59and 39 untranslated regions (6, 38, 43, 48). More recent workhas seen the generation of chimeric yellow fever viruses (YF),containing the prM and E genes of Japanese encephalitis virus(JE) (8) or dengue virus type 2 (DEN-2) (23). In primates,these chimeric viruses provide solid protection against heter-
ologous virus challenge and demonstrate great potential foruse as flavivirus vaccines (23, 24, 49, 50).
The envelope (E) protein of flaviviruses plays a significantrole in viral entry and possesses an interesting structural andfunctional biology. It mediates attachment of the virus to hostcells, as well as fusion of the viral and cellular membranes afterreceptor-mediated endocytosis. In addition, it is the majortarget of neutralizing antibodies in the host and plays a signif-icant role in both viral tropism and pathogenesis. The three-dimensional structure of the ectodomain of the E protein hasbeen determined for tick-borne encephalitis virus (TBE) (60),and it serves as a useful model for other flaviviruses due to thehigh amino acid sequence homology observed throughout thegenus. The protein forms head-to-tail dimers on the virionsurface, and each monomer consists of three domains, referredto as domain I (central domain), domain II (dimerization do-main), and domain III (immunoglobulin-like domain).
Molecular determinants of virulence on the flavivirus E pro-tein form three distinct clusters and are likely to affect viru-lence by disrupting the functional biology of the protein (60).The first cluster, located in a putative hinge region linking do-mains I and II, appears to affect the pH-dependent conforma-tional change required for endosomal fusion. In MVE, muta-tions in this region at amino acid position 277 [E-277 (Ser3Ile)] inhibit the hemagglutination (HA) and fusion propertiesof the virus and cause a loss of neuroinvasiveness (NI) in mice(46, 47). Similarly, mutations in the second cluster, located atthe tip of domain II in a highly conserved “fusion peptide,”also appear to disrupt fusion. In contrast, mutations in thethird cluster, located on the lateral face of domain III, are
* Corresponding author. Mailing address: Virology Division, TVWTelethon Institute of Child Health Research, Subiaco WA 6008, Aus-tralia. Phone: 61 8 9489 7896. Fax: 61 8 9489 7700. E-mail: [email protected].
predicted to disrupt receptor binding. In some mosquito-borneflaviviruses, the presence of an Arg-Gly-Asp (RGD) motif inthis region has led some to suggest that integrin binding may beimportant for virus entry (40). The involvement of an RGDmotif in cell entry has been described for a number of viruses,including adenovirus type 2 (18, 73, 74), human rotavirus RV-5(17), and foot and mouth disease virus (FMDV) (32). Muta-tions in this motif have been shown to affect virus infectivityand virulence in both MVE (39, 40) and YF (69).
In this study, two panels of mutant MVE viruses with sub-stitutions at E-277 (Ser) or E-390 (Asp) were created to inves-tigate the influence of these mutations on virulence. For eachvirus, obvious effects on plaque phenotype, growth kinetics incell culture, genetic stability, and virulence in mice were ob-served. Interestingly, mutations at E-277 or E-390 did notaffect the absorption or penetration rates of any of the virusesnor did they affect the relative rates of inhibition of virusbinding by soluble heparin. However, some mutations caused acomplete loss of NI and/or a reduced ability to agglutinate redblood cells (RBCs). Furthermore, some mutant viruses dis-played either large- or small-plaque phenotypes, correlatingwith altered growth kinetics in Vero cells.
MATERIALS AND METHODS
Virus and infectious cDNA clone. All mutant viruses were derived from aninfectious cDNA clone of MVE virus prototype strain MVE-1-51 (designatedpMVE-1-51). Thorough genotypic and phenotypic characterization of virus de-rived from this clone (CDV-1-51) has been described previously (31), and itscomplete genomic sequence is known (GenBank accession no. AF161266). Site-directed mutations of the E protein (E-277 or E-390) were engineered into clonepMVE-1-51 by using the QuikChange Site Directed Mutagenesis (SDM) kit(Stratagene). The nucleotide sequences of polyacrylamide gel electrophoresis-purified sense primers used for SDM are shown in Table 1.
After SDM and transformation into a bacterial host, three separate cDNAclones of each of the seven mutants were selected and subjected to sequenceanalysis over the entire E gene (approximately 15% of the entire genome) forcomparison to the known sequence of MVE-1-51 (31). This was performed bothto confirm the site-specific change and to ensure that other mutations had notbeen inadvertently introduced during the cycling protocol. In every clone se-quenced (n 5 21), only the site-specific change was present. No other mutationswere identified in any of the clones, confirming the high fidelity of the Pfu DNApolymerase enzyme used in the cycling reaction. One cDNA clone was selectedfor each mutant and used as a template for the production of RNA and infec-tious virus.
Virus recovery and stock preparation. Site-directed mutants of clone pMVE-1-51 were linearized with XbaI and used as templates for subsequent transcrip-tion with T7 RNA polymerase. The generation of genome-length RNA and itstransfection into BHK-21 cells by electroporation was performed as describedpreviously (31). Four to six days after electroporation, cell culture supernatantsshowed obvious signs of cytopathic effect and were assayed for virus by plaqueassay on Vero cells. In order to generate stocks of sufficient titer for subsequentassays, transfected BHK-21 cell culture supernatants were passaged three times
in C6/36 mosquito cells and sequenced across the entire E gene as describedbelow.
Sequence analysis. Sequencing was performed using an ABI–Perkin-Elmerautomated sequencing system, which incorporates fluorescently labeled dideoxy-nucleotides. All site-directed mutant clones derived from clone pMVE-1-51 weresubjected to sequence analysis over the entire E gene. Mutant virus stocks (C6/36cell culture supernatants), as well as virus present in the brains of two enceph-alitic mice infected with each mutant virus, were similarly sequenced across theE gene. For these analyses, genomic RNA was purified using QIAamp ViralRNA spin columns (Qiagen) and amplified by reverse transcriptase PCR asdescribed previously (31). Sequencing primers were supplied by Life Technolo-gies (Gibco BRL) and were used at a final concentration of 0.8 pmol/ml. Detailsof primer sequences are available by request.
Cell culture. Vero (ATCC CCL81 P130-P145), BHK-21 (ATCC CCL10 P56-P59), and Aedes albopictus C6/36 (ATCC CRL1660 P5-P20) cells were grown inM199 medium supplemented with 2 mM L-glutamine and 10% fetal calf serumand were incubated at either 37°C (Vero, BHK-21) or 28°C (C6/36) in anatmosphere containing 5% CO2. For plaque assays, subconfluent monolayers ofVero cells in 12-well tissue culture trays were inoculated with virus and incubatedfor 1 h. Virus was then removed, and cells were overlaid with methylcellulosecontaining 2% fetal calf serum in M199 media. Cells were cultured for 4 to 6 daysat 37°C (5% CO2) and stained with methylene blue to visualize plaques (1%[wt/vol] methylene blue, 10% formaldehyde). To test for temperature sensitivity,plaque assays were performed separately at 37°C and at an elevated temperatureof 39°C, and the number and morphology of plaques were compared. For virusgrowth assays, monolayers of Vero cells in 60-mm2 tissue culture dishes wereinfected with virus at a multiplicity of infection between 1 and 5 (standardized foreach assay). Aliquots of cell culture supernatant (500 ml) were then collected at6, 12, 18, 24, and 30 h postinfection (p.i.) and replaced with an equal volume offresh media. The titer of virus in each sample was subsequently determined byplaque assay. All virus growth assays were performed in duplicate.
Absorption and penetration assays. Monolayers of Vero cells were infected intriplicate according to the adsorption assay method described by Khromykh andWestaway (37) or the penetration assay method described by Hung et al. (30).For adsorption assays, approximately 100 PFU of virus was added to each welland allowed to adsorb for 30, 60, or 90 min at 37°C. Cells were then washed twicewith phosphate-buffered saline, overlaid with growth medium, and incubated for4 to 6 days as per standard plaque assay. For penetration assays, approximately100 PFU of virus was added to each well and allowed to adsorb for 30, 60, or 90min at 37°C. Cells were then incubated in acid glycine (pH 3.0) for 3 min toinactivate noneclipsed virus. Acid glycine was aspirated, and cells were overlaidwith growth medium as outlined for adsorption assays above. The rate of ad-sorption or penetration was calculated as the ratio of the average number ofplaques at 30 or 60 min p.i. relative to the average number of plaques at 90 minp.i. and was expressed as a percentage.
HA assays. Virus from infected C6/36 cell supernatants was used as a sourceof hemagglutinin. HA assays were performed using a modified protocol of Clarkand Casals (14) as described previously (46). Titers were recorded as the recip-rocal of the highest dilution which yielded complete agglutination of ganderRBCs.
Heparin inhibition assays. Inhibition of virus binding by soluble heparin wasperformed using modified protocols based on those described by Chen et al. (13),Hung et al. (30), and Lee and Lobigs (39). Approximately 100 PFU of virus waspreincubated in Hank’s borate-buffered saline (Gibco BRL) containing 200 mg ofheparin (Sigma)/ml at 4°C for 1 h. Virus was then inoculated onto monolayers ofprechilled Vero cells (30 min at 4°C) and incubated for a further 1 h at 4°C toallow virus binding. The inoculum was then removed and cell monolayers were
TABLE 1. Sense primers used to introduce site-directed mutations into clone pMVE-1-51a
a Antisense complementary primers (not shown) were used in combination with sense primers to form primer pairs for site-directed mutagenesis.b Nucleotide substitutions are underlined and affected codons are highlighted in bold.
washed with cold phosphate-buffered saline prior to the addition of M199/MCand incubation at 37°C under standard conditions for 4 to 6 days. Plaques werevisualized as per standard plaque assay. Percent inhibition by soluble heparin wascalculated according to the following formula: (a 2 b)/a, where a is the numberof plaques on cells incubated with untreated virus and b is the number of plaqueson cells incubated with heparin-treated virus.
Virulence in mice. Litters of five 21-day-old ARC/Swiss mice (Animal Re-sources Centre, Murdoch, Western Australia, Australia) were injected intracra-nially (i.c.) or intraperitoneally (i.p.) with 10 or 50 ml, respectively, of a 10-folddilution of virus. Mice were examined daily for signs of morbidity, and deathswere recorded. “Humane end points” were employed to minimize distress inexperimental animals, a method which does not significantly alter 50% lethaldose values in models of viral encephalitis (75). The 50% humane end point dose(HD50) was calculated for each group by following the 50% lethal dose methoddescribed by Reed and Muench (59). Mean time to death was determined byinjecting mice (10 per group) i.c. or i.p. with 103 PFU of virus and recording thesurvival of mice over a period of 21 days. Statistical significance was determinedusing a paired Student’s t test.
For growth in mouse brain assays, groups of 30 18-day-old ARC/Swiss micewere injected i.c. with 103 PFU of virus. At selected times, three mice from eachgroup were anesthetized (penthrane) and their brains were removed and snapfrozen in liquid nitrogen. Tissues were then stored at 280°C until required. Priorto titration by standard plaque assay, tissues were thawed, weighed, and manuallyhomogenized before being prepared as 10% (wt/vol) suspensions in Hanks’borate-buffered saline. Titers were expressed as PFU per gram of tissue.
For protection assays, groups of 21 18-day-old ARC/Swiss mice were mockinoculated or were inoculated with between 104 and 1021 PFU of clone-derivedvirus by the i.p. or i.c. route. At 17 days p.i., surviving mice in each group werechallenged with 104 i.p. HD50s of clone-derived wild-type virus (CDV-1-51).Mice were observed for a further 21 days for signs of morbidity, and deaths wererecorded. All mouse experiments were undertaken using protocols approved bythe University of Western Australia Animal Experimentation Ethics Committee.Mice were kept on a clean litter of sawdust and given food and water ad libitum.
Protein structure graphics. The three-dimensional structure of a solubleectodomain fragment of the TBE E protein (Brookhaven Protein Databank[PDB] entry 1SVB) (60) was used as the basis for all diagrammatic representa-tions of the E protein. Such representations were made using RASMOL molec-ular visualization software (version 2.6) (62). Homology modeling of the MVE Eprotein was performed using Swiss-PDB-Viewer software (version 3.6b3) (21),where the primary amino acid sequence of the MVE E protein was threadedonto the known structure of the TBE E protein and submitted to the Swiss-Model (ExPASy) server in Geneva, Switzerland, for energy minimization andsubsequent generation of a final model.
RESULTS
Mutagenesis of the hinge region at E-277. Four amino acidsubstitutions at E-277, varying in terms of their size and hy-drophobicity, were selected for this study. At E-277, the hy-drophilic Ser residue was converted to a strongly hydrophobicIle residue (S3I) in order to reconstitute the attenuated virusBHv1 from an earlier study (46). This was performed to ascer-tain whether a single amino acid change in the E protein wasresponsible for the observed phenotype, which included a lowNI in mice and an inability to agglutinate RBCs. In addition, asecond mutant from the McMinn et al. study (46) was recon-stituted, since the Ser3Asn (S3N) mutation in this virus(BHv2) had no observable effect on viral phenotype and rep-resented a conservative amino acid change. Two other substi-tutions at E-277 were also made to produce novel viruses notpreviously reported. These substitutions, Ser3Pro (S3P) andSer3Val (S3V), were chosen because they were intermediatein terms of their hydrophobicity between the Ile and Asnresidues of the first two mutants. It was predicted that suchchanges would have various effects on virus phenotype and thatthese effects may correlate with the level of hydrophobicity ofthe substituted amino acid.
Phenotypes of E-277 (hinge) mutant viruses in vitro. The invitro phenotypes of the four hinge mutant viruses (designatedCDV-1-51v1, -v2, -v3, and -v4; Table 2) were compared andcontrasted to that of clone-derived wild-type virus (CDV-1-51;see reference 31) by a number of means, including plaquemorphology, growth kinetics in Vero cells, temperature sensi-tivity, adsorption and penetration rates, and HA activity. Withthe exception of CDV-1-51v2 (S3N), no change to the wild-type (CDV-1-51) plaque morphology was observed for any ofthe E-277 mutant viruses (see notes to Table 2). CDV-1-51v1(S3I), CDV-1-51v3 (S3V), and CDV-1-51v4 (S3P) all dis-played a normal plaque phenotype, contrasting with the large-plaque phenotype observed for CDV-1-51v2 (S3N).
More obvious differences between the phenotypes of theE-277 mutant viruses became apparent when their relativegrowth kinetics in Vero cells were compared. As shown in Fig.1a, these differences were most prominent at 12 h p.i., withCDV-1-51v1 (S3I), CDV-1-51v4 (S3P), and CDV-1-51v3(S3V) exhibiting the lowest titers at this time point. Interest-ingly, CDV-1-51v2 (S3N) displayed a slightly higher titer thanparental virus at this and all other time points, possibly reflect-ing an improved replicative ability in Vero cells.
To further analyze in vitro phenotype, the temperature sen-sitivities of each of the hinge mutant viruses were determinedby plaque assay at 37 and 39°C (Table 2). Little variability intiter was observed for any of the mutant viruses, suggestingthat the introduced mutation had little to no effect on E pro-tein stability at the elevated temperature. To determine ifobserved differences in growth kinetics were due to altered cellentry, the ability of each of the hinge mutant viruses to bind toand penetrate host cells was determined by adsorption andpenetration assay. As shown in Table 2, no significant differ-ences in the ability of E-277 mutant viruses to bind to andpenetrate host cells were observed. Adsorption rates (ratios)ranging from 43 to 49% were observed at 30 min p.i., increas-ing to a range of 72 to 81% by 60 min p.i. Similarly, penetrationrates ranging from 47 to 51% were observed at 30 min p.i.,increasing to a range of 75 to 82% by 60 min p.i.
Since differences in the binding and penetration of each ofthe hinge mutant viruses were not evident, the ability of each
TABLE 2. Phenotypes of hinge and RGD mutant viruses in vitro
Virus MutationPlaquepheno-typea
Tempsensi-
tivity at39°C
Virus entryb
Absorption Penetration
30:90 60:90 30:90 60:90
CDV-1-51 None Normal None 46 73 47 78CDV-1-51v1 E-277 (S3I) Normal None 43 72 49 75CDV-1-51v2 E-277 (S3N) Large None 48 81 47 82CDV-1-51v3 E-277 (S3V) Normal None 49 79 51 80CDV-1-51v4 E-277 (S3P) Normal None 44 77 49 80CDV-1-51v5 E-390 (D3N) Small None 46 74 50 77CDV-1-51v6 E-390 (D3E) Large None 50 80 51 82CDV-1-51v7 E-390 (D3Y) Small None 44 74 43 77
a Plaque phenotypes were determined by comparison to those observed forCDV-1-51. CDV-1-51 had a normal plaque phenotype, with a diametric size ofapproximately 2.2 mm at day 5 p.i. Viruses with small-plaque phenotypes hadplaques approximately half this size at the same time p.i. Viruses with large-plaque phenotypes had plaques approximately twice the size of normal plaquesat the same time p.i.
b Virus entry rates (ratios) for adsorption and penetration assays are expressedas a percentage, i.e., the number of plaques observed at 30 (30:90) or 60 (60:90)min p.i. divided by the number observed at 90 min p.i. (times 100%).
of these viruses to fuse with RBCs at acidic pH was determinedby HA assay. In flaviviruses, HA is a pH-dependent interactionbetween viral and erythrocyte membranes that is thought toinvolve the mixing of membrane lipids (56). As such, it servesas a measure of the ability of a virus to fuse efficiently with hostcell membranes, specifically those of the endosomal membraneduring fusion. Infected C6/36 cell supernatants, ranging in titerbetween 107 and 108 PFU/ml, were used as a source of HA andassayed for HA activity over the pH range 5.9 to 7.2. Using anoptimum pH of 6.6 (determined for CDV-1-51), the HA titersof each of the hinge mutant viruses were determined as shownin Table 3. In addition, the HA titers of two other MVEvariants, BHv1 and BHv2 [E-277 (S3I) and E-277 (S3N),respectively; see reference 46], were determined, serving aspositive and negative controls for altered HA activity. Inter-estingly, both CDV-1-51v1 (S3I) and BHv1 (S3I) failed toagglutinate RBCs at the lowest dilution tested (1:2) over theentire pH range examined (data not shown), confirming that a
single amino acid substitution at E-277 had been responsiblefor the observed phenotype of BHv1 in a previous study (46).Furthermore, the HA titers for both CDV-1-51v2 (S3N) andBHv2 (S3N) were also identical at 1:320, similar to that ob-served for clone-derived wild-type virus (1:160). The HA titersof the remaining two mutants, CDV-1-51v3 (S3V) and CDV-1-51v4 (S3P), were both 1:40; however, the optimal pH forHA was increased from 6.6 to 6.8 for both viruses. Hydropho-bic amino acid substitutions at E-277 therefore caused either acomplete loss of HA activity [CDV-1-51v1 (S3I)] or a markedreduction of this activity coupled with an increase in optimalpH [CDV-1-51v3 (S3V) and CDV-1-51v4 (S3P)]. In con-trast, a hydrophilic amino acid substitution had no effect onHA activity [CDV-1-51v2 (S3N)].
Phenotypes of E-277 (hinge) mutant viruses in vivo. As anextension of the in vitro analyses described above, the in vivophenotype of each of the hinge mutant viruses was examinedby determining HD50 and mortality profiles and comparingaverage survival times after i.p. or i.c. challenge. In addition,the growth kinetics of each virus in infected mouse brain wasalso determined.
As shown in Table 4, HD50 values for each of the mutantviruses ranged between 2 and 5 PFU by the i.c. route, resultingin 100% mortality and a mean time to death in the range of 4.4to 5.5 days. In comparison to the profile for CDV-1-51 (i.c.HD50 of 1 PFU; 100% mortality; time [mean 6 standard de-viation] to death, 4.6 6 0.5 days), each of the E-277 mutantviruses was subsequently deemed to be of high neurovirulence(NV). With the exception of CDV-1-51v2 (S3N), the NI (de-termined by i.p. inoculation) of each of the hinge mutant vi-ruses was found to be markedly different to that of CDV-1-51.The highly neuroinvasive CDV-1-51v2 (S3N) had an i.p.HD50 of 4 PFU, a mortality of 90%, and a mean time to deathof 7.3 6 1.3 days, similar to the profile observed for clone-derived wild-type virus (i.p. HD50 of 5 PFU; mortality of 100%;time to death, 7.2 6 0.9 days). In contrast, CDV-1-51v1 (S3I)was found to be of low NI, with an i.p. HD50 greater than 104.5
PFU (no observed mortality at the lowest dilution of virustested). The remaining two mutants, CDV-1-51v3 (S3V) andCDV-1-51v4 (S3P), were found to be of intermediate NI, withHD50 values of 620 and 580 PFU, mortalities of 40 and 50%,
FIG. 1. Growth kinetics of hinge (A) and RGD (B) mutant virusesin Vero cells. Monolayers were infected at a multiplicity of infection of5, and samples of cell culture supernatant were collected at the timesindicated. Viral titers were determined by plaque assay on Vero cells.All assays were performed in duplicate.
TABLE 3. HA activity of hinge and RGD mutant viruses
and mean times to death of 8.5 6 1.3 and 10.2 6 3.6 days,respectively.
When analysis of the growth kinetics of hinge mutant virusesin infected mouse brain was performed, attenuation was foundto be similar to that observed in vitro. As shown in Fig. 2a,mean peak titers ranging from 108.3 to 108.7 PFU/g were ob-tained for most viruses by day 3 p.i. In contrast, the titer ofCDV-1-51v1 (S3I) lagged approximately 10-fold behind thatof the other viruses (including CDV-1-51) at the same timepoint, having a titer of 107.4 PFU/g. On completion of the assay(day 5 p.i.), titers for CDV-1-51v3 (S3V), CDV-1-51v4 (S3P), and CDV-1-51v1 (S3I) were observed to be 20-, 8-, and13-fold lower, respectively, than those for both CDV-1-51 andCDV-1-51v2 (S3N).
Mutagenesis of the RGD motif at E-390. Three amino acidsubstitutions at E-390 were selected for this study. At E-390,the negatively charged Asp residue was converted to an un-charged Asn residue [E-390 (D3N)] in order to reconstitutean attenuated virus (P5/Ab10) from a previous study (40).Phenotypic characterization of P5/Ab10 was limited to theobservation that the virus had low NI in mice (40). As such, theE-390 (D3N) mutant created in this study was used to furthercharacterize the nature of the attenuation and to ascertainwhether a single amino acid change in the E protein wasresponsible for the observed phenotype. The second mutant,E-390 (D3E), was constructed for two reasons: first, becauseit represented a conservative amino acid change, and second,because other flaviviruses such as West Nile virus (WNV) (72)and KUN (15) possess an RGE as opposed to an RGD motifin this region of the protein. Substitution of a Glu residue atthe third position of an RGD motif has been shown to abolishRGD-dependent integrin binding by a range of viruses, includ-ing FMDV (44), chimeric hepatitis B virus (64), and echovirustype 9 (76). The last mutant, E-390 (D3Y), was constructedbecause it represented the substitution of a large hydrophobicamino acid residue into a strongly hydrophilic region of theprotein. Like the D3N and D3E substitutions above, such amutation would be predicted to significantly disrupt RGD-dependent integrin binding if this were important in the at-tachment and penetration of MVE into host cells.
Phenotypes of E-390 (RGD) mutant viruses in vitro. As forthe panel of hinge mutant viruses, the in vitro phenotypes ofeach of the RGD mutant viruses (CDV-1-51v5, -v6, and -v7;
Table 2) were compared and contrasted in terms of theirplaque morphology, growth kinetics in Vero cells, temperaturesensitivity, adsorption and penetration rates, and HA activity.All three RGD mutants displayed plaque morphologies differ-ent from that of clone-derived wild-type virus. CDV-1-51v5(D3N) and CDV-1-51v7 (D3Y) exhibited a small-plaquemorphology, while CDV-1-51v6 (D3E) exhibited a large-plaque morphology (see notes to Table 2). These differenceswere found to be reflective of differences in the relativegrowth kinetics of each virus in Vero cells. As shown in Fig.1b, obvious differences in replicative ability were evident at 6 hp.i., with CDV-1-51v5 (D3N) and CDV-1-51v7 (D3Y) dis-playing significantly lower titers at this time point (100- to500-fold lower titers than CDV-1-51). In contrast, the titer ofCDV-1-51v6 (D3E) at this and all other time points wassimilar to that observed for CDV-1-51. At 12 h p.i., the titers ofboth CDV-1-51v5 (D3N) and CDV-1-51v7 (D3Y) continuedto lag behind those of both CDV-1-51 and CDV-1-51v6 (D3E) by the same order of magnitude, narrowing to 50- and10-fold differences, respectively, by 24 h p.i.
Further analysis of in vitro phenotype was performed bytemperature sensitivity testing of each of the RGD mutantviruses (by plaque assay at 37 and 39°C). As for the panel ofhinge mutant viruses, little variability in titer was observed forany of the RGD mutants (Table 2). In addition, no significantdifferences in the adsorption or penetration rates of theseviruses into host cells (Table 2) or their ability to agglutinateRBCs (Table 3) were observed.
Phenotypes of E-390 (RGD) mutant viruses in vivo. As forthe hinge mutant viruses described above, the in vivo pheno-types of each of the RGD mutant viruses (CDV-1-51v5, -v6,and -v7) were compared and contrasted to that of CDV-1-51.The results are shown in Table 4. HD50 values ranged between1 and 18 PFU by the i.c. route, resulting in 100% mortality anda mean time to death between 4.3 and 5.5 days. Each of theE-390 mutant viruses was therefore deemed to be of high NVaccording to the criteria outlined by McMinn et al. (HD50 ofless than 20 PFU) (46).
With the exception of CDV-1-51v6 (D3E), the NI of eachof the RGD mutant viruses was found to be markedly differentfrom that of CDV-1-51. The highly neuroinvasive CDV-1-51v6(D3E) had an i.p. HD50 of 2 PFU, a mortality of 100%, anda mean (6 standard deviation) time to death of 6.7 6 0.8 days,
TABLE 4. Phenotypes of hinge and RGD mutant viruses in vivo
Virus MutationNV parametersa NI parametersb
HD50 % Mortality Days (mean 6 SD) to death NVc HD50 % Mortality Days (mean 6 SD) to death NId
CDV-1-51 None 1 100 4.6 6 0.5 H 5.0 100 7.2 6 0.9 HCDV-1-51v1 E-277 (S3I) 4 100 5.5 6 0.5 H .104.5 0 NAe LCDV-1-51v2 E-277 (S3N) 3 100 4.4 6 0.5 H 4 90 7.3 6 1.3 HCDV-1-51v3 E-277 (S3V) 2 100 4.8 6 0.8 H 620 40 8.5 6 1.3 ICDV-1-51v4 E-277 (S3P) 5 100 4.9 6 0.3 H 580 50 10.2 6 3.6 ICDV-1-51v5 E-390 (D3N) 18 100 4.9 6 0.6 H .104.5 0 NA LCDV-1-51v6 E-390 (D3E) 1 100 4.3 6 0.5 H 2 100 6.7 6 0.8 HCDV-1-51v7 E-390 (D3Y) 10 100 5.5 6 0.5 H .104.5 0 NA L
a NV was determined by i.c. inoculation of virus.b NI was determined by i.p. inoculation of virus.c NV was determined according to the criteria described by McMinn et al. (46). H, high (,20 PFU) level of virus.d NI was determined according to the criteria described by McMinn et al. (46). H, high (, PFU); I, intermediate (,103 PFU); L, low (.103 PFU).e NA, not applicable.
similar to the profile observed for CDV-1-51 (i.p. HD50 of 5PFU; mortality of 100%; mean time to death of 7.2 6 0.9days). In contrast, both CDV-1-51v5 (D3N) and CDV-1-51v7(D3Y) were found to be of low NI, with i.p. HD50 values ofgreater than 104.5 PFU (no observed mortality at the lowestdilution of virus tested).
Analysis of the growth kinetics of RGD mutant viruses ininfected mouse brain showed a slightly different pattern fromthat observed in vitro. As shown in Fig. 2b, the titer of CDV-1-51v6 (D3E) had already reached a peak of 109.4 PFU/g byday 3 p.i., approximately eightfold higher than that observedfor CDV-1-51 (108.5 PFU/g) at the same time point. In con-trast, the titers of CDV-1-51v5 (D3N) and CDV-1-51 (D3Y)lagged behind that of CDV-1-51, reaching 107.7 and 106.1
PFU/g, respectively. On completion of the assay (day 5 p.i.),titers for all three of the RGD mutant viruses were two- tofourfold lower than that of CDV-1-51 (109.7 PFU/g), rangingfrom 109.1 to 109.5 PFU/g.
Inhibition of virus binding by soluble heparin. Previouswork on DEN-1, DEN-2, and MVE had highlighted a potentialrole for glycosaminoglycans (GAGs) in host cell entry, basedon their ability to inhibit virus binding (13, 27, 30, 39). Toascertain whether clone-derived MVE virus or attenuated vari-ants with mutations at E-277 or E-390 were similarly suscep-tible to inhibition by GAGs, mutant viruses were preincubatedwith soluble heparin (200 mg/ml) for 1 h at 4°C prior to theirinoculation onto Vero cells for a further 1 h at the sametemperature. The inoculum was then removed, and virus titerswere determined by plaque assay. As shown in Fig. 3, inhibi-tion of virus infectivity was approximately 40% for CDV-1-51and ranged between 34 and 46% for the E-277 mutant viruses.Similar inhibition was observed for the E-390 mutant viruses,ranging between 38 and 50%. Given that data generated forDEN-2 (27, 30) had shown a much higher inhibition of virusinfectivity (greater than 75%) at the same heparin dose, theseresults suggested that MVE was only moderately inhibitedby heparin. Furthermore, the introduced mutations at E-277and E-390 appeared to have little to no effect on the heparinbinding ability of any of the viruses.
Stability of introduced mutations during in vitro passage.Due to selective pressures conferred by an attenuating muta-tion, it is possible that the amino acid sequence of a mutantvirus may revert to that of wild-type virus. Alternatively, sec-ond-site mutations may arise, serving in some way to stabilizeor compensate for the attenuating mutation. To determine ifthe attenuation of viruses with mutations at E-277 or E-390resulted in a selective pressure for reversion (or the appear-ance of second-site mutations), the stability of each of themutants produced in this study was determined in vitro byexamining the nucleotide sequence of the E protein at differentpassage levels.
Reverse transcriptase PCR-generated cDNA of each viruswas sequenced after one passage (P1) in C6/36 cells and againafter two additional passages in the same cell line (P3). In allFIG. 2. Growth kinetics of hinge (A) and RGD (B) mutant viruses
in infected mouse brain. Mice were inoculated i.c. with 103 PFU ofvirus, and brains were collected at the times indicated. Viral titers weredetermined by plaque assay of 10% homogenates on Vero cells. Allassays were performed in duplicate.
FIG. 3. Inhibition of virus binding by soluble heparin. Rates ofinhibition were calculated using the following formula: (a 2 b)/a,where a is the number of plaques on cells incubated with untreatedvirus and b is the number of plaques on cells incubated with heparin-treated virus. CDV-1-51 is shown in black, E-277 mutant viruses are inwhite, and E-390 mutant viruses are in gray. Average results from twoseparate experiments are shown.
four of the hinge mutant viruses, the sequence of the E proteinwas unchanged. Similarly, in two of the three RGD mutants[CDV-1-51v5 (D3N) and CDV-1-51v6 (D3E)], the E proteinsequence was also unaffected. Interestingly, the E gene of CDV-1-51v7 (D3Y) contained a single point mutation at E-497,resulting in a Thr3Ile (T3I) substitution. This mutation wasfound in both the P1 and P3 viruses and must therefore havearisen prior to or during initial amplification of the virus inBHK-21 cells after transfection.
During titration of E-277 and E-390 virus stocks by plaqueassay on Vero cells, virus with a normal plaque phenotype wasobserved at a frequency of approximately 0.5% (1 plaque in200) in the P3 stock of CDV-1-51v5 (D3N). To ascertainwhether this or any other mutant virus stock contained rever-tants at low frequency, the P3 stock of each virus was passagedthree times more in either C6/36 cells (P4-P6) or Vero cells
(VP1-VP3). With the exception of CDV-1-51v5 (D3N), allviruses retained their respective plaque phenotypes during pas-sage in both cell lines. As shown in Fig. 4, however, the CDV-1-51v5 (D3N) mutant (which retained its small-plaque phe-notype throughout passage in C6/36 cells) reverted over thecourse of three passages in Vero cells to a normal plaquephenotype. This normal plaque phenotype constituted approx-imately 10% of the viral population at VP1, 80% of the viralpopulation at VP2, and 100% of the viral population at VP3.Nucleotide sequencing of the E gene of CDV-1-51v5 (D3N)at VP3 confirmed that the Asn (N) residue at E-390 hadreverted to the Asp (D) residue of the wild-type virus. Similarsequencing of the VP3 stocks of CDV-1-51v6 (D3E) andCDV-1-51v7 (D3Y) revealed no reversion of the mutatedE-390 residue to that of the wild type.
Reversion of the CDV-1-51v5 (D3N) mutant may be sug-gestive of an overall intolerance of MVE to mutations atE-390, evidenced by reversion of the CDV-1-51v5 (D3N)mutant in cell culture and the appearance of a second-sitemutation at E-497 of the CDV-1-51v7 (D3Y) mutant. Suchpressure was unlikely for the CDV-1-51v6 (D3E) mutant be-cause the amino acid change was conservative and because thereplicative ability of the virus was unaffected (see in vitro andin vivo phenotypes above).
DISCUSSION
Mutations in the hinge region. The effects of mutations inthe hinge region of the MVE E protein on mouse NI highlightthe importance of this region in viral virulence. Previous stud-ies with JE, YF, or JE/YF chimeras have shown that mutationsin the polar interface linking domains I and II cause significantattenuation. This interface, shown diagrammatically in Fig. 5,includes residues E-52 to E-54 (hereafter referred to as hinge1), E-129 to E-136 (hinge 2), E-191 to E-200 (hinge 3), andE-266 to E-284 (hinge 4; MVE numbering based on the TBEmodel described by Rey et al. [60]). A range of different mu-tations in these regions can cause a loss of either NI or NV. Forexample, a mutation in hinge 1 of JE [E-52 (Q3R/K)] causesa loss of NI (26), while a similar mutation in YF [E-52 (G3R)]contributes to a loss of NV (63). Mutations in hinges 2 and 4
FIG. 4. Plaque phenotype of CDV-1-51v5 (D3N) after multiplepassage in Vero cells. A third passage C6/36 stock was progressivelypassaged to generate Vero passage 1 (VP1) through passage 3 (VP3)stocks. (A) Original C6/36 P3 stock; (B) CDV-1-51v5 (D3N) VP1; (C)CDV-1-51v5 (D3N) VP2; (D) CDV-1-51v5 (D3N) VP3.
FIG. 5. The hinge region of the MVE E protein, based on the known three-dimensional structure of the TBE E protein (60). In MVE, the polarinterface linking domains I and II includes residues E-52 to E-54 (hinge 1 [green]), E-129 to E-136 (hinge 2 [orange]), E-191 to E-200 (hinge 3[red]), and E-266 to E-284 (hinge 4 [yellow]). Hinges 1 through 4 correspond to regions D0-a, e-E0, H0-f, and aB-I0, respectively, according to thenomenclature described by Rey et al. (60). Residue E-277, the residue selected for SDM, is highlighted in black.
also contribute to a loss of NI and/or NV in JE [E-270 (I3S)and E-138 (E3K)] and JE/YF chimeras [E-138 (E3K)] (2, 7,8, 11, 68).
In addition to those described above, mutations in hinge 4also cause a loss or reduction of NI in MVE [E-277 (S3I, V,or P); see reference 46 and work described in this study]. Inthese cases, the introduction of hydrophobic amino acid resi-dues into an otherwise hydrophilic region of the E proteinappears to disrupt the function of the protein at a stage fol-lowing adsorption and penetration of the host cell. Interest-ingly, the introduction of a strongly hydrophobic Ile residueinto hinge 4 abolishes both HA activity and NI; however, theintroduction of a Val residue (which is also strongly hydropho-bic but has a smaller side chain) or a Pro residue (weakly hy-drophobic) reduces but does not abolish HA activity and NI.Given that adsorption and penetration rates for all hinge mu-tant viruses were unperturbed and that inhibition of theseviruses by soluble heparin was similar to that observed forCDV-1-51, evidence from this and other studies suggests thatdisruption of E protein function may be caused not by alteredbinding but by structural instability of the E protein. Thisinstability appears to occur at a point after viral entry and isexacerbated by an increase in the hydrophobicity of the region,as evidenced by the phenotypes of the hinge mutant virusesdescribed above. CDV-1-51v1 (S3I), the most attenuated ofthe four hinge mutant viruses constructed, contained the mosthydrophobic of the substituted amino acids and displayed acomplete loss of NI in mice, coupled with reduced replicationkinetics in both Vero cells and infected mouse brain. Similarly,CDV-1-51v3 (S3V) and CDV-1-51v4 (S3P) contained sub-stituted amino acids of intermediate hydrophobicity, correlat-ing with intermediate levels of both NI and replication in vitroand in vivo. Concomitantly, the substitution of an Asn residueat E-277 [(CDV-1-51v2 (S3N)], which has little effect on thehydrophobicity of the region, correspondingly had no effect onthe phenotype of the virus (high NI and normal growth kineticsin vitro and in vivo).
E-277 forms part of a Ser-Ser-Ser-Thr (SSST) motif at po-sitions E-275 to E-278 of the MVE E protein, a strongly hy-drophilic b-turn (linking b-sheets k and l) which is relativelywell conserved throughout the JEV serocomplex of flavivi-ruses. Interestingly, both WNV and KUN contain a hydro-philic Asn residue at E-277, making the resultant SSNT motifidentical to that present in CDV-1-51v2 (S3N). All of theseviruses are of high NI and NV in mice. It therefore appearslikely that the functional basis for attenuation caused by mu-tations at E-277 is disruption of the b-turn, which on the basisof the three-dimensional structure of the protein appears toform an integral part of the hinge region.
Despite the inferences drawn above, the specific effect ofmutations at E-277 is difficult to ascertain. The inability ofCDV-1-51v1 (S3I) to hemagglutinate RBCs at low pH andthe observed reduction of this ability in both CDV-1-51v3 (S3V) and CDV-1-51v4 (S3P) suggests that a defect in fusionactivity suggested by McMinn et al. (47) is responsible for theobserved phenotypes. In the aforementioned study, BHv1[E-277 (S3I)] was also observed to be less efficient in fusion-from-within assays, lending further support for reduced fusionactivity in viruses with hydrophobic amino acid substitutions atE-277.
With the three-dimensional structure of the TBE E proteinnow known (60), it is possible to use this structure as a basis formodeling similar proteins from related flaviviruses, such asMVE. Threading of the MVE E protein sequence onto theknown structure of the TBE E protein and subsequent energyminimization of this basic model shows a very high degree ofstructural similarity between the two proteins. This structuralhomology is likely to be paralleled by a functional homologywhich includes a substantial rearrangement of the protein dur-ing its transition from dimer to trimer at low pH. In the mech-anism proposed by Rey et al. (60), mobility in the hinge regionis likely to project the conserved hydrophobic fusion peptideupwards for participation in fusion with the endosomal mem-brane, a mechanism analogous to that proposed for membersof other virus families, including paramyxoviruses (3), ortho-myxoviruses (5), and filoviruses (71).
Similarities between the model proposed by Rey et al. (60)for TBE and that observed for other viruses are evident. Viralfusion proteins appear to undergo significant structural rear-rangement at low pH, including a shift from dimer to trimer. Inaddition, mutations in regions which are reorganized duringthe dimer-to-trimer transition can increase the optimal pH forfusion in both viruses, reflecting a disruption of normal fusionactivity. For example, substitutions at positions 55 or 71 of theHA2 subunit of the influenza A virus HA protein are sufficientto shift the optimal pH for fusion 0.6 point up from 5.1 to 5.7(58, 66). Similarly, mutations at E-153 of DEN-2 have beenshown to elevate the pH threshold of fusion (22), as did mu-tations at E-277 of MVE (S3V and S3P) in this study. It ispossible that these substitutions disrupt the stability of theprotein, lowering the energy barrier between the metastablenative state and the more stable final conformation and allow-ing conformational change to occur at higher pH.
By combining the model for dimer-to-trimer transition out-lined by Allison et al. (1) with that of defective fusion in theendosome at low pH described by McMinn (45), hypotheticalmodels for the basis of attenuation caused by mutations in thehinge region can be postulated. For example, limited mobilityin the hinge region (E-277) may prevent correct presentationof the buried fusion peptide on the tip of each protein in thehomotrimer. Alternatively, undue stress may be placed on thereceptor binding site, causing premature release of the virionfrom the receptor just prior to fusion. Receptor binding is animportant part of the viral entry process and is likely to beessential for holding the viral and cellular membranes in closeproximity for initiation of the fusion reaction. In human para-influenza virus type 3, the binding of hemagglutinin-neura-minidase to its sialic acid receptor is essential for the fusionprocess (51). Furthermore, increased avidity of the receptor-ligand interaction correlates with increased fusogenicity, asevidenced by the introduction of site-directed mutations intothe sialic acid binding site of hemagglutinin-neuraminidase(52). In contrast, cleavage of the hemagglutinin-esterase pro-tein of influenza C virus from its receptor is a prerequisite forthe low pH-triggered conformational change required for fu-sion (67). Whether this is the case in flaviviruses or whetherongoing receptor binding in the endosome is required for fu-sion to occur remains to be determined. McMinn (45) andCorver et al. (16) have, however, shown that the presence of areceptor is not required for fusion of MVE or TBE with target
liposomes. Furthermore, the attenuated BHv1 strain of MVEwas able to fuse to liposomes with equal efficiency to wild-typevirus, suggesting that the defective fusion phenotype of BHv1may be influenced by a dynamic interaction between the Eprotein, the receptor, and the target membrane (45). Giventhat preexposure of TBE to acidic pH results in a loss ofinfectivity and fusogenicity (presumably due to an irreversiblestructural rearrangement of the E protein) (16), evidence sug-gests that the fusion active state of the E protein is transientand that the receptor, which is dispensable for the fusion pro-cess itself, may be required to orient the endosomal membranewith a transitional form of the E protein. If this were the case,premature release of the virion from its receptor would allowthe E protein to assume a final “fusion-inactive” conformationbefore it had a chance to interact with the endosomal mem-brane.
Mutations in the RGD motif. Mutations were introducedinto the RGD motif at position E-390 of MVE (on the lateralface of domain III of the E protein) to gain further insight intothe role of this region in receptor binding. Evidence for theinvolvement of the lateral face of domain III in receptor bind-ing is compelling but for the most part is based solely on theobserved structure of the region (60). Direct experimentalevidence is limited to the observation that partial-length C-terminal E protein constructs of DEN-2 (including residues281 to 423) have potent cell binding activity, while N-terminalconstructs do not (12). Whether the RGD motif in mosquito-borne flaviviruses plays an important role in binding to hostcell GAGs or integrins and whether these serve as receptorsfor virus entry is difficult to ascertain. The RGD motif is notpresent in any of the DEN viruses, and these viruses are ableto bind heparin with greater affinity than MVE (see reference39 and work presented in this study). Integrin binding is fur-ther complicated by the fact that motifs other than RGD havebeen implicated in the process. Motifs such as EILDV (29) andIDAPS (70) are involved in the binding of fibronectin to a4b1
integrins. Similarly, DGEA-containing peptides are capable ofinhibiting the binding of collagen to a2b1 integrins, suggestingthat this motif may also play a role (65).
Regardless of whether integrins are involved in flavivirusentry, it is clear that RGD-dependent binding to host cellintegrins is not an important determinant of virulence in fla-viviruses. While the D3N and D3Y substitutions at E-390 ofMVE caused a complete loss of NI in the mouse model, cou-pled with attenuated replication kinetics in vitro and in vivo,the D3E substitution had no effect on viral phenotype. Inte-grin binding should be severely affected by such a mutation, asevidenced by studies of FMDV where disruption of the RGDmotif by R3K and/or D3E substitutions has been shown todisrupt RGD-dependent binding to host cell integrins (44).The presence of an RGE motif in WNV (72) and KUN (15),both of which are highly virulent in mice, plus the observationthat all of the E-390 mutants derived in this study were able toadsorb and penetrate host cells with equal efficiency to wild-type virus, suggests that the RGD motif is not essential forvirus binding. This does not rule out integrin binding as ameans of host cell entry by flaviviruses; rather, it suggests thatRGD-dependent binding to host cell integrins is not a majorpathway for flavivirus entry.
In contrast to the hinge region of the E protein, the lateral
face of domain III is strongly hydrophilic. As such, it is possi-ble that the introduction of a large hydrophobic Tyr residue[CDV-1-51v7 (D3Y)] may destabilize the FG loop (nomen-clature of Rey et al. [60]). Alternatively, the replacement of anegatively charged Asp residue with an uncharged Tyr or Asnresidue could affect the formation of a functionally importantsalt bridge. Formation of such a bridge has recently beenshown to be an important determinant of virulence in TBE.Based on the three-dimensional structure of the TBE E pro-tein, an Asp residue at E-308 (Asp-308) is believed to form asalt bridge with a Lys residue at E-311 (60). Replacement ofAsp-308 with a Lys residue resulted in a virus which was highlyunstable, and passage of this virus in baby mouse brain result-ed in spontaneous reversion of the replacement Lys to a Gluresidue (42). Interestingly, reversion to a negatively chargedGlu residue required only a single nucleotide change, whilereversion to the wild-type Asp would have required two suchchanges. It therefore seems evident that the virus took theshortest route possible to restore the salt bridge between res-idues E-308 and E-311. Confirmation of whether a salt bridgeinvolving E-390 (in MVE) exists will require elucidation of thethree-dimensional structure of the MVE E protein; however,at least three positively charged His residues are in close prox-imity to E-390: His-395, His-396, and His-398. Furthermore,the Arg residue which forms part of the RGD motif is alsopositively charged. It is entirely possible that one of these fourresidues could participate in the formation of a functionallyimportant salt bridge in the MVE E protein. The instability ofthe Asn substitution in CDV-1-51v5 (D3N), as evidenced byits reversion to a wild-type Asp residue after passage in Verocells, is reminiscent of the reversion observed in the Mandl etal. study (42). Furthermore, if a functionally important saltbridge were playing a role in this region of the MVE E protein,a Glu residue at E-390 would be expected to functionallysubstitute for an Asp residue, as demonstrated for CDV-1-51v6 (D3E), which had an almost identical phenotype to thatof clone-derived wild-type virus.
Since mutations in the RGD motif of MVE do not appear toaffect the ability of virus to bind host cells, it seems likely thatthis motif is not functionally important in receptor binding.Disruption of the motif had little to no effect on the heparinbinding ability of the virus, as evidenced by heparin inhibitionassays which showed approximately 40% inhibition for all vi-ruses produced during the study. Incomplete inhibition by hep-arin suggests that receptors other than GAGs can serve asmediators of viral entry.
Variation in the amount of HS expressed on different celltypes, as well as the degree of sulfation of HS, may go someway towards explaining the different levels of inhibition byheparin seen in previous studies. Lee and Lobigs (39) haveshown that inhibition of MVE binding by soluble heparin islow in Vero cells and high in BHK-21 cells. In addition, Chenet al. (13) have shown that highly sulfated HS is capable ofsignificantly inhibiting the binding of DEN-2, while low-sulfateHS is not. Similar work on respiratory syncytial virus has shownthat the interaction of virus with cell surface GAGs is notbased on a simple charge interaction but rather a specific GAGstructural configuration including N-sulfation and iduronicacid (25). Thus, binding of flaviviruses to host cells is likely tobe a complicated process involving multiple receptors with
various levels of affinity. The use of such receptors may dependon their relative availability on the host cell, as well as whetherthese receptors have been posttranslationally modified in a wayamenable to virus binding.
Aside from receptor binding, it is possible that mutations atE-390 could affect virulence by disrupting the conformation ofthe E protein in much the same way as that described for thehinge region. Recent work by van der Most et al. (69), involv-ing SDM of the RGD motif of YF, has shown that virusescontaining Arg3Thr, Gly3Ala, or Asp3Glu/Ser substitu-tions at E-390 are able to bind to and infect both SW-13 andC6/36 cells. In addition, RGD-containing peptides did not in-hibit infection of primary chicken embryo fibroblasts by YF-17D (Preugschat and Strauss, unpublished results cited by vander Most et al. [69]). In the van der Most et al. study (69), theexplanation for the observed phenotypes was instability in theE protein. Intracellular levels of the E protein were consider-ably lower in viruses with RGD motif mutations; however, thisinstability could be overcome by incubation of infected cell cul-tures at a suboptimal temperature (30°C) (69). This promptedthe authors to suggest that the nature of attenuating mutationsin the FG loop of flaviviruses may be to cause incorrect foldingof the protein at a posttranslational stage. Incorrectly folded Eproteins may be defective in their ability to interact with prMor another chaperone protein, resulting in their retention inthe endoplasmic reticulum and eventual degradation. Suchretention has been observed in cells expressing misfolded HAof influenza A virus (19). The suggestion that attenuation offlaviviruses caused by mutations in the RGD motif are causednot by altered receptor binding but by posttranslational insta-bility of the E protein is an interesting one and warrants fur-ther investigation. It may be that the attenuation of MVEmutants with substitutions at E-390 has the same functionalbasis as that described for similar mutants of YF.
REFERENCES
1. Allison, S. L., J. Schalich, K. Stiasny, C. W. Mandl, C. Kunz, and F. X.Heinz. 1995. Oligomeric rearrangement of tick-borne encephalitis virus en-velope proteins induced by an acidic pH. J. Virol. 69:695–700.
2. Arroyo, J., F. Guirakhoo, S. Fenner, Z. X. Zhang, T. P. Monath, and T. J.Chambers. 2001. Molecular basis for attenuation of neurovirulence of ayellow fever virus/Japanese encephalitis virus chimera vaccine (ChimeriVax-JE). J. Virol. 75:934–942.
3. Baker, K. A., R. E. Dutch, R. A. Lamb, and T. S. Jardetzky. 1999. Structuralbasis for paramyxovirus-mediated membrane fusion. Mol. Cell 3:309–319.
4. Bray, M., R. Men, I. Tokimatsu, and C. J. Lai. 1998. Genetic determinantsresponsible for acquisition of dengue type 2 virus mouse neurovirulence.J. Virol. 72:1647–1651.
5. Bullough, P. A., F. M. Hughson, J. J. Skehel, and D. C. Wiley. 1994. Structureof influenza haemagglutinin at the pH of membrane fusion. Nature 371:37–43.
6. Cahour, A., A. Pletnev, M. Vazielle-Falcoz, L. Rosen, and C. J. Lai. 1995.Growth-restricted dengue virus mutants containing deletions in the 59 non-coding region of the RNA genome. Virology 207:68–76.
7. Cecilia, D., and E. A. Gould. 1991. Nucleotide changes responsible for loss ofneuroinvasiveness in Japanese encephalitis virus neutralization-resistant mu-tants. Virology 181:70–77.
8. Chambers, T. J., A. Nestorowicz, P. W. Mason, and C. M. Rice. 1999. Yellowfever Japanese encephalitis chimeric viruses: construction and biologicalproperties. J. Virol. 73:3095–3101.
9. Chambers, T. J., A. Nestorowicz, and C. M. Rice. 1995. Mutagenesis of theyellow fever virus NS2B/3 cleavage site: determinants of cleavage site spec-ificity and effects on polyprotein processing and viral replication. J. Virol.69:1600–1605.
10. Chambers, T. J., R. C. Weir, A. Grakoui, D. W. McCourt, J. F. Bazan, R. J.Fletterick, and C. M. Rice. 1990. Evidence that the N-terminal domain ofnonstructural protein NS3 from yellow fever virus is a serine protease re-
sponsible for site-specific cleavages in the viral polyprotein. Proc. Natl. Acad.Sci. USA 87:8898–8902.
11. Chen, L. K., Y. L. Lin, C. L. Liao, C. G. Lin, Y. L. Huang, C. T. Yeh, S. C.Lai, J. T. Jan, and C. Chin. 1996. Generation and characterization of organ-tropism mutants of Japanese encephalitis virus in vivo and in vitro. Virology223:79–88.
12. Chen, Y., T. Maguire, and R. M. Marks. 1996. Demonstration of binding ofdengue virus envelope protein to target cells. J. Virol. 70:8765–8772.
13. Chen, Y. P., T. Maguire, R. E. Hileman, J. R. Fromm, J. D. Esko, R. J.Linhardt, and R. M. Marks. 1997. Dengue virus infectivity depends onenvelope protein binding to target cell heparan sulfate. Nat. Med. 3:866–871.
14. Clark, D. H., and J. Casals. 1958. Techniques for hemagglutination andhemagglutination-inhibition with arthropod-borne viruses. Am. J. Trop.Med. Hyg. 7:561–573.
15. Coia, G., M. D. Parker, G. Speight, M. E. Byrne, and E. G. Westaway. 1988.Nucleotide and complete amino acid sequences of Kunjin virus: definitivegene order and characteristics of the virus-specified proteins. J. Gen. Virol.69:1–21.
16. Corver, J., A. Ortiz, S. L. Allison, J. Schalich, F. X. Heinz, and J. Wilschut.2000. Membrane fusion activity of tick-borne encephalitis virus and recom-binant subviral particles in a liposomal model system. Virology 269:37–46.
17. Coulson, B. S., S. L. Londrigan, and D. J. Lee. 1997. Rotavirus containsintegrin ligand sequences and a disintegrin-like domain that are implicatedin virus entry into cells. Proc. Natl. Acad. Sci. USA 94:5389–5394.
18. Dmitriev, I., V. Krasnykh, C. R. Miller, M. Wang, E. Kashentseva, G.Mikheeva, N. Belousova, and D. T. Curiel. 1998. An adenovirus vector withgenetically modified fibers demonstrates expanded tropism via utilization ofa coxsackievirus and adenovirus receptor-independent cell entry mechanism.J. Virol. 72:9706–9713.
19. Gething, M. J., K. McCammon, and J. Sambrook. 1986. Expression ofwild-type and mutant forms of influenza hemagglutinin: the role of folding inintracellular transport. Cell 46:939–950.
20. Gualano, R. C., M. J. Pryor, M. R. Cauchi, P. J. Wright, and A. D. Davidson.1998. Identification of a major determinant of mouse neurovirulence ofdengue virus type 2 using stably cloned genomic-length cDNA. J. Gen. Virol.79:437–446.
21. Guex, N., and M. C. Peitsch. 1997. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis18:2714–2723.
22. Guirakhoo, F., A. R. Hunt, J. G. Lewis, and J. T. Roehrig. 1993. Selectionand partial characterization of dengue 2 virus mutants that induce fusion atelevated pH. Virology 194:219–223.
23. Guirakhoo, F., R. Weltzin, T. J. Chambers, Z. X. Zhang, K. Soike, M.Ratterree, J. Arroyo, K. Georgakopoulos, J. Catalan, and T. P. Monath.2000. Recombinant chimeric yellow fever-dengue type 2 virus is immuno-genic and protective in nonhuman primates. J. Virol. 74:5477–5485.
24. Guirakhoo, F., Z. X. Zhang, T. J. Chambers, S. Delagrave, J. Arroyo, A. D. T.Barrett, and T. P. Monath. 1999. Immunogenicity, genetic stability, andprotective efficacy of a recombinant, chimeric yellow fever-Japanese enceph-alitis virus (ChimeriVax-JE) as a live, attenuated vaccine candidate againstJapanese encephalitis. Virology 257:363–372.
25. Hallak, L. K., D. Spillmann, P. L. Collins, and M. E. Peeples. 2000. Glycos-aminoglycan sulfation requirements for respiratory syncytial virus infection.J. Virol. 74:10508–10513.
26. Hasegawa, H., M. Yoshida, T. Shiosaka, S. Fujita, and Y. Kobayashi. 1992.Mutations in the envelope protein of Japanese encephalitis virus affect entryinto cultured cells and virulence in mice. Virology 191:158–165.
27. Hilgard, P., and R. Stockert. 2000. Heparan sulfate proteoglycans initiatedengue virus infection of hepatocytes. Hepatology 32:1069–1077.
28. Hiramatsu, K., M. Tadano, R. Men, and C. J. Lai. 1996. Mutational analysisof a neutralization epitope on the dengue type 2 virus (DEN2) envelopeprotein: monoclonal antibody resistant DEN2/DEN4 chimeras exhibit re-duced mouse neurovirulence. Virology 224:437–445.
29. Humphries, M. J., S. K. Akiyama, A. Komoriya, K. Olden, and K. M.Yamada. 1988. Neurite extension of chicken peripheral nervous system neu-rons on fibronectin: relative importance of specific adhesion sites in thecentral cell-binding domain and the alternatively spliced type III connectingsegment. J. Cell Biol. 106:1289–1297.
30. Hung, S. L., P. L. Lee, H. W. Chen, L. K. Chen, C. L. Kao, and C. C. King.1999. Analysis of the steps involved in dengue virus entry into host cells.Virology 257:156–167.
31. Hurrelbrink, R. J., A. Nestorowicz, and P. C. McMinn. 1999. Characteriza-tion of infectious Murray Valley encephalitis virus derived from a stablycloned genome-length cDNA. J. Gen. Virol. 80:3115–3125.
32. Jackson, T., A. Sharma, R. Abughazaleh, W. E. Blakemore, F. M. Ellard,D. L. Simmons, J. W. I. Newman, D. I. Stuart, and A. M. Q. King. 1997.Arginine-glycine aspartic acid-specific binding by foot-and-mouth diseaseviruses to the purified integrin alpha-v-beta-3 in vitro. J. Virol. 71:8357–8361.
33. Kawano, H., V. Rostapshov, L. Rosen, and C. J. Lai. 1993. Genetic deter-minants of dengue type 4 virus neurovirulence for mice. J. Virol. 67:6567–6575.
34. Khromykh, A. A., M. T. Kenney, and E. G. Westaway. 1998. trans-Comple-
mentation of flavivirus RNA polymerase gene NS5 by using Kunjin virusreplicon-expressing BHK cells. J. Virol. 72:7270–7279.
35. Khromykh, A. A., P. L. Sedlak, K. J. Guyatt, R. A. Hall, and E. G. Westaway.1999. Efficient trans-complementation of the flavivirus Kunjin NS5 proteinbut not of the NS1 protein requires its coexpression with other componentsof the viral replicase. J. Virol. 73:10272–10280.
36. Khromykh, A. A., P. L. Sedlak, and E. G. Westaway. 1999. trans-Comple-mentation analysis of the flavivirus Kunjin NS5 gene reveals an essential rolefor translation of its N-terminal half in RNA replication. J. Virol. 73:9247–9255.
37. Khromykh, A. A., and E. G. Westaway. 1994. Completion of Kunjin virusRNA sequence and recovery of an infectious RNA transcribed from stablycloned full-length cDNA. J. Virol. 68:4580–4588.
38. Lai, C. J., M. Bray, R. Men, A. Cahour, W. Chen, H. Kawano, M. Tadano, K.Hiramatsu, I. Tokimatsu, A. Pletnev, S. Arakai, G. Shameem, and M.Rinaudo. 1998. Evaluation of molecular strategies to develop a live denguevaccine. Clin. Diagn. Virol. 10:173–179.
39. Lee, E., and M. Lobigs. 2000. Substitutions at the putative receptor-bindingsite of an encephalitic flavivirus alter virulence and host cell tropism andreveal a role for glycosaminoglycans in entry. J. Virol. 74:8867–8875.
40. Lobigs, M., R. Usha, A. Nestorowicz, I. D. Marshall, R. C. Weir, and L.Dalgarno. 1990. Host cell selection of Murray Valley encephalitis virusvariants altered at an RGD sequence in the envelope protein and in mousevirulence. Virology 176:587–595.
41. Mackenzie, J. S., and A. K. Broom. 1995. Australian X disease, MurrayValley encephalitis and the French connection. Vet. Microbiol. 46:79–90.
42. Mandl, C. W., S. L. Allison, H. Holzmann, T. Meixner, and F. X. Heinz. 2000.Attenuation of tick-borne encephalitis virus by structure-based site-specificmutagenesis of a putative flavivirus receptor binding site. J. Virol. 74:9601–9609.
43. Mandl, C. W., H. Holzmann, T. Meixner, S. Rauscher, P. F. Stadler, S. L.Allison, and F. X. Heinz. 1998. Spontaneous and engineered deletions in the39 noncoding region of tick-borne encephalitis virus—construction of highlyattenuated mutants of a flavivirus. J. Virol. 72:2132–2140.
44. Mason, P. W., E. Rieder, and B. Baxt. 1994. RGD sequence of foot-and-mouth disease virus is essential for infecting cells via the natural receptor butcan be bypassed by an antibody-dependent enhancement pathway. Proc.Natl. Acad. Sci. USA 91:1932–1936.
45. McMinn, P. C. 1994. Studies on the molecular pathogenesis of MurrayValley encephalitis infection of mice. Ph.D. thesis. Australian National Uni-versity, Canberra.
46. McMinn, P. C., E. Lee, S. Hartley, J. T. Roehrig, L. Dalgarno, and R. C.Weir. 1995. Murray valley encephalitis virus envelope protein antigenic vari-ants with altered hemagglutination properties and reduced neuroinvasive-ness in mice. Virology 211:10–20.
47. McMinn, P. C., R. C. Weir, and L. Dalgarno. 1996. A mouse-attenuatedenvelope protein variant of Murray Valley encephalitis virus with alteredfusion activity. J. Gen. Virol. 77:2085–2088.
48. Men, R., M. Bray, D. Clark, R. M. Chanock, and C. J. Lai. 1996. Denguetype 4 virus mutants containing deletions in the 39 noncoding region of theRNA genome: analysis of growth restriction in cell culture and alteredviremia pattern and immunogenicity in rhesus monkeys. J. Virol. 70:3930–3937.
49. Monath, T. P., I. Levenbook, K. Soike, Z. X. Zhang, M. Ratterree, K. Draper,A. D. Barrett, R. Nichols, R. Weltzin, J. Arroyo, and F. Guirakhoo. 2000.Chimeric yellow fever virus 17D-Japanese encephalitis virus vaccine: dose-response effectiveness and extended safety testing in rhesus monkeys. J. Vi-rol. 74:1742–1751.
50. Monath, T. P., K. Soike, I. Levenbook, Z. X. Zhang, J. Arroyo, S. Delagrave,G. Myers, A. D. T. Barrett, R. E. Shope, M. Ratterree, T. J. Chambers, andF. Guirakhoo. 1999. Recombinant, chimaeric live, attenuated vaccine (Chi-meriVax (TM)) incorporating the envelope genes of Japanese encephalitis(SA14-14-2) virus and the capsid and nonstructural genes of yellow fever(17D) virus is safe, immunogenic and protective in nonhuman primates.Vaccine 17:1869–1882.
51. Moscona, A., and R. W. Peluso. 1991. Fusion properties of cells persistentlyinfected with human parainfluenza virus type 3: participation of hemagglu-tinin-neuraminidase in membrane fusion. J. Virol. 65:2773–2777.
52. Moscona, A., and R. W. Peluso. 1993. Relative affinity of the human para-influenza virus type 3 hemagglutinin-neuraminidase for sialic acid correlateswith virus-induced fusion activity. J. Virol. 67:6463–6468.
53. Muylaert, I. R., T. J. Chambers, R. Galler, and C. M. Rice. 1996. Mutagen-esis of the N-linked glycosylation sites of the yellow fever virus NS1 protein:effects on virus replication and mouse neurovirulence. Virology 222:159–168.
54. Nestorowicz, A., T. J. Chambers, and C. M. Rice. 1994. Mutagenesis of the
yellow fever virus NS2A/2B cleavage site: effects on proteolytic processing,viral replication, and evidence for alternative processing of the NS2A pro-tein. Virology 199:114–123.
55. Pletnev, A. G., M. Bray, and C. J. Lai. 1993. Chimeric tick-borne encephalitisand dengue type 4 viruses: effects of mutations on neurovirulence in mice.J. Virol. 67:4956–4963.
56. Porterfield, J. S., and C. E. Rowe. 1960. Haemagglutination with arthropod-borne viruses and its inhibition with certain phospholipids. Virology 188:160–167.
57. Pryor, M. J., R. C. Gualano, B. Lin, A. D. Davidson, and P. J. Wright. 1998.Growth restriction of dengue virus type 2 by site-specific mutagenesis ofvirus-encoded glycoproteins. J. Gen. Virol 79:2631–2639.
58. Qiao, H., S. L. Pelletier, L. Hoffman, J. Hacker, R. T. Armstrong, and J. M.White. 1998. Specific single or double proline substitutions in the “spring-loaded” coiled-coil region of the influenza hemagglutinin impair or abolishmembrane fusion activity. J. Cell Biol. 141:1335–1347.
59. Reed, L. J., and H. Muench. 1938. A simple method of estimating fiftypercent endpoints. Am. J. Hyg. 27:493–497.
60. Rey, F. A., F. X. Heinz, C. Mandl, C. Kunz, and S. C. Harrison. 1995. Theenvelope glycoprotein from tick-borne encephalitis virus at 2 Å resolution.Nature 375:291–298.
61. Rice, C. M. 1996. Flaviviridae: the viruses and their replication, p. 931–959.In B. N. Fields, D. M. Knipe, and P. M. Howley (ed.), Fields virology, 3rd ed.Lippincott-Raven, Philadelphia, Pa.
62. Sayle, R. A., and E. J. Milner-White. 1995. RASMOL: biomolecular graphicsfor all. Trends Biochem. Sci. 20:374.
63. Schlesinger, J. J., S. Chapman, A. Nestorowicz, C. M. Rice, T. E. Ginocchio,and T. J. Chambers. 1996. Replication of yellow fever virus in the mousecentral nervous system: comparison of neuroadapted and non-neuroadaptedvirus and partial sequence analysis of the neuroadapted strain. J. Gen. Virol.77:1277–1285.
64. Sharma, A., Z. Rao, E. Fry, T. Booth, E. Y. Jones, D. J. Rowlands, D. L.Simmons, and D. I. Stuart. 1997. Specific interactions between human inte-grin alpha v beta 3 and chimeric hepatitis B virus core particles bearing thereceptor-binding epitope of foot-and-mouth disease virus. Virology 239:150–157.
65. Staatz, W. D., K. F. Fok, M. M. Zutter, S. P. Adams, B. A. Rodriguez, andS. A. Santoro. 1991. Identification of a tetrapeptide recognition sequence forthe alpha 2 beta 1 integrin in collagen. J. Biol. Chem. 266:7363–7367.
66. Steinhauer, D. A., J. Martin, Y. P. Lin, S. A. Wharton, M. B. Oldstone, J. J.Skehel, and D. C. Wiley. 1996. Studies using double mutants of the confor-mational transitions in influenza hemagglutinin required for its membranefusion activity. Proc. Natl. Acad. Sci. USA 93:12873–12878.
67. Strobl, B., and R. Vlasak. 1993. The receptor-destroying enzyme of influenzaC virus is required for entry into target cells. Virology 192:679–682.
68. Sumiyoshi, H., G. H. Tignor, and R. E. Shope. 1995. Characterization of ahighly attenuated Japanese encephalitis virus generated from molecularlycloned cDNA. J. Infect. Dis. 171:1144–1151.
69. van der Most, R. G., J. Corver, and J. H. Strauss. 1999. Mutagenesis of theRGD motif in the yellow fever virus 17D envelope protein. Virology 265:83–95.
70. Wayner, E. A., A. Garcia-Pardo, M. J. Humphries, J. A. McDonald, andW. G. Carter. 1989. Identification and characterization of the T lymphocyteadhesion receptor for an alternative cell attachment domain (CS-1) inplasma fibronectin. J. Cell Biol. 109:1321–1330.
71. Weissenhorn, W., A. Dessen, L. J. Calder, S. C. Harrison, J. J. Skehel, andD. C. Wiley. 1999. Structural basis for membrane fusion by enveloped vi-ruses. Mol. Membr. Biol. 16:3–9.
72. Wengler, G., E. Castle, U. Leidner, T. Nowak, and G. Wengler. 1985. Se-quence analysis of the membrane protein V3 of the flavivirus West Nile virusand of its gene. Virology 147:264–274.
73. Wickham, T. J., M. E. Carrion, and I. Kovesdi. 1995. Targeting of adenoviruspenton base to new receptors through replacement of its RGD motif withother receptor-specific peptide motifs. Gene Ther. 2:750–756.
74. Wickham, T. J., P. Mathias, D. A. Cheresh, and G. R. Nemerow. 1993.Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internaliza-tion but not virus attachment. Cell 73:309–319.
75. Wright, A. J., and R. J. Phillpotts. 1998. Humane endpoints are an objectivemeasure of morbidity in Venezuelan encephalomyelitis virus infection ofmice. Arch. Virol. 143:1155–1162.
76. Zimmermann, H., H. J. Eggers, and B. Nelsen-Salz. 1997. Cell attachmentand mouse virulence of echovirus 9 correlate with an RGD motif in thecapsid protein VP1. Virology 233:149–156.
