expression of avian reticuloendotheliosis virus envelope confers host resistance

VIROLOGY 173,167-l 77 (1989)

Expression of Avian Reticuloendotheliosis Virus Envelope Confers Host Resistance

MARK J. FEDERSPIEL,*+’ LYMAN B. CRIll-ENDEN,t AND STEPHEN H. HUGHES**2,3

*BRI-Basic Research Program, NCI-Frederick Cancer Research Facility, P. 0. Box B, Frederick, Maryland 2 170 1; and WSDA Regional Poultry Research Laboratory, 3606 East Mount Hope Road, East Lansing, Michigan 48823

Received February 27, 1989; accepted June 27, 1989

We constructed two reticuloendotheliosis virus (REV) envelope gene expression plasmids, one containing the REV- A envelope gene, the other the spleen necrosis virus (SNV) envelope gene. Cell lines were generated by transfecting each of the REV envelope plasmids into D17 cells, a canine cell line. The levels of REV envelope glycoprotein in the cell

lines were assayed by immunoprecipitating the envelope glycoproteins from lysates of cells that were labeled with [%]methionine. Virological challenge assays determined the degree of resistance of each of the cell lines to REV-A or SNV infection. The expression of either envelope gene protected the cells from infection by either REV-A or SNV virus.

Several cell lines were significantly more resistant to REV infection than the parental D17 cells, and two lines were 25,000-fold more resistant, approaching the resistance of REV-infected D17 cells to reinfection. The resistant cell lines

were not able to confer resistance to susceptible cells by cocultivation. The level of resistance was correlated with the uniformity of expression of the REV envelope glycoproteins by the individual cells in a cell line and not with the absolute

level of expression by the population of cells. 0 1989Academic press, IIIC.

INTRODUCTION

The life cycle of retroviruses begins with the binding of the virion to a specific receptor host cell and penetration of the cell membrane by the virion. This interaction between the viral envelope glycoprotein and a specific host cell surface receptor is the first step that deter- mines whether a cell is susceptible to infection by a particular retrovirus (Weiss, 1982). Retroviral particles absorb to both susceptible and resistant cells, but the virion successfully penetrates only those cells that express the specific receptor on the cell surface (Critten- den, 1968; Piraino, 1967). Cell surface resistance to infection can occur because (1) the cell is genetically resistant; e.g., the specific receptor is not exposed on the surface of the cell or (2) the receptors are saturated with glycoprotein physically blocking the receptor, a phenomenon known as receptor interference (Steck and Rubin, 1966; Vogt and Ishizaki, 1966). Envelope glycoproteins produced from an exogenously acquired provirus or from an endogenous provirus can block the corresponding host cell receptor. Detailed host susceptibility and resistance studies have been done with avian and murine retroviruses (Crittenden, 1968; Hart- leyeta/., 1970, 1977; Levy, 1973, 1975; Piraino, 1967; Steck and Rubin, 1966; Vogt and Ishizaki, 1966; Weiss,

’ Present address at NCI-Frederick Cancer Research Facility. ’ To whom requests for reprints should be addressed. 3 The contents of this publication do not necessarily reflect the

views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organiza-

tions imply endorsement by the U.S. Government.

1982). The host range of both avian and murine C-type retroviruses are determined by envelope-receptor interactions and can be classified into interference groups.

Avian leukosis viruses (ALVs) have been classified into five envelope subgroups (A-E) based on host- range, neutralizing antibody specificity, and cross-interference of receptors (Weiss, 1982). ALV-infected chicken cells are resistant to reinfection with the same subgroup ALV, presumably because the envelope glycoproteins physically interfere with cell receptor binding and prevent virus penetration. Chicken cells that carry defective subgroup E endogenous proviruses (ev3 and ev6) that express only the envelope glycoproteins are highly resistant to subgroup E ALV infection both in vitro and in vivo (Robinson eta/., 1981). In 1986, Crittenden and Salter proposed a method for producing resistance to subgroup A ALV, the most common subgroup found in field strains of ALV, based on this endogenous ALV interference model.

We have previously described the generation of transgenic chicken lines that carry either a recombinant or wild-type subgroup A ALV provirus (Salter et al., 1987; Crittenden et al., 1989). One transgenic line, alv6, carries a defective recombinant ALV that expresses only the subgroup A envelope glycoprotein (Salter and Crittenden, 1989). a/v6 chicken embryo fi- broblasts (CEF) show a 5000-fold increase in resistance to infection by subgroup A Rous sarcoma virus (RSV). a/v6 chickens that were inoculated at 1 day of age with a subgroup A ALV field strain and constantly

167 0042-6822189 $3.00 CopyrIght 0 1989 by Academic Press. Inc. All rights of reproducton in any form reserved.

168 FEDERSPIEL, CRITTENDEN, AND HUGHES

exposed to pathogenic subgroup A virus showed no evidence of infection by the exogenous subgroup A ALV or related pathology to 40 weeks of age. Presum- ably, the mechanism for the resistance is receptor interference (Robinson et a/., 1981). The ah6 interference is specifically limited to the subgroup A receptors, since a/v6 birds are susceptible to subgroup B ALVs. The objective of the work described here was to determine if viral interference caused by expressing only the envelope gene can be used to induce high levels of resistance to other avian retroviruses, specifically reticuloendotheliosis virus (REV).

The REV family is a group of closely related retroviruses that have a common morphology, share anti- genie determinants (Kang eta/., 1975) and have exten- sive nucleotide sequence homology (Kang and Temin, 1973). Despite the fact that they replicate in avian spe- cies, this group is more closely related to mammalian retroviruses (Barbacid et al,, 1979; Charman et a/., 1979) than to the ALV group of avian viruses (Kang and Temin, 1973; Maldonado and Bose, 1973; Theilen et al., 1966). REVS have not been clearly divided into func- tional subgroups with different host range and serologi- cal properties, although the different isolates can be distinguished with monoclonal antibodies (Cui et a/., 1986). The REVS include one replication-defective transforming virus, REV-T, that carries the oncogene re/(Robinson and Twiehaus, 1974). The defective REV- T virus, when inoculated into chicks, causes acute re- ticulum cell neoplasia causing death after 3 to 21 days postinoculation (Witter, 1984). There are more than 26 biologically cloned, nondefective REV isolates (Chen et a/., 1987). Two isolates that have been studied extensively are REV-A, the helper virus originally isolated with REV-T (Hoelzer et a/., 1979) and spleen necrosis virus (SNV) (Trager, 1959). Nondefective REVS cause a wide spectrum of slow developing (latencies greater than 8 weeks) pathologic lesions in poultry including splenomegaly, spleen necrosis, lymphoproliferative nerve lesions, B-cell and T-cell lymphomas, and ane- mia (Witter, 1984). The nondefective REVS also rapidly and severely depress the cellular immune response in infected birds (Rup et a/., 1979, 1982; Smith and van Eldik, 1978). REVS can be propagated in a variety of primary avian cells and in several permissive dog, mink, and rat cell lines (Keshet and Temin, 1979; Witter, 1984).

The REV envelope gene is 1750 base pairs (bp) in length and encodes two glycoproteins (Tsai et al., 1986). The primary polyprotein precursor, gPr77e”“, is glycosylated and converted into the secondary polyprotein precursor gPr1 1 gen”, that is rapidly processed proteolytically to gp90, the surface glycoprotein, and gPr22(E) (Tsai and Oroszlan, 1988). The final modifica-

tion of gPr22(E), the transmembrane glycoprotein precursor, to the mature gp20 occurs after incorporation into the virion (Tsai and Oroszlan, 1988).

We have constructed two expression plasmids, one containing the REV-A envelope gene, the other the SNV envelope gene. Cell lines were generated by transfecting each of the REV envelope expression plasmids into D17 cells, a canine cell line. Several cell lines were significantly more resistant to REV infection than to the parental D17 cells, and two lines were 25,000- fold more resistant. The expression of either the REV- A or SNV envelope gene protected the cells against infection by either REV-A or SNV virus. The resistant cell lines did not confer resistance to REV infection to susceptible cells in trans. The level of resistance correlated with the uniformity of expression of the REV envelope glycoprotein by every cell in the cell line and not with the level of expression of the total population of cells measured by immunoprecipitation.

MATERIALS AND METHODS

Enzymes and chemicals

All enzymes were purchased from either New En- gland Biolabs or Boehringer-Mannheim Biochemicals and used under conditions recommended by the man- ufacturer. SeaKem LE and SeaPlaque agarose were purchased from the FMC Corporation. Ultrapure acrylamide was purchased from Bio-Rad Laboratories.

Cell lines and viruses

The D17 cell line (American Type Culture Collection CCL 183) a continuous cell line derived from a canine osteosarcoma, was grown in Fl O-l 99 medium (F-l 0 Nutrient Mixture and Media 199 from GIBCO containing 0.15% (w/v) tryptose phosphate broth, penicillin, streptomycin, fungizone, and nystatin) plus 109/o fetal bovine serum. Line 0 CEF (C/E, ev gene negative) (As- trin et a/., 1979) was grown in Fl O-l 99 medium plus 4% calf serum. The D17-C3-21-A2 cell line (Watanabe and Temin, 1983) a gift from H. Temin (University of Wisconsin-Madison), was grown in Fl O-l 99 medium plus 10% fetal bovine serum and 100 pglml hygromycin B (Calbiochem). Thevirus, ID21 5HYRAM, is produced by the D17-C3-21 -A2 cell line. The JD215- HYRAM proviral genome contains an SNV LTR, the hygromycin resistance gene, an RSV LTR, the neomycin resistance gene with an amber mutation, and an SNV LTR. The D17-C3-21 -A2 cell supernatants were collected as the JD215HYRAM viral stock and stored at -70”. REV-A and SNV viral stocks were aliquots from cloned viral stocks (Chen et a/., 1987) obtained from R. L. Witter (USDA-RPRL, East Lansing, MI).

REV ENVELOPE INDUCES RESISTANCE 169

Construction of the REV envelope expression vectors and cell lines

The REV-A envelope gene was isolated from the plasmid pSW283 (Watanabe and Temin, 1983) and the SNV envelope gene from the plasmid pPB101 (Ban- dyopadhyay and Temin, 1984) both gifts from H. Temin, and were then inserted into the adaptor plasmid, pCLA12NCO (Hughes et al., 1987) in two steps. pCLA12NCO is a pBR327-derived plasmid that contains a polylinker region and a eukaryotic transcription leader flanked by C/al sites. An oligonucleotide replac- ing the segment of REVenvfrom the initiator ATG (position 1359) (Shimotohno et al., 1980; Wilhelmsen et al., 1984) to the Hincll site (position 1398) of the envelope gene was synthesized and ligated to the env segment from Hincll (position 1398) to BamHl (position 2033) in the presence of the large Ncol-BarnHI segment from pCLAl2NCO. The resulting plasmid was digested with BarnHI and Sacl and ligated to the env segment from BarnHI (position 2033) to Sacl (position 3143) to complete the envelope adaptor construction.

The REV envelope genes were excised from the adaptor plasmids by C/al digestion and subcloned into the expression vector TFANEO (S. Hughes and J. Greenhouse, unpublished) and named TFREVAE and TFSNVE (Fig. 1). TFANEO is an ampicillin-selectable plasmid that contains the neo gene (Jorgensen et a/., 1979) expressed because it is linked to a 340-bp segment containing the chicken P-actin promotor (C. Or- dahl, University of California, San Francisco). The expression cassette of TFANEO consists of two LTRs derived from Schmidt-Rupin A (SR-A) Rous sarcoma virus (RSV) that provides both a transcriptional promotor and polyadenylation site. A unique C/al cloning site lies between the two LTRs. A region containing several restriction enzyme sites separates the neo gene and the LTRs. These sites were used to linearize the plasmid prior to transfection, providing a known breakpoint in the DNA for integration into the host genome.

Twenty neomycin-resistant cell lines were isolated, 10 from TFREVAE and 10 from TFSNVE DNA transfections. In a standard transfection, 5 pg of CsCI, banded DNA was digested with /Votl to linearize the plasmid. The linear plasmid was introduced into D17 cells by CaPO, transfection (Graham and van der Eb, 1973; Wigler et a/., 1979). The transfected cells were grown with 500 pg/ml G418 to select for neomycin-resistant cells. Single colonies were isolated and maintained in the presence of 200 pglml G418.

Southern transfer

DNA was isolated from confluent cells by extraction with 250 pg/ml pronase (Calbiochem), 0.5% sodium

dodecyl sulfate (SDS), 100 mlVl NaCI, 20 mAITris-HCI, 10 mM EDTA, pH 8.0. Samples were prepared for Southern transfer to Nytran (Schleicher & Schuell) and probed with 32P-labeled, nick-translated DNA accord- ing to standard procedures (Maniatis et al., 1982).

Radiolabeling of cell cultures

Confluent cell monolayers in 60-mm plates were incubated for 2 hr at 37” in methionine-free RPMI 1640 (GIBCO) plus 5% dialyzed calf serum and then labeled with 60 &i/ml [35S]methionine (New England Nuclear) for 6 hr (Silva and Lee, 1984). The cells were lysed in 2 ml lysis buffer (1 o/o Triton X-l 00, 1% sodium deoxycho- late, 150 mn/r NaCI, 50 mM Tris-HCI, pH 7.5) at 4” for 15 min. The cell debris was removed by centrifugation at 12,000 g (Eppendorf microfuge) and SDS added to the supernatant to a final concentration of 0.1% (Bra- dac and Hunter, 1984). The incorporated [35S]- methionine was quantitated bytrichloroacetic acid pre- cipitation (Maniatis et al., 1982).

lmmunoprecipitations

In a standard immunoprecipitation (Bradac and Hunter, 1984) 1.2 X 10’ cpm of 35S-labeled cell lysate was mixed with 40 /*I anti-REV-A rabbit antisera (Cui et a/., 1986) and incubated at 37” for 1 hr, and the immune complexes were collected with 100 ~1 of a 1 Oq/o cell suspension of fixed, killed Staphylococcus aureus (Boehringer-Mannheim) at 25” for 30 min. The precipi- tate was washed three times with 1 ml lysis buffer.

SDS-PAGE and autoradiography

The immunoprecipitates were resuspended in 50 ~1 of gel sample buffer(75 mMTris-HCI, pH 6.8,2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 10 pg/ml brom- phenol blue), heated to 100°C for 2 min to dissociate the immune complexes from the bacteria, cooled, and fractionated on Laemmli SDS-polyacrylamide gels (Laemmli, 1970) with a 4% stacking and a 12% resolving gel. The gels were stained with 0.25% brilliant blue R (Sigma) and destained. The gels were then soaked in 5 vol of Enlightning (New England Nuclear) for 30 min with agitation, dried, and exposed to Kodak X-OMAT film.

Indirect fluorescent antibody virus assay

In a standard assay 2 X 1 O5 D17-REV envelope cells were plated onto a 35-mm plate, infected with either REV-A or SNV at a multiplicity of infection (m.o.i.) of 0.5, and incubated 6 days with one media change, and the supernatants were collected and stored at -20”. REV virus was assayed by indirect fluorescent antibody as-


say as described previously (Chen et al., 1987). Briefly, IO-fold serial dilutions of the harvested supernatants were plated onto line 0 CEF freshly seeded into wells of 96-well plate (4 X 1 O4 CEF/well) and incubated for 3 days. The cells were fixed with ethanol:methanol (4:6) at 25” for 15 min, and air-dried. The fixed cells were reacted with a 1:ZOO dilution of the monoclonal antibody 1 lA25 (Cui et al., 1986) kindly provided by L. F. Lee (USDA-RPRL, East Lansing, Ml), that binds to gPr22(E), at 37” for 30 min, and then mixed with a 1: 20 dilution of fluorescein-conjugated rabbit anti-mouse IgG (H + L) (ICN Biomedicals) at 37” for 30 min. REV infectious centers were visualized and counted with a Leitz fluorescent microscope with Ploem illumination at a magnification of 100X.

Hygromycin virus assay

In a standard assay, 2 X 1 O5 D 17 cells were plated onto a 35-mm plate and infected with 1 O-fold serial dilutions of JD215HYRAM (undiluted m.o.i. of 1 .O), with 2 pg/ml DEAE-Dextran to improve the efficiency of infection. After 24 hr, the medium was replaced with medium containing 100 pg/ml hygromycin B. The medium was changed several times as needed during the experiment. Hygromycin-resistant colonies (al 0 cells) were counted 14 days postinfection.

Fluorescent photomicroscopy

Confluent monolayers of the REV-A envelope cell lines, grown on four chamber glass slides (Lab-Tek), were washed twice with pH 7.45 phosphate-buffered saline (PBS). The live cells were reacted with a 1:200 dilution of the monoclonal antibody 1 1 Cl 00 (provided by L. F. Lee) which specifically binds to REV-A gp90 (Cui et a/., 1986) at 4’ for 30 min, washed three times with PBS, and then mixed with a 1:20 dilution of fluorescein-conjugated rabbit anti-mouse IgG (H + L) at 4” for 30 min. The cells were washed three times with PBS, fixed with ethanol:methanol (4:6), and covered with PBS:glycerol (1: 1). The labeled cells were photo- graphed with a Leitz fluorescence microscope with Ploem illumination at a magnification of 250X using Ko- dak Tri-X pan 400 film at 1600 ASA.

RESULTS

REV envelope construction

TFREVAE and TFSNVE were constructed by intro- ducing the REV envelope gene into the expression plasmid TFANEO using adaptor plasmids. The restriction enzyme sites used in constructing the REV enve-

EcoRl

TFSNVE

TFREVAE

FIG. 1. Maps of the TFREVAE and TFSNVE plasmids. This figure shows the map of the REV-A envelope plasmid TFREVAE and the SNV envelope plasmid TFSNVE, both constructed in the expression

plasmid TFANEO as described under Materials and Methods. TFA- NE0 is 6.9 kb and TFREVAE and TFSNVE are 8.8 kb in size. The TFANEO plasmid contains the Tn5 neo gene (NEO) expressed from

a 340-bp chicken @actin promotor region (&I) and an expression cassette consisting of two RSV LTRs (II). The plasmid also contains

the amp gene (AMP), an fscherichia co/i origin of replication (ORI),

and linearization region (B) that contains the recognition sites for several restriction enzymes. The arrows designate the direction of transcription. The C/al adaptor fragments containing REV-A env

(TFREVAE) and SNV env (TFSNVE) were inserted into the C/al site of TFANEO (LTR-ENV). The map shows the restriction enzyme sites

used in the envelope adaptor plasmid construction. The REV-A and SNV envgenes have restriction site differences that are not shown.

lope genes are shown in Fig. 1. The initiator ATG is located at the IVcol site and the construction extends to the Sac1 site, 37 bp downstream of the REV envelope gene translation-termination codon. A synthetic oligonucleotide, encoding the first 43 bp of the REV envelope gene, was used to link the initiator ATG to the Ncol site. An authentic eukaryotic initiator ATG (at the A/co1 site) and an acceptable untranslated leader sequence are present between the upstream C/al and the Ncol sites in the adaptor plasmids pCLA12NCO sequence (Hughes eta/., 1987).

REV envelope cell line characterization

Twenty neomycin-resistant cell lines were generated by CaPO, transfection (Graham and van der Eb, 1973; Wigler et a/., 1979) of TFREVAE DNA (Rl-10) and TFS- NVE DNA (Sl-10) into D17 cells. The D17 cell line, which has been used extensively in retroviral research (Watanabe andTemin, 1982,1983) was used since the available continuous chicken cell lines are either in-


fected with avian retroviruses or grow only in suspension. Although RSV will not replicate in most mammalian cells, the RSV LTR promotor is appropriately expressed in mammalian cells (Bauer and Janda, 1967). The chicken fl-actin and RSV LTR promotors of TFA- NE0 were known to exhibit a low to moderate promotor activity in D17 cells (J. Casey, personal communica- tion). The different REV envelope cell lines exhibit mor- phological variation; approximately one-half of the lines produce syncytia (data not shown). We believe this cell fusion results from the interaction between the envelope glycoproteins and cellular receptors (Diglio and Ferrer, 1976; Roizman, 1962; Rowe et al., 1970). Syn- cytia were only detected in cell lines that expressed relatively low levels of REV envelope glycoprotein as estimated by immunoprecipitation and fluorescent staining (data not shown). Only small syncytia (5 to 10 fused cells) were observed and affected less than 1% of the cell population. Cell line R3 was the exception with syncytia affecting approximately 5% of the population (see Discussion).

An analysis of the integrated REV envelope sequences in the neo-resistant cell lines is shown in Fig. 2. TFANEO contains three EcoRl sites, one site upstream of the p-actin promotor, and one site in each RSV LTR (see Fig. 1). EcoRl digestion of TFREVAE and TFSNVE DNA generates a 2.8-kb (kb) fragment that contains the REV envelope gene. Only EcoRl fragments containing REV envelope sequences should be detected when EcoRl digested genomic DNAs are probed with REV envelope sequences in a Southern transfer assay. Host-vector DNA junction fragments will not be detected unless a rearrangement occurs that eliminates a flanking EcoRI site. In addition to cell lines that contained the expected 2.8-kb EcoRl fragment, there were cell lines that contained no detectable REV envelope sequences and others that contained rearranged REV sequences (Fig. 2).

The level of expression of the REV envelope glycoproteins was analyzed by immunoprecipitation (see Fig. 3). Cell lysates, labeled with [35S]methionine for 6 hr(Silva and Lee, 1984) were immunoprecipitated with an anti-REV-A rabbit antisera (Bradac and Hunter, 1984) and the precipitates fractionated on SDS polyacrylamide gels (Laemmli, 1970). Under these conditions, three intracellular REV envelope glycoprotein forms were expected; the mature surface glycoprotein gp90, the primary unglycosylated polyprotein precursor gPr77e”“, and the transmembrane glycoprotein precursor gPr22(E) (Trager, 1959; Tsai et al., 1986). Pre- cipitations of envelope proteins from virally infected cells (lanes D17 + REV-A and D17 + SNV) in Fig. 3 show the three envelope proteins. The cell lines that did not contain detectable levels of REV glycoprotein

FIG. 2. Analysis of the integrated REV envelope sequences in the REV envelope cell lines. The figure shows 10 rg EcoRl digested DNA

from the REV-A envelope cell lines (Rl-1 O), SNV envelope cell lines (Sl-1 0), and the parental D17 cell lines (D17) hybridized with the C/al

fragments containing the REV-A and SNV envelope genes (see Fig. 1). The REV envelope genes contain no EcoRl sites and should ap-

pear as a 2.8.kb fragment that extends from the EcoRl sites in the

flanking LTRs (see Fig. l), if there were no rearrangements during transfection.

(Fig. 3) were those lacking the 2.8-kb EcoRl fragment (see Fig. 2). All cell lines that contain this segment express the REV envelope glycoproteins, although the level of expression varies among the cell lines.

Virological challenge assays

Two different REV virus challenge assays were used to measure the resistance of the REV envelope cell lines relative to the parental D17 line. The indirect fluorescent antibody (FA)virus assay, described under Ma- terials and Methods, measured the REV virus released by the REV cell lines following infection. The second assay measured the efficiency of infection of the cell lines by JD215HYRAM, a defective SNV-based virus that contains the hygromycin resistance gene and is encapsidated into virions that have REV-A envelope glycoproteins. Both assays are measuring resistance


92.5 K

66.2 K

45.0 K

31.0 K

21.5 K

14.4 K

-gPr22(E)

FIG. 3. lmmunoprecipitation of the REV envelope glycoproteins expressed by the REV envelope cell lines, 35S-labeled cell lysates from the REV-A envelope cell lines (Rl-1 0), SNV envelope cell lines (Sl-1 0), parental D17 cells (D17), and D17 cells infected with REV-A (D17 + REV-A)

or SNV (D17 + SNV) were prepared as described under Materials and Methods. Samples were reacted with anti-REV-A rabbit antisera, precipitated with Sfaphlococcus aureus, washed, and analyzed on SDS-PAGE (4% stacking and 12% resolving gels). The REV envelope glycoproteins are indicated: the primary polyprotein precursor gPr77env, the mature surface glycoprotein gp90, and the transmembrane glycoprotein precursor gPr22(E). The 30.kDa protein that is seen in lanes that contain immunoprecipitates from infected cells migrates at the position expected for

the major gag protein of REV.

as a decrease in virus or colony production. The results of the two virus challenge assays are summarized in Table 1.

Since the susceptibility of the cell lines to either REV- A or SNV infection were similar, the virus JD215- HYRAM was used to generate quantitative data on the resistance of all 20 cell lines. Since the hygromycin virus is defective and is released from a helper cell in the absence of a replication competent helper virus, the hygromycin assay measures only the entry and integration of the JD215HYRAM virus into the cell genome from the initial infection. The infection of 2 X 1 O5 D17 cells with JD215HYRAM (m.o.i. of 1 .O), upon selection with hygromycin, resulted in a confluent monolayer of resistant cells. No background resistant colonies were obtained from hygromycin-selected D17 cells. The ti- ters of hygromycin-resistant colonies obtained from a representative experiment, and the relative resistance of each cell line compared to D17 cells, are shown in Table 1. In several cell lines, S2, S5, S6, and S8, the relative resistance levels measured by the two assays were different (see Discussion). The Rl, R5, R6, R7, R9, and S4 cell lines were clearly resistant to REV infection in both assays. Lines R2 and R4 do not contain detectable REV envelope sequences in their genomes (Fig. 2) do not produce detectable REV glycoprotein (Fig. 3) and show low resistance. Several lines, R3, S2,

S5, and S6, produced large quantities of REV glycoprotein as measured by immunoprecipitation (Fig. 3) yet were relatively susceptible to infection (see Discus- sion).

The two most resistant cell lines, R6 and S4, were tested for their ability to confer resistance to REV infection to susceptible cells in trans. Serial 1 O-fold dilutions of R6 and S4 were mixed with D17 cells and infected with JD21 SHYRAM. As shown in Table 2, the presence of cells producing the envelope glycoprotein did not confer significant resistance to D17 cells in the same culture.

Fluorescent microscopy

To analyze the relative amount of REV envelope glycoprotein on the surface of individual cells, unfixed confluent monolayers of the 10 REV-A cell lines were fluorescently labeled with the monoclonal antibody 1 1 Cl 00, which binds specifically to REV-A gp90 (see Materials and Methods). The levels of glycoprotein expression in 9 of the 10 REV-A cell lines, as measured by FA staining, were judged similar to the expression levels detected by immunoprecipitation in Fig. 3 (data not shown). The one exception was line R3. Fluores- cent micrographs of three representative REV-A cell lines, R3, R6 and R7, are shown in Fig. 4. The R3 line,


TABLE 1

VIROLOGICAL ASSAYS TO DETERMINE THE RESISTANCE OF THE REV ENVELOPE CELL LINES’

Cell line

REV virus Hygromycin colony

production after number after infection with infection with

Relative

REV-Ab SNVb JD21 5HYRAMC resistanced

Rl 3 0 10 10,000 R2 1 x 10’ 7x 10’ 3x103 33

R3 2x10* 10 1 x103 100

R4 1x102 80 4x lo3 25

R5 20 0 90 1,100

R6 2 0 4 25,000

R7 12 0 30 3,300

R8 5x102 20 2x lo3 50 R9 2 0 30 3,300

RlO 8x10’ 4x10’ 9x lo4 0 Sl 2X lo3 8X 10’ 7x lo5 0

s2 0 0 1 x104 10

s3 2x10” 70 3x lo3 33 s4 0 1 4 25,000

s5 30 0 7x lo3 14

S6 0 0 3x lo3 33

s7 1x104 2x103 1 x1$ 10

58 60 40 1 x105 0 s9 2x 10’ 3x lo2 1 x lo5 0

SlO 1 x10* 7 1 x lo4 10 D17 4x lo3 5x lo* 1 x105 D 1 7/REVAe 1 100,000 ’

a The experiments were repeated twice. The data presented are from one experiment of each type.

b The cell lines were infected at an m.o.i. of 0.5. REV virus production was determined by the indirect fluorescent antibody assay of REV infectious centers described under Materials and Methods.

‘The cell lines were infected at an m.o.i. of 1 .O. d The hygromycin resistance data were used to determine the rela-

tive resistance of the REV envelope cell lines compared to the parental D17 cell line.

e D17 cells were infected with REV-Aat an m.o.i. of 1 .O 6 days prior

to JD215HYRAM challenge. ‘The relative resistance of D17/REV-A was estimated from only

one colony but was similar to other reported levels (Delwert and Pan- ganiban, 1989).

which was relatively susceptible to REV infection (Ta- ble l), appeared to have two different cell populations as shown by FA labeling; (1) cells that expressed high levels of REV glycoprotein and (2) cells that expressed little or no REV glycoprotein (Fig. 4A). Line R6 was ex- tremely resistant to REV infection (Table 1) and uniformly expressed a high level of glycoprotein throughout the cell population (Fig. 4B). Line R7 also had a high relative resistance to REV infection (Table 1). This line expressed a relatively low level of REV glycoprotein uniformly throughout the population (Fig. 4C). Whether every cell in the population uniformly expresses the REV

glycoproteins, even at relatively low levels, appears to determine resistance to infection by REV.

DISCUSSION

The objective of the experiments reported here was to test for increased virus resistance by expressing a cloned, inserted envelope gene in vitro. The envelope genes of two extensively studied REV isolates, REV- A and SNV, were compared for their ability to confer resistance to challenge by REV-A and SNVviruses. We found that by expressing an inserted REV envelope construction in the cell genome, the relative resistance to REV infection was increased up to 25,000-fold in some cell lines. This level of interference approaches the resistance of REV-infected D17 cells to reinfection (see D 17/REVA, Table 1).

The D17-REV envelope cell lines displayed several distinct morphologies. Syncytia were observed in some of the cell lines and were probably caused by the interaction of viral envelope glycoproteins and cellular receptors resulting in fused, polynucleated cells (Diglio and Ferrer, 1976; Roizman, 1962). Syncytia formation is a characteristic of certain retroviral infections in vitro and has been used in quantitative assays to titer viral stocks (Rowe et al., 1970). In the REV cell lines that form syncytia, only a very small proportion of the cells were affected and the isolated cytotoxic effects did not detectably interfere with the viral challenge assays. Small syncytia affecting 1 to 2Ob of the cell population was also observed in experiments in which HIV envelope glycoproteins and CD4 receptor interactions were measured in vitro in relationship to HIV-induced cytotoxic effects (Stevenson et a/., 1988). It may be significant that only the cell lines expressing a low level of REV glycoprotein (as measured by immunoprecipita-

TABLE 2

TESTING REPRESENTATIVE REV env CELL LINES FOR CONFERRING RESISTANCETO REV INFECTION IN tram

Hygromycin- Cell population resistant coloniesa

REV env D17 R6 s4

6x lo5 0 5 6x lo4 5.4 x 1 o5 c9; C 6x lo3 6x105 C C 6X 10’ 6x105 C C 6X 10’ 6x105 C C

0 6x105 C C

a Cell populafions infected with 1 X 1 O4 CFU of JD215HYRAM virus.

b Confluent cell monolayer.

FIG

. 4.

Fluo

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of

cell

surfa

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REV-

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tion and fluorescence) produced syncytia. It is possible that the syncytia were due to incomplete blockade of cellular receptors enabling free receptors on one cell to bind to REV envelope glycoprotein on neighboring cells. Cell line R3 is composed of a mixture of cells that express high and low levels of the REV-A envelope glycoprotein and shows the highest levels of syncytia formation of any of our cell lines, which is consistent with this hypothesis.

The spectrum of integrated TFREVAE and TFSNVE plasmid sequences in the genomes of the various cell lines is characteristic of DNA transfections where rearrangements and deletions often occur (Wigler eT al., 1979). REV envelope glycoproteins were precipitated with anti-REV antisera only from those cell lines that contained detectable REV envelope sequences. REV- A and SNV have distinct pathologies and show some small antigenic differences. Since the anti-REV antisera was raised against REV-A viral antigens, the antisera bound more efficiently to REV-A envelope proteins than to SNV envelope proteins. This may account in part for the differences in the total amounts of REV-A and SNV glycoprotein precipitated, especially gp90 (see Fig. 3). The levels of gPr22(E) did not always correlate with the levels of gp90 and gPr77 env in the immunoprecipitation (see R5, R6, Fig. 3). These discrepancies may be due to variation in the extent of membrane breakdown during cell lysis, causing an incomplete release of membrane proteins.

The relative resistance of the REV cell lines to an REV infection was measured using several different assays. The indirect FA virus assay was used to answer two questions: (1) are there differences in resistance among the different cell lines? and (2) are the resistant cell lines equally resistant to both REV-A and SNV? Re- sults with the FA assay showed that the cell lines have a significant range of resistance to infection by the viruses, and that individual cell lines were equally resistant or susceptible to infection with either REV-A or SNV (Table 1). The resistance of the cell lines was also measured in a second assay in which a viral vector, JD215HYRAM, that carries a gene for resistance to hygromycin, was encapsidated into virions with REV-A envelope glycoproteins.

The resistance levels of several of the cell lines were clearly different as measured by the two assays (Table 1). These discrepancies may have been due to differences in the two methods used to measure resistance. The indirect FA assay depends on two parameters: (1) the extent of REV entry and integration into cells and (2) the subsequent virus production. The hygromycin assay measures only virus entry and integration. In addition, the FA assay is less sensitive than the hygromycin assay. For these reasons we believe that the

hygromycin more accurately measures the levels of resistance induced by expression of the envelope glycoproteins.

We were initially surprised that the level of expression of the envelope glycoproteins, as measured by immunoprecipitation, did not always correlate with the level of resistance to infection. However, since there is no transfer of resistance if cells that produce the glycoprotein are cocultivated with cells that do not, we con- sidered the possibility that some of the cell lines were heterogeneous with respect to the expression of the envelope glycoprotein. Assays of the envelope glycoprotein expression in several cell lines by immunofluo- rescence showed that resistance to viral infection cor- relates with the uniformity of expression of the envelope glycoprotein by the cells. Lines that make relatively large amounts of envelope glycoprotein but show minimal resistance contain significant numbers of cells that express little or no envelope glycoprotein. This heterogeneity could arise in at least two ways. It is possible that the cell lines are not truly clonal. How- ever, it is also possible that the lines are clonal and that the expression is unstable; instability of this type is not uncommon for genes introduced into cultured cells by transfection.

In a recent article, Delwert and Panganiban (1989) reported a 476- and 286-fold increase in resistance to REV infection from two D17 cell lines expressing the SNV envelope gene from an SNV-based expression vector, compared to the parental D17 cells using a hygromycin resistance assay. This is less than 1 o/‘o of the resistance of SNV- or REV-A-infected D 17 cells to reinfection (-80,000-fold). We have found that subclones of the D17 cell line that have survived the transfection process but lack DNA sequences encoding SNV or REV-A envelopes vary more than 1 OO-fold in their relative resistance to viral infection (Table 1). These observations left open the possibility that the expression of the SNV or the REV-A envelope glycoprotein is insuffi- cient to induce the levels of resistance seen in cells infected by the virus and that other viral proteins or components may contribute to the high levels of resistance seen in REV-infected cells.

We have generated cell lines with levels of resistance to viral infection approximating the levels of resistance seen in infected cells (25,000-fold when compared with the parental D17 line). However, because of the variability in the resistance displayed by the subclones that lack envelope DNA, we cannot yet con- clude that this resistance is exclusively due to envelope glycoprotein expression and not partially due to variation in the cells themselves. However, the data do suggest that envelope glycoprotein expression can account for at least a lOOO-fold increase in resistance,


and we suspect, although cannot prove, that the envelope glycoprotein is responsible for most, if not all, of the resistance induced by viral infection.

Our next goal will be to determine if expressing REV envelope in chickens will increase resistance to REV infection. Although it remains to be proven, it is possible that the expression of any one of the REV envgenes can block infection from any of the REV isolates. How- ever, since the expression of the env gene in one cell does not confer resistance on neighboring cells, all the potential REV target cells must express the REV envelope glycoproteins. The REV envelope gene will be tested in chickens using replication-competent and defective ALV vectors. It should be possible to use such vectors to introduce the REV env gene into the germline of chickens.

ACKNOWLEDGMENTS

We are most grateful to Dr. Howard Temin for the gifts of REV-A- and SNV-cloned DNA and the hygromycin vector/helper cell line. We are also grateful to Dr. Lucy Lee for the gifts of REV-recognizing

monoclonal antibodies. We acknowledge the excellent technical as-

sistance of C. Cantwell and B. Riegle, and thank H. Marusiodis for preparing the manuscript. This research was supported in part by

Grant US-8 1 l-84 from BARD, the United States-Israel Binational Ag- ricultural Research and Development Fund, Grant 88-37266-4141 from the U.S. Department of Agriculture, and the National Cancer

Institute, DHHS, undercontract No. NOl-CO-74101 with BRI.

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