VIROLOGY

187, 18-32 (1992)

Neutralizing Epitopes on Herpes Simplex Virus-l -Expressed Rotavirus Are Dependent on Coexpression of Other Rotavirus Proteins

VP7

PHILIP R. DORMITZER,*+’ DORA Y. HO,* ERICH R. MACKOW,+$* EDWARD S. MOCARSKI,+ AND HARRY B. GREENBERG*++ *The Division of Gastroenterology, tProgram in Cancer Biology, and +Department of Microbiology and Immunology, Stanford University, Stanford, California 94305; and the SPalo Alto Veterans Administration Medical Center, 380 1 Miranda Avenue, Palo Alto, California 94304 Received August 9, 199 1; accepted

November

13, 199 1

We constructed a recombinant thymidine kinase-negative herpes simplex virus type 1 (HSV-1) that expressed the rotavirus major outer capsid glycoprotein, VP7. In the recombinant HSV-1, a promoter from the 5’ noncoding region of the HSV-1 glycoprotein B locus regulated the expression of VP7 as a HSV-1 yl gene product. HSV-l-expressed VP7 resembled rotavirus-expressed VP7 in its SDS-PAGE mobility, high mannose-type glycosylation, disulfide bonding, perinuclear to cytoplasmic localization, intracellular retention, and reactivity with polyclonal antisera and nonneutralizing antibodies. Unlike rotavirus-expressed VP7, HSV-1 -expressed VP7 lacked several neutralizing epitopes by immunohistochemical staining and by ELISA. One neutralizing epitope identified on HSV-l-expressed VP7 by ELISA was masked by paraformaldehyde fixation of recombinant HSV-l- but not rotavirus-infected cells. Neutralizing epitopes were restored to HSV-1 -expressed VP7 by coinfection of cells with the HSV-1 recombinant and a heterologous rotavirus that lacked the neutralizing epitopes. The recovered neutralizing epitopes were detected on double-shelled rotavirus particles produced in the coinfected cells. This study indicates that the formation of several neutralizing epitopes on rotavirus VP7 requires interaction of VP7 with other rotavirus proteins. In addition, HSV-1 was a useful vector for studying the localization, processing, and antigenicity of an RNA virus glycoprotein. o 1992Academic PNS, I~C.

INTRODUCTION

In rotavirus-infected cells, VP7, the outer capsid glycoprotein, is targeted to and retained in the lumen of the endoplasmic reticulum (ER), where it receives a high mannose-type glycosylation (Both eta/., 1983; Ericson et al., 1983). VP7 is incorporated into mature, double-shelled rotavirus particles by a complex process. A single-shelled rotavirus particle, which contains no VP7, buds from the cytoplasm into the lumen of the ER, transiently acquiring an envelope (Adams and Kraft, 1967). During maturation in the ER lumen, a nascent rotavirus particle loses its envelope and acquires an outer capsid, consisting of VP7 and VP4. The ability of HSV-1 -expressed VP7 to undergo proper processing and to participate in the assembly of a doubleshelled rotavirus particle is a rigorous test of HSV-l’s ability to express a properly processed and targeted foreign glycoprotein. Neutralization of rotavirus in cell culture and protection from rotavirus disease in a mouse model can be mediated by monoclonal antibodies (mAbs) directed at either of the two outer capsid proteins, VP4 and VP7 (Greenberg et al., 1983; Matsui et al., 1989; Offit et al., 1986). On the virion, the serotype-determining glycoprotein, VP7, contains complex, conformationally determined epitopes that are recognized by serotypespecific and heterotypic neutralizing antibodies (Greenberg et al., 1983; Mackow et al., 1988; Shaw et al., 1986; Taniguchi et a/., 1988).

Herpes simplex virus type 1 (HSV-1) is a potentially useful expression vector for studying protein processing, localization, and antigenicity in cell culture and immunogenicity in experimentally infected animals. HSV1 can be engineered to carry foreign genes, has a large excess packaging capacity, infects a variety of tissues, and has a broad host range (Arsenakis and Roizman, 1985). In addition, its characteristic neurovirulence can be attenuated, raising the possibility of its use as a vaccine vector (Meignier et a/., 1988). HSV-1 recombinants have expressed other DNA virus proteins, such as hepatitis B virus surface antigen (Shih et al., 1984) and Epstein-Barr virus nuclear antigen 1 (Hummel eta/., 1986). In addition, HSV-1 recombinants have expressed a bacterial protein, Escherichia co/i P-galactosidase (E. co/i P-gal), and a retrovirus protein, the human immunodeficiency virus type 1 gag protein (Ho and Mocarski, 1988, 1989; RosenWolff et a/., 1990). In this study, we tested the ability of a recombinant HSV-1 to express, target, and posttranslationally process an antigenically complex RNA virus glycoprotein, rotavirus VP7. ’ To whom reprint requests should be addressed. 2 Present address: Department of Medicine, State University New York at Stonybrook. Stonybrook, NY 11794. 0042-6822/92

$3.00

Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.

of

18

SAll

Because infection with rotavirus can confer resistance to disease upon reexposure (Bishop eta/., 1983; Chiba et a/., 1986), it should be possible to immunize children against rotavirus in order to reduce the considerable morbidity and mortality caused by rotavirus disease. Nevertheless, attempts to protect children from rotavirus disease by immunizing with attenuated rotaviruses have, thus far, had mixed results (De Mol et a/., 1986; Hanlon et a/., 1987; Santosham et a/., 1991). Therefore, there is considerable interest in developing recombinant vaccines in which rotavirus neutralization antigens are expressed by live recombinant viral or bacterial vectors. In this respect, it is important to determine whether VP7 expressed by a recombinant vector bears the epitopes recognized by neutralizing antibodies. MATERIALS Cells, viruses,

AND METHODS

and antibodies

Vero, MA104, and COS cells were grown in Dulbecco’s modified Eagle’s medium or medium 199 with Earle’s salts supplemented with 10% fetal calf serum (FCS). HSV-1, strain KOS, was prepared on Vero or COS cells and titered on Vero cells. HSV-1 stocks were prepared from infected cells by 1: 1 dilution in sterile skim milk and sonication. Rotavirus strains SAl 1, RRV, NCDV, and AR2 were grown and titered on MA104 cells. Rotavirus was prepared from infected cells by freeze-thawing, sonication, trichlorotrifluoroethane (Freon) extraction, and concentration by pelleting (142,000 g, 1 hr). The following mAbs were used in this work: Ml 29 and M60 recognize cross-reacting, nonneutralizing epitopes on VP7 (Shaw et a/., 1986); M 159, 4F8, 4C3, 5H3, 96, 4G2, and 3 recognize VP7 and neutralize serotype 3 rotavirus (Greenberg et al., 1983; Shaw et al,, 1986); 57-8, which is of the IgM class, recognizes VP7 and neutralizes several serotypes of rotavirus (Benfield et a/., 1987); and H233 recognizes gB of HSV (Pereira eta/., 1981). GP8, GP22, and GP962 are hyperimmune guinea pig antisera raised against purified rotavirus strains OSU, SAI 1, and RRV, respectively. Peroxidaselabeled goat antibodies to mouse IgG, mouse IgM, and guinea pig IgG were obtained from Kirkegaardt and Perry (Gaithersburg, MD). Construction

of recombinant

19

ROTAVIRUS VP7 EXPRESSED BY HSV-1

viruses

The recombinant viruses RH133, which expresses P-gal, and RH135, which expresses SAll VP7, were constructed using described techniques (Mocarski et al,, 1980; Spaete and Mocarski, 1987; Ho and Mocarski, 1988). The coding sequences for the expressed

proteins were E. co/i/acZin RH 133 and a cloned cDNA copy of SAll gene segment 9 (Poruchynsky et al., 1985) in RH135. The glycoprotein B (gB) promoter used in this study, a 140-bp DNA fragment corresponding to bases -101 to +39 relative to the transcription start site of the HSV-1 strain F gB locus (Pellet et a/., 1985), was placed upstream of each foreign gene. In each recombinant virus, the gB promoter and foreign coding sequence cassette was inserted into the thymidine kinase (TK) locus, interrupting TK expression. Details of the construction strategy are available on request. Infection

and coinfection

of cells

Rotavirus samples were activated with 5 pg/ml of trypsin, and cells were infected with rotavirus in serumfree medium. Rotavirus-infected cells were maintained in serum-free medium with 0.5 pg/ml trypsin for production of infectious virus and with 0.5 fig/ml trypsin and 0.7% agarose added for plaquing. HSV-1 infections were performed in medium containing 100/o FCS. MA104 cells were coinfected with NCDV and RH 133 or RH135 at 4 days after cell splitting. One hour after infection with a recombinant HSV-1 at a multiplicity of infection (m.0.i.) of 12 in medium containing 10% FCS, the cells were washed with serum-free medium and infected with NCDV at a m.o.i. of 1 for grossly visible cell staining or antigen production or at a m.o.i. of 3.3 when preservation of cellular architecture was desired. For microscopy of coinfected syncitia, cells were infected with NCDV (m.o.i. of 3.3) 5 hr after infection with the HSV-1 recombinant. Cells were fed with medium containing 10% FCS 1 hr after NCDV infection. Monolayers were fixed or harvested for antigen 13.5 hr after the HSV-1 infection. The optimal m.o.i.s, timing of infection, and timing of fixation or harvest were determined empirically. To produce infectious rotavirus from a RH 135- and NCDV-coinfected monolayer, MA1 04 cells were infected with RH135 at a m.o.i. of 10 and infected with NCDV 1 hr later at a m.o.i. of 1. One hour after NCDV infection, the monolayer was washed once with medium containing 57-8, a mAb that neutralizes NCDV, washed extensively with medium to remove the mAb, maintained in serum-free medium, and harvested at 1 or 20 hr after the NCDV infection. Freon extraction of the coinfected cell lysate eliminated infectious HSV-1 before the determination of rotavirus titer. Radiolabeling

of virus-infected

cells

Metabolic labeling of rotavirus- or HSV-1 -infected cell layers with [35S]methionine was performed as described (Harlow and Lane, 1988). Cell layers were in-

20

DORMITZER ET AL.

fected at a m.o.i. of 10, unless otherwise noted. [35S]Methionine was added to culture medium at a concentration of 100 &i/ml, following a 1- to 2-hr methionine depletion period. Unless otherwise noted, HSV-1 -infected cells were radiolabeled from 8 to 18 hr after infection, and SAl 1-infected cells were radiolabeled from 3 to 9 hr after infection. Cell monolayers were harvested in radioimmunoprecipitation assay (RIPA) buffer (1% deoxycholate, 1% Triton X-l 00,0.3 M NaCI, 0.1% SDS, and 1 mM phenylmethylsulfonyl fluoride). Lysates were centrifuged at 150,000 g for 40 min to remove particulate debris. Analysis

of proteins

lmmunoprecipitation was performed as described (Harlow and Lane, 1988). Sera and ascites fluid were added to lysates at a dilution of approximately 1:50 and hybridoma tissue culture supernatant was added at 1: 1. Antigen-antibody complexes, precipitated with protein A-conjugated Sepharose beads (Sigma Chemical Co., St. Louis, MO), were washed with RIPA buffer, 1% bovine serum albumin (BSA); phosphate-buffered saline (PBS), 1% BSA; and 0.05 MTris-Cl, pH 6.8. To cleave N-linked high mannose-type oligosaccharides, immunoprecipitated proteins were digested with endoglycosidase H (endo H) overnight at 37” in 0.05 M Tris-Cl, pH 6.8, 0.1% SDS (Tarentino and Maley, 1974). Samples were reduced by boiling for 2 min in a sample buffer containing 2% ,&mercaptoethanol before electrophoresis on 10% SDS-PAGE gels as described (Hames, 1990). To examine the disulfide bonding of RH135-expressed VP7, samples were electrophoresed under nonreducing conditions by omitting ,&mercaptoethanol from the sample buffer as described (Allore and Barber, 1984). Cell fixation

and immunoperoxidase

staining

Cells were fixed by treatment with ice-cold methanol or 3% paraformaldehyde in PBS for 30 min. Paraformaldehyde-fixed cells were permeabilized with 1O/O Triton X-100 in PBS for 5 min, except when cell surface staining was desired. Unless otherwise noted, rotavirus-infected cells were fixed at 12.5 hr after infection, and HSV-l-infected cells were fixed at 13.5 hr after infection. PBS, 1% BSA, 0.2% polyoxyethlenesorbitan monolaurate (Tween 20) was used to dilute antibodies and to wash fixed cell layers during staining. For cell surface staining, Tween 20 was omitted from the dilution and washing buffer. Cell layers were incubated with the primary antibody for 1 hr at room temperature, washed, incubated with peroxidase-labeled goat antibody to mouse IgG or IgM for 1 hr at room temperature, washed, and stained with aminoethylcarbazole as de-

scribed (Harlow and Lane, 1988). For both cell staining and enzyme-linked immunosorbent assay (ELISA), optimal antibody dilutions were determined empirically. ELISA Antigen for ELISA was prepared by infecting Vero or MA1 04 cells with RH133 (m.o.i. of lo), RH135 (m.o.i. of lo), or SAl 1 (m.o.i. of 0.05). Infected cell layers were harvested in 1 ml cell culture medium at 22 hr after infection. These samples were freeze-thawed, sonicated, and cleared of cell debris by centrifugation (12,000 rpm for 10 min in a microfuge). Antigen capture ELISA plates were prepared as described using mAbs against VP7 for antigen capture and GP962 and peroxidase-linked goat antibody to guinea pig IgG for detection of captured antigen (Padilla-Noriega et a/., 1990). Antibodies were diluted in PBS, 10% FCS, 0.2% Tween 20, and wells were washed with PBS, 0.2% Tween 20 between the addition of each reagent. An ELISA reading (A& that was reproducibly greater than 0.10 in magnitude and 3 standard deviations above the mean of negative control samples was scored as positive. In the direct coat ELISA, antigen, diluted in PBS, 0.05% sodium azide, was incubated in uncoated ELISA plates overnight at 4” before blocking with PBS, 0.050/o sodium azide, 10% FCS. Bound antigen was detected as described for the mAb capture ELISA. Sucrose pelleting and density infected cell lysates

gradient

analysis

of

Infected or coinfected MA1 04 cell monolayers were harvested at 13.5 hr after infection and processed as described above for ELISA antigen. In addition, the pellet of the low-speed spin was resuspended in a small volume of 0.01 MTris-Cl, pH 7.5, 0.1 M NaCI, 1.5 mM CaCI, (TNC) or cell culture medium and extracted with Freon. The Freon extraction supernatant and the lowspeed spin supernatant were pooled. For sucrose pelleting, 500 ~1 of this preparation was made 3% in sucrose and layered over 1 ml of 18% sucrose in TNC in an SW55 tube, which was balanced with TNC. Samples were centrifuged at 100,000 g for 1 hr at 4“, pelleting particles with sedimentation coefficients greater than approximately 95s (calculated by formulas and tables in Rickwood, 1984). Pellets were resuspended in 60 yl tissue culture medium containing 10% FCS and used undiluted for ELISA. For CsCl gradient analysis, the antigen preparations were layered over a 1.37 g/ml CsCl solution in TNC and centrifuged at 1 10,000 g for 20 hr at 20”. The refractive index of gradient fractions was measured, and fractions were used as antigen for ELISA or in hemagglutination assays as described (Greenberg et a/., 1983).

SAll

Quantitation

ROTAVIRUS

of protein expression

RH133- or RH135-infected (m.o.i. of 10) MA1 04 cell lysates were prepared at 22 hr after infection as described above for ELISA. The VP7 content of a RHl35infected cell lysate was compared to that of purified, double-shelled SAl 1 particles by M60 capture ELISA of serial dilutions. The dilution buffer was made 10 mM in EGTA to uncoat SAl 1 virions, solubilizing VP7 (Cohen et al,, 1979). Known quantities of protein standards (BSA and carbonic anhydrase) and serial dilutions of the purified SAl 1 sample were compared by SDS-PAGE and sequential staining with Comassie and silver stain (Wray et al., 1981). The dual-staining technique has high sensitivity and relatively low interprotein variability (Hames, 1990). Because glycoproteins often stain poorly by this technique, we used published estimates of virion protein composition (Estes, 1990) to estimate the VP7 content of the purified SAl 1 sample from the measured content of VP2 and VP6. To quantitate P-gal in a RHl33-infected cell lysate, the o-nitrophenyl+b-galactopyranoside assay was used (Rotman, 1970). Total protein in the infected cell lysates was determined by the Bradford protein assay (Bio-Rad Laboratories, Richmond, CA). RESULTS lmmunoprecipitation of VP7 from lysates of RH135-infected cells Recombinant HSV-1 -expressed rotavirus VP7 was detected by immunoprecipitation from [35S]methionine-labeled lysates of RHl35-infected Vero cells. Immunoprecipitation with the anti-SAl 1 rotavirus hyperimmune antiserum GP22 (Fig. 1) and with the nonneutralizing anti-VP7 mAbs Ml 29 (Fig. 2) and M60 (not shown) yielded a protein that comigrated by SDSPAGE with authentic SAl 1 VP7 (Figs. 1 and 2) having an apparent molecular weight of 37 kDa (not shown). This protein was not detected in lysates infected with the ,&gal-expressing HSV-1, RH133 (Fig. 1). Immunoprecipitated RH135- and SAl 1-expressed VP7 electrophoresed under nonreducing conditions (Allore and Barber, 1984) had the same increased mobility, indicating similar disulfide bonding of RH 135- and SAl lexpressed VP7 (not shown). In addition, VP7 was not detected by immunoprecipitation from the tissue culture medium of RH135-infected cell cultures, indicating that the expressed VP7 was not secreted (not shown). Temporal

21

VP7 EXPRESSED BY HSV-1

regulation

of VP7 in RH135infected

cells

The time course of VP7 expression in RH135-infected cells was determined to verify that VP7 expres-

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

c@

VP6 VP7 -

FIG. 1. Time course of VP7 and gB expression in RH135infected Vero cells. Autoradiogram of immunoprecipitated proteins analyzed by SDS-PAGE. RH135- or RH133-infected Vero cells (m.o.i. of 4) were methionine depleted for 1 hr and metabolically pulse-labeled with [35S]methionine for 2 hr before harvesting; uninfected Vero cells were harvested at 1 1 hr after infection; SAl 1-infected MA1 04 cells were harvested at 9 hr after infection; an RH135 infection was performed in the presence of phosphonoacetic acid (PAA). Rotavirus proteins were immunoprecipitated by the anti-SAl 1 hyperimmune antiserum GP22, and gB was immunoprecipitated by mAb H233, as indicated above the brackets. Time of harvest, use of PAA, and infecting virus, if other than RH135, are indicated above each lane. VP6 is seen in the lane of the SAl 1 sample. Some spill-over of proteins from lane SAl 1 into lane 11 hr/RH133 is apparent.

sion was controlled by the gB promoter fragment. VP7 and gB were first detected by immunoprecipitation from RHl35-infected Vero cell lysates pulse-labeled with [35S]methionine between 5 and 7 hr after infection. (Fig. 1). Both glycoproteins continued to be expressed through at least 15 hr after infection (not shown). Expression of the proteins at 1 1 hr after infection was not prevented when viral DNA replication was inhibited by adding 100 pg/ml phosphonoacetic acid at the time of infection. The upper bands detected in the H233 immunoprecipitations correspond to precursor and mature forms of gB (Eberle and Courtney, 1980) while the lower bands are cleavage products of gB produced by host-encoded proteases in Vero cells (Pereira et a/., 1982). Thus, gB and VP7 were expressed

22

DORMITZER

ET AL.

cells had the same mobility by SDS-PAGE: the nonglycosylated VP7 immunoprecipitated from lysates of tunicamycin-treated SAl l- or RH 135-infected cells also comigrated but had increased mobility by SDS-PAGE. VP7 immunoprecipitated from RHl35- or SAl l-infected cell lysates also comigrated and had increased SDS-PAGE mobility when digested with endo H, an enzyme that specifically cleaves high mannose-type oligosaccharides (not shown; Tarentino and Maley, 1974). These results indicate that VP7 was processed by the addition of an N-linked high mannose-type oligosaccharide side chain in both SAl 1- and RH 135-infected cells. Effect of fixation rotavirus-infected VP6

-

VP7 Unglycosylated

VP7

FIG. 2. VP7 glycosylation inhibition by tunicamycin in SAl l- and RHl35-infected cells. Autoradiogram of immunoprecipitated proteins analyzed by SDS-PAGE. [%]Methionine-labeled MA1 04 and Vero cells, infected with SAl 1 rotavirus or RHl35, respectively, in the presence or absence of tunicamycin, were harvested at 7 hrafter infection (SAl 1) or 11 hr after infection (RH135). Rotavirus proteins were immunoprecipitated with the antiSA 1 hyperimmune antiserum GP22, and VP7 was specifically immunoprecipitated by mAb M 129. The infecting virus and the presence or absence of tunicamytin are indicated above each lane. The immunoprecipitating antiserum or mAb is indicated above the brackets.

late in infection, and expression was not dependent on DNA replication. This pattern of expression is consistent with regulation of both gB and VP7 as yl HSV-1 gene products (Roizman and Sears, 1990). Glycosylation

of VP7 in RH135-infected

cells

The effect of tunicamycin, an inhibitor of N-linked glycosylation (Tkacz and Lampen, 1975) on RH135expressed VP7 was examined to verify proper ER processing. [35S]Methionine-labeled lysates were prepared from MA1 04 or Vero cells infected with SAl 1 or RH 135, respectively, in the presence or absence of 2 pg/ml tunicamycin (Fig. 2). Glycosylated VP7 immunoprecipitated from lysates of SAl l- or RH 135-infected

on neutralizing cells

epitopes

in

To test a technique for rapidly determining the presence or absence of epitopes on VP7, rotavirus strain SAl 1- or RRV-infected Vero cells were fixed with methanol and immunoperoxidase stained with the VP7reactive mAbs M60, M 129, M 159,4F8,4C3, and 57-8 (summarized in Table 1; examples illustrated in Fig. 3). The nonneutralizing mAbs M60 and M 129 detected VP7 distributed in a perinuclear to cytoplasmic pattern in SAl l- or RRV-infected, methanol-fixed cells (Fig. 3a). In contrast, the neutralizing mAbs Ml 59, 4F8, 4C3, and 57-8 failed to detect VP7 in SAl l- or RRV-infected, methanol-fixed cells (Fig. 3b). To determine whether lack of reactivity with methanol-fixed VP7 was a general property of VP7-specific neutralizing mAbs, the neutralizing mAbs 5H3, 96, 4G2, and 3, which recognize RRV VP7 more strongly than SAll VP7, were used to immunoperoxidase stain RRV-infected, methanol-fixed cells. These neutralizing mAbs also failed to detect rotavirus-expressed VP7 in methanol-fixed cells (not shown). In contrast to the results in methanol-fixed cells, when SAl 1- or RRV-infected cells were fixed with paraformaldehyde and permeabilized with Triton X-l 00, all of the tested neutralizing and nonneutralizing mAbs detected VP7 (Fig. 3~). lmmunoperoxidase staining of the rotavirus-infected cells with any of the eight neutralizing mAbs (Ml 59,4F8,4C3, 57-8, 5H3,96,4G2, or 3) spared the cell nucleus, was most intense immediately surrounding the nucleus, and was present more diffusely toward the periphery of the cell. Lack of neutralizing VP7 in fixed cells

epitopes

on RH135-expressed

The nonneutralizing mAbs M60 and M 129 detected VP7 in RHl35-infected Vero cells by immunoperoxidase staining, regardless of whether the infected cells

SAll

ROTAVIRUS

23

VP7 EXPRESSED BY HSV-1

FIG. 3. Effect of fixation on epitopes of rotavirus-expressed VP7. RRV-infected Vero cells were fixed with methanol or paraformaldehyde and immunoperoxidase stained. Photographed at 200X magnification. (a) Fixed with methanol and stained with M 129. (b) Fixed with methanol and stained with M 159. (c) Fixed with paraformaldehyde, permeabilized with Triton X-l 00, and stained with Ml 59.

were fixed with methanol or paraformaldehyde (summarized in Table 1; examples illustrated in Fig. 4). M60 and M 129 stained the RHl35-infected cells less intensely than rotavirus-infected cells, and the RH 135infected cells had rounded up due to HSV-1 cytopathic

effects (Fig. 4a). Nevertheless, the stained VP7 in RH 135-infected cells was distributed in a perinuclear to cytoplasmic pattern. M60 immunoperoxidase staining of RH 135-infected, paraformaldehyde-fixed cells was only observed after the cells were permeabilized

TABLE 1 REACTIVITYOF VP7-SPECIFIC mAbs WITH RH135, SAl 1, AND NCDV Assay and antigen preparation lmmunoperoxidase

staininga ELISA

mAb

SAl 1

SAl 1 MeOHc

M60 Ml29 Ml59 4F8 4c3 57-8

+ + + + + +

+ + -

RH135

NCDV

+ + -

+ + +

a Unless otherwise noted, infected cells were fixed with paraformaldehyde b Neutralization data from Shaw et al. (1986) and Mackow et al. (1988). ’ Infected cells were fixed with methanol. d Cells were coinfected with RH135 and NCDV.

RH135 + NCDVd + + + + + + and permeabilized

SAll + + + + + +

Neutralizationb RH135

SAll

+ + + -

-

with Triton X-l 00

+ + + +

24

DORMITZER

ET AL.

FIG. 4. Recognition of RH135-expressed VP7 by immunoperoxidase staining. RH135-, RHl33-, or SAl l-infected Vero cells were fixed with paraformaldehyde, permeabilized with Triton X-l 00, and immunoperoxidase stained. No syncitium formation is seen in the recombinant HSV-linfected cell layers because of the relatively low m.o.i. (approximately 0.1). Photographed at 200X magnification. (a) RH135infected cells, stained with Ml 29. (b) SAl l-infected cells, stained with 4F8. (c) RHl35-infected cells, stained with 4F8. (d) RH133-infected cells, stained with M129.

with Triton X-l 00, indicating the absence of expressed VP7 from the cell surface, as expected. In contrast to their staining of rotavirus-infected, paraformaldehyde-fixed cells (Fig. 4b), the neutralizing mAbs Ml 59, 4F8, 4C3, and 57-8 did not stain VP7 in RH135-infected cells, regardless of fixation technique (Fig. 4~). Thus, while nonneutralizing mAbs against VP7 stained both rotavirus- and RH l35-expressed VP7, whether fixed with methanol or paraformaldehyde, neutralizing mAbs against VP7 would only stain rotavirus-expressed VP7 fixed with paraformaldehyde. None of the mAbs stained cells infected with RHI 33, the ,&gal-expressing HSV-1 (Fig. 4d).

ELISA reactivities expressed VP7

of SAl 1- and RHI 35-

The ELISA reactivities of SAl l- and RHl35-expressed VP7 were compared using mAbs to VP7 for antigen capture (Padilla-Noriega et al., 1990). The m.o.i.s used for lysate production were adjusted so that lysates of RH 135-infected Vero cells contained at

least as much VP7 as lysates of SAl l-infected Vero cells, as judged by reactivity in M60 capture ELISA. RH 133-infected cell lysates and PBS, 10% FCS were used as negative control antigen preparations. As shown in Table 1, SAl l-expressed VP7 was detected by ELISA when any of the SAl 1-staining mAbs, M129, M60, Ml 59, 4F8, 4C3, or 57-8, were used to capture antigen. RH 135-expressed VP7 reacted strongly by capture ELISA with the nonneutralizing mAbs M60 and Ml 29. In contrast to the immunoperoxidase staining results, the neutralizing mAb Ml 59 also recognized RHl35-expressed VP7 by capture ELISA. The reactivity of RH 135-expressed VP7 in the M 159 capture ELISA was not diminished by including 1% Triton X-100 in the antigen binding buffer or by washing bound antigen with PBS, 1% Triton X-l 00. On the other hand, consistent with the immunoperoxidase staining results, RH135-expressed VP7 showed no reactivity above background by ELISA using neutralizing mAbs 4F8, 4C3, or 57-8 for antigen capture (Table 1). Omitting Tween 20 from the antigen binding or washing buffers did not affect the reactivity of VP7 with any mAb.

SAll

20 hr

1 hr

0 hr

Time

after

NCDV

ROTAVIRUS VP7 EXPRESSED BY HSV-1

infection

FIG. 5. Yield of NCDV from an infection of MA1 04 cells with NCDV and from a coinfection of MA1 04 cells with NCDV and RH135. Yield at 0 hr is inDut virus.

Replication monolayer

of rotavirus

in a HSV-l-infected

To assess the possibility of insinuating RH135-expressed VP7 into the replication cycle of a coinfecting rotavirus, we tested the ability of rotavirus to replicate in a HSV-l-infected monolayer. In a monolayer of MA1 04 cells that was infected with RH 135 at a m.o.i. of 10 and coinfected 1 hr later with rotavirus strain NCDV at a m.o.i. of 1, a HSV-l-induced syncitium formed over 90% of the monolayer. This mixed infection produced 4 X lo7 PFU of NCDV at 20 hr after NCDV infection (Fig. 5). The yield of NCDV from the mixed infection was 1 log lower than the yield of NCDV from a monolayer infected with NCDV alone. Nevertheless, the 4-log rise in titer in the coinfected cell layer from 1 hr after NCDV infection to 20 hr after NCDV infection indicates that 99.99% of the rotavirus recovered at the 20-hr time point was produced de nova in the coinfected monolayer. Recovery of neutralizing epitopes on RH135expressed VP7 by coinfection with a heterologous rotavirus (Summarized in Table 1; examples illustrated in Figs. 6 and 7.) Like RH 135-expressed VP7, rotavirus strain NCDV-expressed VP7 (serotype 6) is not recognized by the serotype 3specific neutralizing mAbs M 159, 4C3, or 4F8 by immunoperoxidase staining (Fig. 6, wells 1 and 3; Figs. 7a and 7b). Nevertheless, by immunoperoxidase staining, these neutralizing mAbs reproducibly

25

recognized their epitopes in MA1 04 cells that were infected at a m.o.i. of 12 with RH 135, coinfected at a m.o.i. of 3.3 with NCDV, and fixed with paraformaldehyde 13.5 hr after the initial infection (Fig. 6, well 2; Figs. 7c, 7d, and 7e). 4C3 staining of a RH 135- and NCDV-coinfected monolayer (Fig. 6, well 2) had roughly 1% of the intensity of M60 staining of a RH133and NCDV-coinfected monolayer (Fig. 6, well 6), indicating that a modest proportion of the VP7 in a coinfected monolayer had become recognizable by 4C3. No 4C3 staining was observed in a monolayer coinfected with NCDV and RHl33, the P-gal-expressing HSV-1 (Fig. 6, well 5). On microscopic examination of RH 135- and NCDVcoinfected monolayers, individual cells were stained darkly by the neutralizing mAbs, with granular inclusions of stain, typical of rotavirus-infected cells, visible (Fig. 7e). In some staining coinfected cells, the region of 4C3 staining surrounded the nucleus completely (Fig. 7c), as it does in a rotavirus-infected cell (Fig. 7f). In other cells, the stain formed partial arcs around the nucleus, leaving portions of the. perinuclear area unstained (Fig. 7~). Based on the Poisson distribution, in a monolayer coinfected with RH135 at a m.o.i. of 12 and NCDV at a m.o.i. of 3.3, approximately 96% of the cells should be infected with both viruses. Nevertheless, a relatively small proportion of cells displayed the recovered epitope (Figs. 7c and 7e). RHl35-infected (m.o.i. of 12) monolayers formed syncitia (Fig. 7b). Coinfection of a RHl35-infected monolayer 1 hr later with rotavirus (m.o.i. of 3.3) suppressed syncitium formation (Figs. 7c and 7e). Increasing the delay between RH 135 and NCDV infection to 5 hr increased the amount of syncitium formation (Fig. 7d). Neutralizing epitopes were detected on RH135expressed VP7 in both syncitial and cellular regions of coinfected monolayers (Figs. 7c, 7d, and 7e). ELISA reactivity of particles pelleted from lysates of RH135 and NCDV-coinfected MA1 04 cells MA1 04 cells were infected with SAl 1, NCDV, or RH135, or coinfected with RH135 and NCDV. At 13.5 hr after initial infection, infected cells were fixed with paraformaldehyde, permeabilized with Triton X-l 00, and immunoperoxidase stained with 4C3, giving results equivalent to those in Fig. 6. Lysates prepared from duplicate cultures were used as antigen for 4C3 capture ELISA. The 4C3 epitope was detected by the ELISA in a SAl l-infected cell lysate, but not in a lofold dilution of the SAl l-infected cell lysate nor in RH135- or NCDV-infected cell lysates. The RH135and NCDV-coinfected cell lysate also failed to give consistently positive results by 4C3 capture ELBA.

26

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ET AL.

I “, FIG. 6. lmmunoperoxidase staining of infected and coinfected MA1 04 cell monolayers. MA1 04 cell monolayers were infected with RH 133 or RH135 (m.o.i. of 12) and/or NCDV (m.o.i. of 3.3). The infected monolayers were fixed with paraformaldehyde, permeabilized with Triton X-l 00, and immunoperoxidase stained. Well 1. Infected with NCDV, stained with 4C3. Well 2. Coinfected with RH135 and NCDV, stained with 4C3. Well 3. Infected with RHI 35, stained with 4C3. Well 4. Infected with RH133, stained with M60. Well 5. Coinfected with RH133 and NCDV, stained with 4C3. Well 6. Coinfected with RH133 and NCDV, stained with M60.

Particles with sedimentation coefficients greater than approximately 95s were pelleted from 500 ~1 of each lysate and resuspended in 60 ~1 of tissue culture medium. Particles pelleted from the 1O-fold dilution of the SAl 1 lysate reacted by 4C3 capture ELISA, indicating approximately 4-fold concentration of SAl 1-expressed 4C3 antigen by pelleting. Although no 4C3reactive VP7 was detected in particles pelleted from RH 135- or NCDV-infected cell lysates, particles pelleted from the RH 135- and NCDV-coinfected cell lysate had consistently positive 4C3 reactivity, reflecting approximately 3-fold concentration of the 4C3-reactive antigen by pelleting. In contrast, VP7 detected by M60 capture ELISA in a lysate of cells infected with RH135 alone was not pelleted under the same conditions. ELBA reactivity of CsCl gradient fractions of RHl35 and NCDV-coinfected MA1 04 cells To determine the density of particles bearing the recovered 4C3 epitope in RH 135- and NCDV-coinfected cells, infected and coinfected cell lysates were frac-

tionated on CsCl gradients. The fractions were analyzed by M60 capture ELISA, a direct coat ELISA, and 4C3 capture ELISA. The RH 135-infected cell lysate only reacted by the M60 capture ELISA, which efficiently detects soluble VP7. The MGO-reactive antigen in fractions of the RH 135-infected cell lysate was detected at densities of 1.28 g/ml and below, peaking at 1.24 to 1.25 g/ml, corresponding to soluble VP7 (Fig. 8a). In the direct coat ELISA, antigen binds to ELISA plates directly, without capture antibody. Because soluble VP7 competes with other soluble cellular proteins for binding to the plate, it is not detected efficiently by direct coat ELISA. Because of their high density, rotavirus particles are contained in CsCl gradient fractions with few cellular proteins and can efficiently coat the ELISA plate. In addition, the detection reagent, GP962, an anti-RRV hyperimmune antiserum, reacts efficiently with group A rotavirus particles (single- and doubleshelled) that contain the cross-reactive and highly immunogenic inner capsid protein, VP6. The direct coat ELISA detected two peaks of reactivity in NCDV lysate

SAll

ROTAVIRUS

27

VP7 EXPRESSED BY HSV-1

FIG. 7.4C3 staining of infected and coinfected MA1 04 cells. MA1 04 cells were infected with SAl 1 (m.o.i. of 0.05), RH135 (m.o.i. of 12), and/or NCDV (m.o.i. of 3.3). The infected monolayers were fixed with paraformaldehyde, permeabilized with Triton X-l 00, and immunoperoxidase stained with 4C3. Unless otherwise indicated, there was a 1-hr lag between RH135 and NCDV infection, and photographs were shot at 200x magnification. (a) NCDV-infected cells. (b) RH135-infected cells. (c) RHl35- and NCDV-doinfected cells. (d) RH135- and NCDV-coinfected cells, 5-hr lag between RH135 and NCDV infection. (e) RH135- and NCDV-coinfected cells, shot at 400x magnification. (f) SAl 1-infected cells.

fractions, one at 1.29 to 1.30 g/ml, corresponding in density to empty particles (without a genome), and one at 1.35 g/ml, corresponding in density to full (genomecontaining) particles (Fig. 8b; Rodger et al., 1975). NCDV particles in fractions with peaks of direct coat ELISA reactivity hemagglutinated, confirming the presence of double-shelled virions in the empty and full particle peaks. As expected based on the immunoperoxidase staining results, 4C3 capture ELISA detected no reactivity in CsCl gradient fractions of RH 135- or NCDV-infected cell lysates. However, 4C3 capture ELISA detected a single modest peak of reactivity at 1.29 g/ml in fractions of the RH135- and NCDV-coinfected cells (Fig. 8~). This peak corresponds in density to the NCDV empty double-shelled particle peak detected by direct coat ELISA (Fig. 8b). The modest level of 4C3 reactivity in the coinfected cell lysate (note scale for A,,, in Fig. 8c) corresponds to the limited intensity of 4C3 immunoperoxidase staining of RHl35- and NCDV-coinfected cell layers (Fig. 6, well 2). The direct coat ELISA detected fewer full doubleshelled particles in the RH 135- and NCDV-coinfected

cell lysate than in the NCDV-infected cell lysate, but an equivalent amount of empty double-shelled particles in each (Fig. 8d). Fractions of an SAl 1-infected cell lysate had peaks of 4C3 capture ELISA reactivity at the densities of empty and full double-shelled particles, indicating that 4C3 can detect VP7 in both types of particle (not shown).

Quantitation of foreign protein expression by RH133 and RH135 In a lysate of RH133-infected MA1 04 cells (m.o.i. of IO) harvested at 22 hr after infection, enzymatically active P-gal made up 0.014% of total cell protein. In a similarly prepared lysate of RH 135-infected MA1 04 cells, MGO-reactive VP7 made up approximately 0.058% of total cell protein.

DISCUSSION We undertook this study to determine whether a recombinant HSV-1 can express a properly processed and localized rotavirus VP7 glycoprotein in infected cells. We also examined the antigenicity of HSV-1 -ex-

28

DORMITZER

ET AL

6

1.4

z t

1.3 1.2

h 2C

1 .o

1.1

ti

0.9

ii K

0.8 0.7

a cn 3 W

0.5

0.6

ti

0.3

0.4

6

0.2

t;2

0.1 0.0

.. ...................................-.........

Fraction B 2

0.22 0.20

wa .= .E z

0.1a 0.16 0.14 0.12 I

/

Density

(g/ml)

1.4 1.3-

1

d

1.2 1.1 1.0 0.9

1

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Fraction

Density

(g/ml)

Fraction

Density

(g/ml)

FIG. 8. ELISA reactivity (A,,,) of density gradient fractions of infected and coinfected MA1 04 cell lysates. Baseline is reactivity with uninfected MA1 04 cells. (a) RH 135infected cell lysate reactivity by M60 capture ELISA. (b) NCDV-infected cell lysate reactivity by direct coat ELISA. (c) RH 135. and NCDV-coinfected cell lysate reactivity by 4C3 capture ELISA. Each data point is the sum of two ELISA readings for a fraction. (d) RHl35- and NCDV-coinfected cell lysate reactivity by direct coat ELISA.

pressed VP7 and the effect of interaction with other rotavirus proteins on this antigenicity. This is the first report of the expression of an RNAvirus gene by HSV-1 and the first evaluation of the gB promoter’s ability to drive and regulate the expression of a foreign gene. In the cytoplasm of rotavirus-infected cells, a viral polymerase transcribes the viral double-stranded RNA gene segments (Estes, 1990). In the nuclei of RHl35 infected cells, HSV-1 signals directed transcription of a cloned cDNA copy of a rotavirus gene and export of the resulting mRNA to the cytoplasm. The 140-bp gB promoter sequence (from bases -101 to +39 relative to the gB transcription start site) regulated rotavirus VP7

as a yl HSV-1 gene product. The promoter contains a TATA box (sequence ATATAlT) at position -27 and two potential CAAT boxes at positions -97 (sequence GCGAATT) and -60 (sequence CGGAATA; Bzik et a/., 1984; Pellet et al., 1985). While P-gal and VP7 accumulated to moderate levels in RH133- and RH135-infected cells (0.014 and 0.058% of cell protein, respectively), they were not expressed as efficiently as gB, which makes up approximately 0.32% of total protein in HSV-1 -infected COS cells (Norrild and Vestergaard, 1977). In HSV-1 -infected cells, the nuclear, ER, and plasma membranes are modified by HSV-1 gene products, and

SAll

ROTAVIRUS VP7 EXPRESSED BY HSV-1

many viral proteins are transported to the cell nucleus (Roizman and Sears, 1990). Nevertheless, RH135-expressed rotavirus VP7 received proper ERtargeting, as indicated by the glycoprotein’s high mannose-type side chain, intracellular retention, and perinuclear to cytoplasmic staining pattern. This finding complements the observation that hepatitis B virus (HBV) surface antigen, a transmembrane protein that buds in spherical particles from HBV-infected cells, also forms spherical particles when expressed from the HSV-1 genome (Shih et a/., 1984). Because some VP7-specific neutralizing mAbs react poorly or not at all by Western blot and immunoprecipitation (Shaw eta/., 1986) we used immunohistochemical staining to detect neutralizing epitopes on VP7. The dehydrating and denaturing fixative methanol destroyed all VP7 neutralizing epitopes tested, including epitopes mapping to the A and C regions of the glycoprotein (Mackow et al., 1988). In contrast, fixation with the cross-linking agent paraformaldehyde preserved all VP7-specific epitopes examined on rotavirus-expressed VP7. This finding indicates that the conformation of neutralizing epitopes on rotavirus-expressed VP7 requires the maintenance of noncovalent interactions. Although RH135-expressed VP7 was recognized by polyclonal antisera and nonneutralizing mAbs and was properly localized, glycosylated, and disulfide bonded, neutralizing mAbs failed to detect the RH135-expressed protein by immunoperoxidase staining, regardless of fixation technique. We considered three explanations for RH135-expressed VP7’s lack of reactivity with neutralizing mAbs. First, RHl35-expressed VP7 might simply lack neutralizing epitopes; second, neutralizing epitopes on RH135-expressed VP7, but not on rotavirus-expressed VP7, might be destroyed by paraformaldehyde fixation or Triton X-l 00 permeabilization; third, neutralizing mAbs might be less sensitive than nonneutralizing mAbs for detecting small amounts of VP7. To distinguish between these explanations, we set up a sensitive and specific capture ELISA as described (Padilla-Noriega et a/., 1990). Antigen was prepared by freeze-thawing and sonicating infected cells, eliminating destruction of epitopes by fixation or permeabilization. In addition, the m.o.i. was adjusted so that RH135-infected cell lysates contained at least as much VP7 as SAl l-infected cell lysates, as measured by M60 capture ELISA. The capture ELISA showed that the neutralizing mAbs 4C3, 4F8, and 57-8 did not recognize RHl35-expressed VP7, even when Tween 20 was omitted from the assay. Therefore, RH135-expressed VP7 lacked several neutralizing epitopes that were present on SAl 1-expressed VP7.

29

In contrast, Ml 59 capture ELISA detected RH135expressed VP7 as well as it detected SAl 1 rotavirusexpressed VP7. Since Ml 59 reactivity was not reduced by treating antigen with Triton X-100, we concluded that in fixed cells, paraformaldehyde crosslinking had masked the M 159 epitope on RH135-expressed VP7, but not on SAl l-expressed VP7. In published competition ELISA studies, prebound Ml 59 blocked six of seven other VP7-specific neutralizing mAbs from binding to rotavirus strain RRV,but no other mAb could block Ml 59 binding (Shaw et al., 1986). Perhaps M 159 binds its conformational epitope avidly enough to block binding of other neutralizing mAbs and to stabilize free VP7 in a folded conformation. In this case, cross-linking RH135-expressed VP7 might prevent it from transiently folding into the proper conformation for M 159 binding, causing the observed loss of M 159 reactivity upon paraformaldehyde fixation. The complexity of VP7 neutralization domains is emphasized by the observation that, while mAbs 4C3, M 159, and 57-8 all select the same amino acid (number 94) in escape mutant studies with RRV(Mackow et al., 1988), all three mAbs have distinct binding characteristics: 57-8 is the only heterotypically neutralizing mAb of the three (Benfield et al,, 1987), M 159 is the only mAb that recognized its epitope on RH135-expressed VP7, and 4C3 neither neutralizes heterotypitally nor recognized RH135-expressed VP7 (Shaw et al., 1986). Because rotavirus replicated in an RHl35-infected monolayer, we could determine whether the antigenicity of RH135-expressed VP7 was altered by interaction with proteins of a heterologous rotavirus in coinfected cells. Although the neutralizing mAbs Ml 59, 4F8, and 4C3 did not immunostain their conformational epitopes in MA1 04 cells infected with RH135 or rotavirus strain NCDV alone, they all recognized their epitopes in RHl35- and NCDV-coinfected cells. Apparently, through interaction with other rotavirus proteins, RHl35-expressed VP7 acquired the 4C3 and 4F8 epitopes, and its Ml 59 epitope became resistant to masking by paraformaldehyde cross-linking. This epitope recovery was not limited to coinfections with rotavirus strain NCDV, since some recovery of the 57-8 neutralizing epitope has also been observed in cells coinfected with RH135 and rotavirus strain AR2, an avian strain that does not bear the 57-8 epitope (not shown). Gaps in the perinuclear staining of individual coinfected c’ells and the absence of recovered epitopes in most coinfected cells indicate that the interaction between RH 135 and NCDV is complex. Interference between the viruses due to host shut-off functions or partitioning of a coinfected cell could explain these obser-

30

DORMITZER

vations. Suppression of HSV-1 -induced syncitium formation by coinfection with rotavirus is an observable form of interference between the viruses. The mechanism of syncitium suppression is not simply elimination of the HSV-1 infection, since NCDV proteins interacted with VP7 expressed from a HSV-1 yl promoter in coinfected single cells and in syncitia. The reduction in the proportion of full double-shelled NCDV particles relative to empty double-shelled NCDV particles produced in a coinfected monolayer could reflect HSV-1 interference with rotavirus genome packaging. Further studies will examine the viral interactions in greater detail. Kabcenell et al. (1988) reported pulse-chase experiments on SAl l-infected cells in which virion-associated VP7, recognized by a neutralizing mAb (72A4), was detected 15 min after membrane-associated VP7, recognized by an antiserum against gel-purified VP7 (Kabcenell et a/., 1988). The authors suggested that the membrane-associated VP7 was the precursor to virion-associated VP7. We considered the possibility that RH 135-expressed VP7 acquired neutralizing epitopes as it became associated with NCDV virions. In fact, unlike the MGO-reactive VP7 in a RHl35-infected cell lysate, the 4C3-reactive VP7 in a RHl35- and NCDV-coinfected cell lysate could be concentrated in a pellet of particles with sedimentation coefficients greater than approximately 955. The lack of quantitative concentration of the 4C3 epitope by pelleting could indicate the presence of additional nonpelletable 4C3-reactive antigen, incomplete resuspension of the pellets, or destruction of particles during the procedure. By CsCl density gradient analysis, the recovered 4C3 epitope in RH135- and NCDV-coinfected cells was detected on particles with a density of 1.29 g/ml, corresponding to empty double-shelled NCDV particles. In contrast, in RHl35-infected cells there was no 4C3-detectable VP7, and the MGO-detectable VP7 had a density of 1.28 g/ml or less, with M60 reactivity peaking in fractions from 1.24 to 1.25 g/ml. Thus, in coinfected cells, the RH 135expressed VP7 that acquired the 4C3 epitope had shifted from the density of soluble protein to a density consistent with incorporation into empty, double-shelled rotavirus particles. The lack of 4C3 reactivity at the density of full double-shelled partcles in the coinfected cell lysate may reflect either a specific lack of incorporation of RH 135-expressed VP7 into full double-shelled particles orthe reduced production of full double-shelled particles in coinfected cells noted by direct coat ELISA. These findings imply that VP7 acquires its full complement of neutralizing epitopes as it assembles into double-shelled particles. If VP7 neutralizing epitopes form on less stable or less abundant intermediates be-

ET AL.

tween free VP7 and double-shelled particles, they could be overlooked by ELISA of sucrose pelleted material or CsCl gradient fractions. In fact, the amount of RH 135-expressed VP7 incorporated into rotavirus particles was modest, as indicated by the limited 4C3 capture ELISA reactivity of the coinfected cell lysate and by the inability of SAl 1-specific neutralizing mAbs to detectably inhibit hemagglutination or infection by rotavirus particles produced in the coinfections (not shown). This study has practical implications for rotavirus vaccine development. It indicates that a recombinant vaccine expressing VP7 alone may present certain neutralizing epitopes, such as that recognized by M 159, to the immune system; however, to present the full complement of neutralizing epitopes, VP7 will either have to be expressed together with other rotavirus proteins or be engineered to fold into a mature conformation. Indeed, this study may provide an explanation for the rather poor neutralizing antibody response elicited by immunization of animals with expressed native VP7 (Andrew et al., 1987). This study also demonstrates that HSV-1 is a useful vector for studying protein processing, localization, and antigenicity. Directions for future research include determining the proteins with which VP7 must interact to attain its mature conformation and testing the usefulness of recombinant HSV-1 vectors for studying protein immunogenicity.

ACKNOWLEDGMENTS We thank Richard Bellamy for generously providing the cloned copy of SAl 1 gene segment 9 and Lenore Pereira for mAb H233 and the gB promoter. We thank Koki Taniguchi for mAb YO-lE2. We thank Jack Stevens for the KOS strain of HSV-1. We thank Phuoc Vo for tissue culture work. This investigation was supported by PHS Grant NRSA 5T32 CA 09302, awarded by the National Cancer Institute, DHHS; by NIH Grants R03 Al30346 and R22 Al21 362, awarded by the National Institute of Allergy and Infectious Disease, DHHS; and by a student fellowship from the American Gastroenterological Association. Note added in proof. The VP7 C region-specific neutralizing mAb YO-1 E2 (Nishikawa ef a/., 1989) was used in the following assays: immunoperoxidase staining of SAl 1-infected, methanol- or paraformaldehyde-fixed MA1 04 cells; immunoperoxidase staining of RH135infected, NCDV-infected, or RH135- and NCDV-coinfected, paraformaldehyde-fixed MA104 cells; and ELISA of SAl l- or RH135infected MA1 04 cell lysates. By all assays, YO-1 E2 behaved like the VP7 A region-specific neutralizing mAbs 4F8 and 4C3, indicating that the conformation of both the A and C regions of VP7 are dependent on interaction with other rotavirus proteins.

REFERENCES W. R., and KRAFT, L. M. (1967). Electron-microscopic study of the intestinal epithelium of mice infected with the agent of epi-

ADAMS,

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VP7 EXPRESSED BY HSV-1

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