Variants of Venezuelan equine encephalitis virus that resist neutralization define a domain of the E2 glycoprotein

VIROLOGY

177,676-683

(1990)

Variants

of Venezuelan Equine Encephalitis Virus That Resist Neutralization Define a Domain of the E2 Glycoprotein

BARBARA

J. B. JOHNSON,’

Division

JOHN R. BRUBAKER,

JOHN T. ROEHRIG,

AND

of Vector-Borne Viral Diseases, Center for Infectious Diseases, Centers for Disease U.S. Department of Health and Human Services, P. 0. Box 2087, Fort Collins, Received

January

26, 1990; accepted

April

DENNIS W. TRENT

Control, Colorado

Public Health 80522

Service,

18, 1990

Stable neutralization (N) escape variants of Venezuelan equine encephalitis (VEE) virus were selected by anti-E2 glycoprotein monoclonal antibodies (MAbs) that neutralize viral infectivity, block viral hemagglutination, and passively protect mice. The nucleotide sequence of the El, E2, and E3 genes of four variants revealed a clustering of single mutations in a domain spanning E2-182 to E2-207. The conformation of this short linear sequence affects antigenicity in the N domain because reduction and alkylation of virus disrupted binding of some E2 neutralizing MAbs. Serologic evidence for interaction of E2 epitopes also was obtained. Mutations in the N domain of VEE virus did not alter the kinetics of binding to Vero cells. They did, in some cases, produce attenuation of virulence in mice. o 1990 Academic Press,

Inc.

activity is correlated with its distance from E2”, the hub of the critical N site (Roehrig and Mathews, 1985; Fig. 1). MAbs to E2”, E2’, E2g, and E2h neutralize viral infectivity, block viral hemagglutination, and passively protect mice against virulent VEE virus challenge (Mathews and Roehrig, 1982; Roehrig and Mathews, 1985). Four MAbs recognizing epitopes E2”, E2’, E2g, and E2h were used to select variants of the live, attenuated vaccine strain of VEE virus that escape N. The nucleotide sequences of the El, E2, and E3 genes of four stable variants were determined. From the deduced amino acid sequences of these viruses, four amino acids involved in the N of VEE virus were mapped to a single 26-residue-long domain of the E2 protein. The kinetics of binding these variants to cultured cells and the virulence of the viruses in mice are described. This domain of VEE virus spans essentially the same region of the E2 glycoprotein as that defined by N escape variants of Sindbis (SIN) virus (Strauss et al., 1987; Davis et a/., 1987) and is near, but distinct from, a major antigenie site involved in N of Ross River (RR) virus (Vrati et al., 1988).

INTRODUCTION Venezuelan equine encephalitis (VEE) virus is an arthropod-borne member of the family Togaviridae, genus Alphavirus. Alphaviruses contain a positive-sense, single-stranded RNA genome enclosed within an icosahedral nucleocapsid. The nucleocapsid is surrounded by a lipid envelope, derived from the plasma membrane of the infected cell, in which glycoprotein El and E2 heterodimers are embedded as trimers (Garoff, 1982; Fuller, 1987). The antigenic structure of the El and E2 surface proteins of VEE virus has been extensively investigated. The E2 glycoprotein is the more potent in eliciting virus-specific neutralizing monoclonal antibodies (MAbs) (Roehrig and Mathews, 1985; Roehrig eta/., 1988). The El glycoprotein elicits groupspecific MAbs that cross-react with other alphaviruses; one anti-El neutralizing MAb has been produced, but it has a low neutralization (N) titer (Roehrig et al., 1982). Monospecific polyclonal antisera raised against purified El and E2 proteins implicate the E2 glycoprotein in hemagglutination; both proteins induce hemagglutination-inhibiting (HI) MAbs (France et al., 1979; Roehrig et al., 1982). The spatial arrangement of epitopes on the El and E2 proteins (four and eight epitopes, respectively) has been mapped with MAbs by competitive binding assay (CBA) (Roehrig et al., 1982; Roehrig and Mathews, 1985). Six of the epitopes (five on E2 and one on El) cluster in CBA to form a major N domain (Fig. 1). The ability of an epitope to elicit a MAb with neutralizing ’ To whom 0042-6822190

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METHODS Virus and cultured

cells

The VEE virus used for selecting variants was the vaccine strain, TC-83. The passage history, cDNA cloning, and sequencing of this virus have been previously described (McKinney, 1972; Johnson et a/., 1986; Kinney et a/., 1989). Viruses were plaque-puri-

be addressed. 676

NEUTRALIZATION

DOMAIN

OF

VEE VIRUS

Serologic a 0 El

FIG. 1. Map of VEE virus glycoprotein spike. Squares are E2 domains; circles are El domains. MAb competition is indicated by overlap of domains. The shaded area represents the virus N site.

fied and grown in monolayers of Vero cells. Viruses were purified by polyethylene glycol precipitation of culture fluids and centrifugation in glycerol tartrate and sucrose gradients as described by Obijeski et a/. (1976). Antibodies The MAbs used in this study were elicited by viruses belonging to subtype 1AB of the VEE serologic complex (variants TC-83, Trinidad donkey (TRD), and PTF39; Young and Johnson, 1969). These MAbs define epitopes on the E2 surface glycoprotein and possess both N and HI activities (Roehrig er al., 1982; Roehrig and Mathews, 1985). Polyclonal antibody to VEE virus, strain TC-83, was hyperimmune mouse ascitic fluid (HIAF) prepared as described by Chappell et a/. (1974). Commercial alkaline phosphatase-conjugated goat anti-mouse immunoglobulin G (IgG) was from Jackson Laboratories.* Selection

of variant VEE viruses

Cloned TC-83 virus (100 PFU in Eagle’s minimal essential medium (MEM) containing 5% fetal bovine serum (FBS)) was incubated with individual MAb ascitic fluids (diluted 1: 100) for 1 hr at 37” and plaqued on Vero cell monolayers grown in 6-well plates. Well-isolated plaques resulting from N-resistant virus were cored and incubated overnight at 4” in 1 ml of MEM containing 5% FBS. These viruses were again incubated with their selecting MAb by mixing 0.5 ml of eluted virus with a 1: 10 dilution of ascitic fluid for 1 hr at 37” and then added to Vero cells (25-cm2 flasks) containing 10% MAb in cell culture medium. Following this second selection cycle, the resultant virus seed was passed once in Vero cells without MAb to test for mutant stability. Viruses that were genetically stable and resistant to N by selecting antibody were characterized in this study. * Use of trade names and commercial sources is for identification only and does not imply endorsement by the Public Health Service or by the U.S. Department of Health and Human Services.

677

assays

Plaque reduction neutralization assays were performed in Vero cells using 709/o N of 70-l 50 PFU as endpoints (Hunt and Calisher, 1979). The enzymelinked immunosorbent assay (ELISA) was carried out with alkaline phosphatase-conjugated goat antimouse IgG as described by Voller et al. (1976) and modified by Roehrig et al. (1980). HI tests were done at pH 5.9 with goose erthrocytes by the procedure of Clarke and Casals (1958). Gradient-purified virus antigen was standardized to 4-8 units/test. Assessment

of virulence

of variant viruses

The 50% median lethal doses of variant viruses administered intracerebrally (ICLDSO) or intraperitoneally (IPLD& were determined by the method of Reed and Muench (1938) in 2-day-old NIH Swiss mice. Six suckling mice were used for each viral dose in the titrations. Kinetics

of binding of variant viruses

to Vero cells

Vero cell monolayers were inoculated with VEE virus (50 PFU in 0.2 ml of MEM plus 10% FBS/35 mm well) and incubated at 37”. After intervals of 0, 5, 10, 15, 20, 30, 45, 60, 90, 120, and 150 min, viral inocula were removed, and the cell sheets were rinsed three times with 2 ml of PBS. Agar overlay medium was immediately added to the rinsed cells, and the cultures were incubated at 37“ in 5% CO, for 3 days. A second overlay containing neutral red was applied, and after an additional day, plaques were counted. In a single experiment, three cell monolayers were used for each time point. The mean time to adsorb 50% of the input plaque-forming units was calculated from three independent experiments for each virus. Sequencing

of viral RNAs

Genomic RNAs were extracted from gradient-purified virions suspended in TNE (0.01 h/l Tris-HCI, pH 7.5, 0.15 M NaCI, 0.001 M EDTA). Viral suspensions were digested with proteinase K (0.2 mg/ml, Boehringer-Mannheim) for 30 min at 37”, solubilized with 1% sodium dodecyl sulfate (SDS) for an additional 15 min at 37”, then extracted twice with TNE-saturated phenol/chloroform/isoamyl alcohol (25/24/l, v/v/v), and precipitated from ethyl alcohol. cDNA to the E2hvariant (Vl A3B-7) was prepared as previously described (Kinney et a/., 1986) and sequenced by the dideoxynucleotide chain termination method (Sanger et a/., 1977) using ol-[32P]dCTP and the synthetic DNA primers listed below. Genomic RNAs of the E2”, E2’, and E2g variants (V3B4C-4, VlA4D-1, and VlA3A-9, respectively) were sequenced directly by the Sanger method using (Y-

676

JOHNSON ET AL

[35S]dCTP as label (Biggin et al., 1983) in the reverse transcriptase-catalyzed extension of synthetic DNA primers (Johnson et a/., 1986). Sequence resolution was improved in some areas by incorporating 7-deazadGTP in the dideoxynucleotide mixtures (Mizusawa et a/., 1986) or by labeling primers directly with T-[~‘P]ATP (Maniatis et al., 1982). DNA primers 19 or 20 residues long were synthesized by an Applied Biosystems Model 380A DNA synthesizer’ and purified by preparative gel electrophoresis. The genes and 26 S RNA nucleotide positions in VEE TC-83 26 S RNA (Johnson et al., 1986) complementary to the 3’-nucleotide of the DNA primers were as follows: E2,1092; E2,1417; E2,1683; E2,1947; E2,2179; 6K,2412; El ,2595; El ,2788; El ,299l; El ,3209; El ,3418; El ,3615; and 3’-noncoding,3830.

Antigenic

analysis of alkylated

proteins

Purified TC-83 virus (300 pg, 1 PgIpl in 1 MTris-HCI, pH 9.0, 2% SDS) was sonicated to dissociate aggregates. To reduce proteins, 2-mercaptoethanol was added to 0.28 M and the mixture was incubated for 4 hr at 37”. A nonreduced control was prepared by incubating a similar viral mixture for 4 hr at 37” without 2mercaptoethanol. Reduced and nonreduced viral proteins were alkylated by adding iodoacetamide (16.5 mg) and incubating for 1 hr at 37”. Excess iodoacetamide and 2-mercaptoethanol were removed by dialysis in 50 mM Tris-HCI, pH 7.4, 0.1 o/oSDS, followed by dialysis in 20 mM Tris-HCI, pH 7.4, 0.10/b SDS, 109/o sucrose. Proteins were analyzed by SDS-polyacrylamide gel electrophoresis on a 10% gel. Reactivity of purified MAbs was determined by ELISA using 1 pg of treated virus per well.

Computer

analysis

Surface sites in the N domain of the E2 protein were predicted using SURFACE PLOT 1988, version 1.20, developed for Synthetic Peptides, Inc., Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. SURFACE PLOT calculates a composite surface profile using a hydrophilicity scale derived from retention times of model synthetic peptides subjected to high-performance liquid chromatography (Parker eta/., 1986) surface accessibility parameters of Janin (1979) and B values for flexibility using the algorithm of Karplus and Schultz (1985). Hydrophilic, accessible, or mobile regions predicted by SURFACE PLOT correlate well with known antigenic sites for several proteins (Parker et al., 1986).

RESULTS Serologic characterization of N-resistant variants of VEE virus TC-83

antigenic

The four MAbs that we used to select N-resistant variants of VEE virus TC-83 define discrete epitopes on the E2 glycoprotein (E2”, E2’, E2g, and E2h; Fig. 1). Stable variant viruses were derived that escape N by each of their selecting MAbs (Table 1). All four variants also demonstrated reduced titers in ELISA and HI with their homologous antibodies. Each variant was neutralized by anti-VEE virus HIAF; however, N of the E2h variant was significantly suppressed (>8-fold) when compared to its TC-83 virus parent, suggesting that the E2h epitope is important in virus N in v&o. This reduction in sensitivity to N of the E2hvariant by HIAF was reflected in lower N with all four MAbs. Increased N and HI activity was seen with some antibody/virus pairs, even though no increase in binding activity was observed. The anti-E2’ and -E29 MAbs demonstrated reciprocal increases in both N and HI titers with its heterologous variant, as well as with the E2” variant. HI activity could be dissociated from N activity, as has been previously observed (Roehrig and Mathews, 1985). Only the variation at the E2h epitope lowered ELISA binding of heterologous MAbs (anti-E2C and -E2g).

Location of an antigenic VEE virus E2 protein

domain in the

The TC-83 virus variants were analyzed by sequencing the genes encoding the El, E2, E3, and 6K proteins and comparing these sequences with those of the parent virus (Johnson et a/., 1986). A single nucleotide change in the E2 gene was detected for each of the variant viruses. No mutations were detected in the El, E3, or 6K genes. The sequence of the 6K gene could not be read unambiguously due to several severe compressions of the sequence ladder. The pattern of these compressions, however, did not vary between the mutant viruses. The predicted consequence of each nucleotide change is a nonconservative amino acid substitution, as shown in Table 2. All four deduced amino acid changes cluster in a single region of the E2 protein spanning positions 182 to 207. Two of the predicted amino acid substitutions (Ser+Arg, E2-182; and Glu+Lys, E2-199) alter the charge of the E2 glycoprotein. One mutation (Gly+Val, E2-183) renders it substantially more hydrophobic. Another mutation (Ile+ Phe, E2-207) may result in perturbation of the surface topography of the E2 glycoprotein by insertion of a more bulky hydrophobic side chain. Each of the variant VEE viruses was examined by the method of Parker et a/. (1986) to calculate the effect of

NEUTRALIZATION

DOMAIN TABLE

CHARACTERIZATION

OF

679

VEE VIRUS

1

OF VEE VIRUS VARIANTS Viruses

Epitope

Antibodies

Assays

-

TC-83 HIAF

ELISA HI NT

+ 200 2800

E2”

3B4C-4

ELISA HI NT

>320,000 a-8.1 92 a800

ELISA HI NT

>320,000

z=320,000 2,048 3800

<10,000

32 100

ELISA HI NT

>320,000 1,024 200

z-320,000 >8,192 3800

>320,000 >8,192 21,600

ELISA HI NT

>320,000

E2’

lA4D-1

E2Q

1A3A-9

E2”

lA3B-7

TC-83

V3B4C-4

128 400

the mutations on the hydrophilicity, accessibility, and mobility of the E2 N domain. In composite surface profiles, TC-83 virus amino acids E2-182 and E2-183 received high surface scores; residues E2-199 and E2207 had intermediate values. Mutation at E2-183 from Gly to Val dramatically altered the surface profile, making this local region less likely to be on the surface of the virion. The predicted perturbations in surface structure due to the other mutations were far less marked (plots not shown). Conformational

stability

of epitopes

in the N domain

Putative binding sites for four N MAbs were localized to a single 26-amino acid domain of the E2 protein. To determine whether the E2”, E2’, E2g, and E2h epitopes are linear or are dependent on the conformation of the polypeptide, MAb reactivities with TC-83 proteins that had been denatured by reduction and alkylation were measured. Previously we defined nonconformational

TABLE

2

LOCATION OF ANTIGENIC SITES IN VEE VIRUS TC-83

Virus V3B4C4 VlA4D-1 V 1A3A-9 Vl A3B-7

Mutation AGC GGC GAA AUC

+ CGC -. GTC +AfL4 + UUC

Amino acid change (position) Ser Gly Glu Ile

+ -* + +

Arg Val Lys Phe

(E2-182) (E2-183) (E2-199) (E2-207)

VlA4D-1

+ 400 a-800 <10,000 4 <25

160,000 256 <25

Vl A3A-9

+ 200 21,600 160,000 2,048 al ,600

4 <50

160,000 2,048 400

+ 100 a800 >320,000 >8,192 a800 160,000 512 3800 <10,000 16 <25 160,000 16 a-800

VlA3B-7 + 400 100 80,000 128 100 160,000 2,048 <50 80,000 2,048 <25 <10,000 4 <25

epitopes on the E2 glycoprotein of,TC-83 virus by immunoblotting reduced viral proteins (Roehrig and Mathews, 1985; Roehrig, 1986). Subsequent studies with flaviviruses indicated that reduced disulfide bonds may reassociate during the blotting process (Winkler et al,, 1987). Therefore, we analyzed the reactivities of purified preparations of MAbs with virus that had been reduced and alkylated with iodoacetamide. ’ After alkylation in reduced virions, the El and E2 glycoproteins possessed increased apparent molecular weights on SDS-polyacrylamide gels. These proteins have 16 and 18 cysteines, respectively. The electrophoretic migration of the nucleocapsid, which contains only 2 cysteines, was not noticeably altered by reduction and alkylation (data not shown). E2 glycoprotein epitopes were differentially sensitive to reduction and alkylation (Fig. 2). The binding of MAbs defining epitopes E2”, E2C, and E2h was decreased by reduction-alkylation, yet significant binding activity was retained. In contrast, reduction-alkylation abrogated binding of MAbs defining the E2’ and E2g epitopes and enhanced binding of MAbs defining E2d and E2”. Alkylation of native virus also abrogated binding of anti-E2g MAb. Treatment of virus with SDS had no effect on MAb binding with any epitope (data not shown). Identical binding results were found with alkylated native virus and with virus treated with SDS prior to alkylation. Similarly, SDS treatment prior to reduction and alkylation of virus did not alter MAb binding profiles compared with

680

JOHNSON 3.0

a

ET AL TABLE

c

3

KINETICS OF BINDING OF VARIANTS OF TC-83

1.0

,;;.--1

1.0

TC-83 V3B4C4 VlA4D-1 Vl A3A-9 VlA3B-7 TRD

0.0

‘n = 4 for TC-83;

3.0

d

2.0

e : 4:

Time to adsorb mean

Virus

0.0 lY!rsd 2.0

3.0 m

50% of input PFUs + d (min)=

18+ 30*19 27* 22+ 20* 33*

5 9 4 9 2

n = 3 for all others.

f A

2.0 :

1.0

o.o-

50

3.0

26

12.6 6.25 YP ANTIBODY

3.125

1

2.0 1.0

0.0 1 50

VEE VIRUSTO VERO CELLS

HIAF 1

r--d 4

26

12.6

6.26

“(I *NT, BODY

3.126

1.86

1.56 100

1

200 400 800 1600 ANTIBODY DlL”TlON

3200

FIG. 2. Effects of reduction and alkylation on reactivities of anti-VEE virus MAbs in ELISA. Reactivities of purified MAbs (50 rg in first well) were determined on reduced and alkylated TC-83 virus (0) or untreated virus (m). Panel designations refer to E2 epitopes; e.g., panel “a” shows results with anti-E2” MAb. The last panel shows the reactivity of anti-TC-83 hyperimmune ascitic fluid. These reactivities are reported as dilutions of the ascites (n = 3).

50% of the input plaque-forming units (Table 3). This finding is particularly noteworthy in the case of the E2’ variant. Despite the fact that the deduced amino acid substitution dramatically alters the predicted surface profile and that binding of anti-E2’ stabilizes virus-cell interaction (Roehrig et al., 1988), the E2’variant did not display significantly altered kinetics of binding to cultured cells. Virulence

of VEE virus variants

The virulence of the variants was examined in outbred suckling mice by both intracerebral and intraperitoneal inoculation. The E2h variant was >l O-fold more attenuated than TC-83 virus by either route of administration (Table 4). The E2C variant was also attenuated about 1 O-fold when inoculated intracerebrally. The virulence of the E2’ and E2g variants was indistinguishable from that of TC-83 virus. None of the N escape variants was more virulent than its parent. DISCUSSION

those of reduced and alkylated virus. Western blot analysis of these viruses was sufficiently sensitive to detect binding of only the anti-E2” MAb (data not shown). Binding characteristics

of VEE virus variants

One of several mechanisms by which alphaviruses are neutralized involves antibody blocking of virus attachment to cells (Roehrig et a/., 1988). Since the VEE virusvariants possess amino acid substitutions in a domain critical to TC-83 virus N, they were tested to see if their ability to bind to Vero cells had been altered. The kinetics of binding of TC-83 virus, its variants, and the virulent TRD virus were determined over a 150-min period. Although the binding of TRD was significantly slower (P = 0.01) than that of TC-83, the binding behavior of the variants did not differ significantly from that of TC-83 when expressed as the mean time to adsorb

We present serologic and sequence evidence that the immunodominant N site of VEE virus includes the domain from E2-182 to E2-209. In addition to the mutations characterized at E2-182, -183, -199, and -207, two other relevant amino acid substitutions in this re-

TABLE

4

VIRULENCE OF ANTIGENIC VARIANTS OF TC-83

Virus TC-83 V3B4C-4 VlA4D-1 Vl A3A-9 VlA3B-7

VEE VIRUS

Eprtope

Position OfAA change

100 ICLDS,, PW

100 IPLDSO F’W

E2C E2’ E29 E2h

E2-182 E2-183 EZ-199 E2-207

40 500 30 20 500

200 500 80 200 2700

NEUTRALIZATION

VEE

DOMAIN

170

180

190

200

210

I

I

I

I

I

MHLPGSEVDSSLVSLBOSSVTVTPPVGTSALV&ZECGGTKZSKTINKT

SIN

..R.RPHAYT.YLEvE.8GK.YAK..S.KNITY..K..DY.TGTVSTB.

RR

..T.PDIP.RT.L.QTAGN.-KITAG.RTIRYN.T..RDNVGT.STDK

220

230

240

250

260

I

I

I

I

I

VEE

KQFSQCTKKEQCRAYRLQNDKWYNSDKLPKAAGATLKGKLHVPFLL

SIN

BITG-..AIK..V..KSDQT...F..PD.IRHDDH.AQ....L..K.

RR

TINT-.-.ID..H.AVTSI!.:.QFT.PFV.R.PQTTARB..V....P.

FIG. 3. The predicted amino acid sequences of the E2 glycoprotein domains involved in N of three alphaviruses. Amino acids that are altered in viruses which escape N by various monoclonal antibodies are underlined. Dots indicate amino acid identity with VEE virus. The primary sequences are aligned (by introducing dashes) to maximize these identities (Kinney et a/., 1986). SIN, Rice and Strauss (1981); Strauss et a/. (1987). RR, Dalgarno eta/. (1983); Vrati eta/. (1988).

gion have been identified. E2-209 also influences the binding of anti-E2h MAb (Kinney et al., 1988). This was discovered when cDNAs encoding VEE virus structural proteins were expressed in recombinant vaccinia viruses. One construct contained a substitution of Lys for Glu at E2-209 that greatly reduced detection of the E2h epitope in virus-infected cells. This result is consistent with our observation that the E2h variant has an amino acid change at E2-207. E2-192 may also play a role in N. The anti-E2g and -E2h MAbs neutralize TRD VEE virus 10 times more effectively than TC-83 virus, its vaccine derivative (Table 1; Roehrig and Mathews, 1985). An amino acid substitution (VaI(TRD) + Asp(TC83)) one of six in the E2 and El glycoproteins that occurred during attenuation of TRD virus, is located at E2192 (Johnson et al., 1986; Kinney et a/., 1989). This E2192 substitution may contribute to the decreased N of TC-83 virus by anti-E2g and -E2h antibodies. Alphavirus N domains may be formed by conformational interaction of distal linear sequences. The defined linear sequences are composed of amino acids that cluster in the primary sequence of the E2 glycoprotein. This clustering is consistent with CBA data: E2”, E2’, and E2h are indistinguishable spatially from each other and E2g overlaps the E2c-f-hdomain (Roehrig, 1986). Figure 3 compares the predicted amino acid sequences of three alphaviruses through the N domain and notes the relative location of mutations associated with escape from N. (The numerical designation of E2 positions is that of Kinney et al. (1986) which maximizes identities of other alphaviruses with VEE virus.) Neutralization escape mutations of VEE and SIN viruses (Strauss et a/., 1987; Davis et a/., 1987) map to

OF VEE VIRUS

681

essentially identical regions of the E2 glycoprotein; one mutation occurs at the same location (E2-183) in both viruses. The major antigenic site for RR virus in N overlaps the VEE/SIN domain, but most of the variant mutations occurred more toward the carboxy-terminal end of E2 (E2 215-253; Vrati et al., 1988). All four cysteines are conserved over the region depicted in Fig. 3. Conservation of the hydropathy profile in this area among RR, SIN, and Semliki Forest viruses has been previously noted (Vrati eta/., 1988). As would be expected, since N by anti-E2 antibodies is virus-specific, the amino acid sequences of the three alphavirus proteins are not highly homologous in the N domain. The sequence of the VEE virus E2 glycoprotein region shown in Fig. 3 has only 37 and 32% identity with SIN and RR viruses, respectively. This degree of sequence conservation is even lower than that seen for the E2 proteins as a whole, where the comparable identity values are 41 and 389/o. Although this analysis localizes the critical N domain of VEE virus to a short linear sequence, it is possible that mutations which alter MAb binding and N activity do so by inducing distant conformational changes. The observation that ELISA binding activity with these variants may remain constant while N or HI activity may vary suggests spatial rearrangement of peptide sequences in the selected variants. Although five of these epitopes (E2a, E2”, E2d, E2”, and E2h) have a conformational component, they also have a linear aspect because significant MAb binding is retained following reduction-alkylation (Fig. 2). Treatment of virus with SDS has no effect on MAb binding. These results are consistent with the previous observation that MAb binding is conserved in Western blots even when tested with proteolytic fragments of E2 (Roehrig and Mathews, 1985). Two of these epitopes (E2d and E2e) appear to be partially hidden on thevirion surface because reduction-alkylation enhances their recognition. These epitopes map by CBA to similar areas on the TC-83 virus E2 glycoprotein (Roehrig and Mathews, 1985). Epitopes E2’ and E2g were quite sensitive to reductionalkylation. The finding that E2’- and E2”-associated mutations were immediately adjacent in the linear sequence, but their selecting antibodies still reacted with the heterologous virus mutants, probably reflects the conformational nature of the E2’epitope. It is likely that the change in amino acid 183 does not define the true binding site of the anti-E2’MAb. The binding of the antiE2’ is also peculiar. Although virus is neutralized by anti-E2’ MAb, the mechanism of N appears to depend on a conformational alteration which stabilizes the virus-cell receptor complex (Roehrig et a/., 1988). Anti-E2g binds and efficiently neutralizes virus, but does not block virus association with cellular recep-

682

JOHNSON

tom. This MAb may neutralize after virus attachment to cells by inhibiting fusion between the envelope of the virion and prelysosomal endosomes (Roehrig et a/., 1988), as has been described for a flavivirus, West Nile virus (Gollins and Porterfield, 1986). These obsetvations indicate that E2g may be close to, or influence, the El fusion domain (VEE El 75-108, Kinney et a/., 1988; Kielian and Helenius, 1985). E2g is also of special interest because it was destroyed by alkylation of native virus. The reason for this property is unknown; however, the amino acid change associated with this variant (199) is immediately adjacent to two cysteines (residues 200 and 202). It is possible that one or both of these cysteines contain free sulfhydryl groups in the native virus that can be alkylated without prior reduction. E2 regions remote from the domain characterized here have been shown to interact with virus-neutralizing antibodies for other alphaviruses. A RR virus mutant with a deletion spanning E2 55-61 is neutralized by an N MAb a 1OOO-fold less efficiently than the prototype virus (Vrati et al., 1986). This MAb selects for an N escape mutant that encodes an amino acid change at E2234, indicating possible spatial overlap of these nonlinear regions. In general, amino acid substitutions in the immunodominant N domain of VEE virus have only a modest effect on virulence. The E2h variant, however, was >lO-fold more attenuated than TC-83 virus by intraperitoneal inoculation. By intracerebral inoculation, both the E2h and the E2C variants were about lo-fold attenuated. This is the first documentation of an amino acid change in an alphavirus N domain that causes attenuation. In SIN virus, the E2b antigenic site (associated with mutation of E2-216) did not affect neurovirulence (Pence and Johnston, unpublished; cited in Davis et al., 1987).

ACKNOWLEDGMENTS We thank Richard Kinney for synthesizing cDNAfor the E2” variant, Crystle Kost for assistance with RNA sequencing during the early phase of this work, and Richard Bolin for help with the reductionalkylation studies.

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