AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 8, Number 12, 1992 Mary Ann Liebert, Inc., Publishers

Stable

Expression of the Human Immunodeficiency Virus Type Envelope Glycoprotein in Transfected L Cells

CATHERINA

BIRD,1

PAUL A.

GLEESON,1

ALISTAIR

RAMSAY,2 PENG LI,3

and JAMES

1

McCLUSKEY4

ABSTRACT An SV40-based expression vector was used to generate CD4-negative murine L cell lines which stably expressed the human immunodeficiency virus envelope glycoprotein (env). Despite the presence of abundant intracellular envelope glycoprotein, the expression of env gpl20/41 was not detected on the cell surface. Pulse-chase studies showed that the majority of the gpl20 detected at the end of a 20-h chase was in the culture medium. Therefore gpl20 was shed and/or secreted from these cells. Transfected L cells (H-2k) served as targets for specific lysis by CTL raised against vaccinia virus-encoded env gpl60. The discrepancy in relative levels of intracellular versus surface expression of env was probably due to the highly inefficient processing of newly synthesized gpl60, as well as the apparent instability of the gp 120/41 complex in the transfected cell lines. Digestion of immunoprecipitated gpl20 and gpl60 with endoglycosidase H and peptide N-glycosidase F revealed that the envelope glycoprotein in transfected L cells possessed both high mannose and complex N-glycans, analogous to the posttranslational modification of the mature envelope glycoprotein in infected T cells. These studies indicate that the relatively inefficient processing of env gpl60 occurs in the absence of CD4, and that the stable surface expression of envelope gpl20/41 complex may require additional factors not present in transfected cells.

INTRODUCTION the human immunodefivirus type 1 (HIV-1) is the major immunogenic protein encoded by the virus. It is the surface molecule on infectious virions that binds to CD4 and mediates viral fusion with susceptible host cells. '^4 Envelope glycoprotein appears to play a critical role in the killing of HIV-infected, CD4-positive cells.5-7 The HIV-1 envelope glycoprotein is synthesized on the rough endoplasmic reticulum of infected cells, initially as a 160-kD precursor glycoprotein.8 The precursor protein is subsequently cleaved by cellular proteases to generate the 120-kD glycoprotein, gpl20, which is noncovalently associated with a 41-kD glycoprotein, gp41, anchored in the cell membrane.2'910 Shed and/or secreted gpl20 is also found in the culture superna' tants of virally infected, cultured cells' and has been reported in

THEciency

ENVELOPE GLYCOPROTEIN OF

the serum of patients infected with HIV-1.12 In addition to the structural genes, HIV-1 has several regulatory genes that play an important role in the complex life cycle of the virus in infected cells. Among these regulatory genes, both the tat and rev are for env from the expression gene products required viral genome. In addition, several viral proteins including the nef, rev, and tat gene products may influence the rate of RNA initiation,22-25 whereas the precise role of other proteins such as 30 vif, vpu, and vpr is still under investigation.26 Because of the overall complexity of the regulation of env expression in virally infected cells we, among others,5'7'31 34 have developed expression systems suitable for the study of env expression in cell lines stably expressing the envelope glycoprotein. Stable expression of the envelope glycoprotein has been described in human CD4-positive lymphocyte cell lines such as Jurkat and HeLa cells, and Chinese hamster ovary CEM,5 ~

'Department of Pathology and Immunology, Monash Medical School, Commercial Road, Prahran, Victoria, Australia, 3181. 2Division of Cell Biology, Australian National University, John Curtin School of Medical Research, Canberra, Australia Capital Territory. 3AIDS Research Laboratory, Institute of Medical and Veterinary Science, Frome Road, Adelaide, Australia, 5000. "Department of Clinical Immunology, Flinders Medical Centre, Flinders University, Bedford Park, Adelaide, Australia, 5042; and The Australian Red Cross Society, Blood Transfusion Service, 301 Pirie Street, Adelaide, Australia, 5000. 1999

20(H)

BIRD ET AL.

cells.32'34 However, for studies of immune recognition and antigen presentation in mice, the availability of stable murine cell lines have proven to be particularly valuable35 because these

are easy to grow and transfect with multiple genes includhuman ing major histocompatibility complex (MHC) genes which allows them to serve as targets for human as well as murine T cell recognition. Moreover, because L cells do not express CD4, the processing and transport of env can be studied under circumstances where CD4-gp 160 complexes do not form intracellularly. In this report, we describe the stable expression of the HIV-1 envelope glycoprotein in transfected murine L cells and their recognition by env-specific CTL from H-2k mice. In addition, we have characterized the biosynthesis, processing, and pattern of expression of env in these cells. Our findings indicate a marked discrepancy between the level of intracellular and surface env expression detectable by direct assay systems. We suggest that the stable membrane expression of gp 120/41, on the surface of virions or on virally infected cells, may depend upon other viral or cellular proteins absent in transfected L cells.

cells

METHODS Plasmids The

plasmids pSVTenv(tat+rev+) and pSVTenv(tat-rev+)

have:been described previously.3 Briefly, a 3.1-kb Sal I-Xho I fragment of the cloned HIV-1 provirus, pHXBc2, was subcloned into the Sal I site of the expression vector, pSVT7.37The

resulting plasmid,pSVTenv(tat+rev + ), possesses the complete coding regions of tat, rev, and env with intact translational initiation codons ( + ) for all three genes. The plasmid pSVTenv(tat-rev+) was derived from a mutant (mutant 80:tat[-]) of the HIV-1 clone, pHXBc2, in which the translational initiation codon for the to? gene had been removed ( ) by site-directed mutagenesis (the gifts of R. Sadaie and F. WongStaal; 38). To facilitate selection of env transfectants, the dominant selectable marker gene, Ecogpt, was subcloned as a 2.4-kb Pvu II-Pst I fragment (from pSV2gpt; 39) into the unique Cla I site of the pSVTenv constructs, located upstream of the SV40 origin of replication. —

Cells Murine L cells (LTA.5, the gift of M. Sandrin) were maintained as monolayers in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, glutamine, and antibiotics (complete DMEM). Cells were transfected by the calcium phosphate precipitate method40 followed by a glycerol shock. In order to use the selectable marker gene Ecogpt, which requires active thymidine kinase expression, L cells (tk-) were first transfected with the Herpes simplex virus thymidine kinase gene and tk+ transformants were selected in 500 p,M hypoxanthine, 0.4 p,M aminopterin, and 16 p-M thymidine (HAT). The env constructs were then introduced into these cells by transfection, and transformants containing the dominant selectable marker gene, Ecogpt, were selected in medium containing 250 p,g/ml xanthine, 6 p-g/ml mycophenolic acid, and HAT. After drug selection, mycophenolic acid-resistant clones expressing the envelope glycoprotein were isolated by limiting dilution in

96-well trays containing selection medium. Cell growth was monitored regularly and only wells containing a single clone were selected for screening. Because we were unable to demonstrate surface expression of env determinants, cloned cells were screened for intracellular env expression by indirect immunofluorescence of fixed and permeabilized cells grown on glass slides. Cells were stained using pooled HIV-positive sera. The HIV-positive sera were strongly reactive with env gpl60, gpl20, and gp41 by immunoblotting and quantitatively immunoprecipitated recombinant secreted gpl20.

Immunofluorescence analyses For detection of intracellular antigens by indirect immunoflucell monolayers grown on glass slides were fixed for 20 min with 3.7% formaldehyde in PBS containing 0.1 mM CaCl2, and 0.1 mM MgCl, (staining buffer). The monolayers were then quenched with 20 mM ammonium chloride and permeabilized for 15 min with 0.1% Triton X-100 in staining buffer. Env glycoprotein was detected by incubation of the fixed cell monolayers for 30 min with 1:100 dilution of pooled HIV-positive patient sera which had been clarified by centrifugation at 100,000 x g in a Beckman airfuge. The monolayers were then washed and incubated for 30 min with 1:50 dilution of fluorescein isothiocyanate (FITC)-sheep [F(ab')2] anti-human immunoglobulin (Silenus, Melbourne). To detect surface proteins, env- and human CD4-transfected L cells (gift of Rozanne Blok) were grown on slides and placed on ice while incubating with HIV-positive sera and OKT4, respectively. Cells were then fixed as described and incubated with the appropriate FITCconjugated immunoglobulin. Monolayers were mounted in PBS/glycerol (9:1 vol/vol) containing p-phenylenediamine for examination and photomicrography using an Olympus microorescence,

scope.

ForFACS analyses, cells were harvested with 50 mM EDTA,

washed, and incubated with either 1:100 HIV-positive

sera or

1:100 normal human sera in phosphate buffered saline (PBS; containing 0.2% sodium azide) on ice for 30 min. Cells were then washed and incubated for 30 min on ice with FITCconjugated sheep anti-human immunoglobulin. As a positive control, L cells expressing surface human CD4 were incubated for 15 min in medium containing soluble gpl20 (gift of Weisan Chen), then treated with either HIV-positive or normal sera. Syncytial assays were carried out as described.34

Metabolic

labeling of cells

Metabolic labeling and pulse-chase experiments were performed essentially as described.36 Briefly, 5 x 106 cells per 100-mm dish were starved in serum- and methionine-free medium for 30 min. The medium was then removed and replaced with 750 p.1 of fresh medium containing 100 p,Ci of L-[35S]methionine (>800 Ci/mmol, New England Nuclear). At the conclusion of the labeling period, cell extracts were prepared by lysing the cells in 0.5 ml of 1% Nonidet P40 lysis buffer as described.36 To detect shed/secreted gpl20, the culture supernatants were collected after the chase period and clarified by centrifugation. Both culture supernatants and cell lysates were immunoprecipitated with pooled HIV-positive sera as described.36 Precipitates were analyzed by SDS-PAGE on 7.5%

RECOGNITION BY env-SPECIFIC CTL

2001

polyacrylamide gels followed by treatment with the enhancing agent, Amplify (Amersham), and autoradiography at —70°C.

Peptide N-glycosidase F and endoglycosidase H digestion Radiolabeled env determinants were immunoprecipitated as described above and samples were divided into aliquots. As a control, untreated bound radiolabeled protein was released from the protein A-Sepharose pellet by boiling in Laemmli sample buffer. For endoglycosidase H and peptide N-glycosidase F digestions, the radiolabeled protein was first released by boiling for 3 min in buffer containing 0.5% sodium dodecyl sulfate (SDS) and 0.1 M 2-mercaptoethanol prior to enzyme digestion performed as described.4' For each experiment a control incubated in digestion buffer, without enzyme, was included.

Derivation

of cytotoxic

T

lymphocyte (CTL) effectors

CBA/H mice 6-9 wk old were raised under specific pathogenfree conditions in the Animal Services Section of the John Curtin School of Medical Research. Mice were immunized i.v. with 107 plaque-forming units (pfu) of the recombinant vaccinia virus (rVV), VV-gpl60, which encodes the HIV-1 env glycoprotein of the SF-2 isolate and was constructed according to established protocols.42'43 Three weeks later, immune splenocytes were restimulated for 6 days in vitro with rVV-infected syngeneic splenocytes (cells were cultured at 2 x 106/ml in 75-cm2 flasks, with 20% of cells infected with rVV at 20 pfu/cell for 1 h, washed three times, and gamma-irradiated prior to culture).

present for all three genes. Studies in COS-1 cells, using similar

plasmid constructs, showed that removal of the tat initiation codon greatly enhanced env gene expression.36 Therefore a second plasmid pSVTenv(tat—rev+)gpt, was also constructed. Both plasmids were introduced by transfection into tk+ L cells. After selection in medium containing mycophenolic acid, xanthine, and HAT, the cells were metabolically labeled and examined for the presence of env proteins by immunoprecipitation and SDS-PAGE (Fig. 2a). Bands corresponding to env determinants were not detected in pSVTenv(tat + rev + )gpt transformants or in cells transfected with pSV2gpt alone. However, in the cells transfected with the pSVTenv(tat—rev + )gpt construct, labeled proteins corresponding to envelope gp 160 and gpl20 were specifically immunoprecipitated from the cell lysates. Gp41 was difficult to detect due to presence of nonspecific bands in this region of the gel. A band corresponding to secreted or shed gpl20 was detected in the culture medium of the pSVTenv(tat—rev+)gpt-transfected cells after an 18-h chase

period (Fig. 2a). Attempts to demonstrate

surface expression of gp 120/41 these transfected L cells were not successful despite our use of indirect sandwich techniques for staining the cells, including the use of recombinant human CD4 or biotinylated pooled human antisera as first reagents. However, following permeabilization of fixed pSVTenv(tat—rev+)gpt-transfected L cells, intracellular env determinants were detected by indirect immunofluorescence microscopy after staining with pooled human HIV-positive sera (data not shown). As expected, the expression of env determinants was heterogeneous in the uncloned L cell population. determinants

on

Cytotoxicity assays labeled with 50 p,Ci of sodium [5lCr]chromate/106 cells for 90 min at 37°C in a humidified atmosphere of 5% C02 in air, washed three times in DMEM (GIBCO, Grand Island, NY) supplemented with antibiotics and

Target

cells

were

10% heat-inactivated FCS (Flow Laboratories, North Ryde, Australia; complete medium), and resuspended at 2 x 105 cells/ml. For virus-infected targets, cells were infected with rVV at 20 pfu/cell during the labeling period. A standard chromium release assay was carried out in triplicate in round bottom 96-well plates. Target cells (2 x 104) were incubated with the appropriate number of effectors in 200 pJ of complete medium for 6 h at 37°C and specific lysis was determined according to the formula: (experimental release spontaneous release)/ —

(maximal release spontaneous release) release from target cells ranged from 11%

x

100.

Spontaneous

—

to 15%.

RESULTS

Knight et al. (18) have demonstrated an absolute requirement tat and rev gene products for efficient translation of the envelope gene product from its cognate mRNA in virally infected cells. The absolute requirement for rev co-expression in heterologous expression systems has also been well established.5'21'36'44 Therefore,constructs including the to?, rev, and env genes were prepared as shown in Fig. 1. The plasmid pSVTenv(tat+rev + )gpt possesses the complete coding regions for the

of tat,

rev,

and

env

with intact translational initiation codons

Env

expression

in

transfected L

cell clones

Because of the inability to detect surface env determinants on uncloned L cell transfectants, L cell clones were isolated by limit-dilution of the heterogeneous cell population. Individual clones were screened for env expression by indirect immunofluorescence of fixed and permeabilized cells. To examine env expression more quantitatively, env-positive clones were metabolically labeled for 1 h with [35S]methionine (under these labeling conditions only gpl60 is detected). Cell extracts containing approximately equal amounts of total protein were reacted with pooled HIV-positive sera and the immunoprecipitated proteins were analyzed by SDS-PAGE and fluorography (Fig. 2b). An unidentified background band of —200 kD was detected in all cell lysates. Bands corresponding to the gpl60 envelope glycoprotein were detected in significantly greater amounts in clones 1,2,4, and 5 compared with clone 3 and the uncloned L cell population. In order to evaluate whether any of the L cell transfectants expressed surface env determinants, untransfected and cloned cells were harvested with EDTA in the absence of trypsin, incubated with HIV-positive and HIVnegative sera, and analyzed by flow cytometry (Fig. 3). However, surface expression of gpl20 could not be detected on any of the clones (data not shown) including the high expressing env-transfectant, clone 2 (Fig. 3a and b). As a positive control for the detection of surface gp 120, CD4-transected L cells were reacted with soluble gpl20 and then incubated with HIVnegative and HIV-positive sera demonstrating that gpl20 was detectable on the surface of CD4+ cells coated with soluble

2002

BIRD ET AL.

Bam HI

Kpn

I

Hind

Pst I Hind III Bam HI

FIG. 1. Gene constructs used for transfection of murine L cells. A 3.1-kb Sal I-Xho I fragment of the HIV-1 proviral DNA was subcloned into the unique Sal I site of the expression vector pSVT737 to create the plasmid pSVTenv(tat+rev + ). The subcloned DNA (lower panel) contains coding exons of both tat (black boxes) and rev (hatched boxes) as well as the complete coding region of env (open box). An analogous DNA fragment, containing a mutated tat initiation site, was also subcloned into the expression vector thus creating the plasmid pSVTenv(tat—rev + ). The dominant selectable marker gene, Ecogpt, was then subcloned as a 2.4-kb Pvu II—Pst I fragment into the upstream Cla I site. SD, splice donor; SA, splice acceptor.

gpl20 (Fig. 3c and d). Env-transfectants were also negative for expression of gpl20 and gp41 antibody determinants when studied by flow cytometry (data not shown). Therefore, although the env-transfected cells synthesized envelope glycoprotein, this was not detectable by direct assays on the cell surface

of env-transfectants. When clone 2 cells were grown on slides and then examined for surface env expression by fluorescence microscopy, env-positive cells were still not detected (data not shown). This finding is contrasted with the surface expression of human CD4 in transfected L cells stained and examined under the same conditions, (Fig. 4c). However, after permeabilization of fixed clone 2 cells grown on glass slides, intracellular env determinants were detected in env-transfectants (Fig. 4b and d) but not untransfected L cells (Fig. 4a). The localization of env determinants in the majority of permeabilized cells was perinuclear and resembled that of Golgi staining (Fig. 4b). In some

a staining pattern resembling the distribution of the endoplasmic reticulum was also evident (Fig. 4b and d). Despite the lack of detectable surface env expression by direct assay methods, L cells expressing env glycoprotein did form syncytia with CD4-positive HUT 78 cells, however, the number of syncytia varied directly with the level of env expression by individual

cells

cloned transfectants (data not shown). Notably, the staining pattern of intracellular env determinants was heterogeneous even in the cloned cell population. To rule out the possibility that the heterogeneous staining observed for the env L cell clones was due to mixed cell populations, individual clones were recloned by limiting dilution. However, the intracellular expression of env determinants remained heterogeneous within the recloned cell population. A similar pattern of env staining was observed in multiple independent envtransfected L cell clones.

RECOGNITION BY env-SPECIFIC CTL

Processing of env proteins Only

a

transfected L

in

small percentage (5% to 15%) of

2003

cells

newly synthesized

gpl60 in HIV-1-infected T lymphocytes is cleaved to produce the mature gpl20 component, the remainder probably being transferrred to lysosomes where it is degraded.10 In addition, approximately —30% to 50% of the envelope gp 120/41 complex

is secreted into the culture medium of these virally infected cells. Using transfected COS-1 cells, we have shown that the intracellular processing and transport of the envelope glycoprotein in noninfected cells is also very inefficient.36 Therefore, the failure to detect env determinants on the surface of transfected L cells may have been the result of inefficient and/or detective processing and transport of env-protein. To examine this possibility, pulse-chase metabolic labeling of cloned env-transfectants was carried out. Cells were incubated for 30 min in medium containing [35S]methionine then chased in complete DMEM. At + O u

cu

s

M

100 kD

69 kD

46 kD

T^

TT"»C M

—

—

FIG. 2. Metabolic labeling of env proteins in transfected L cells, (a) Analysis of env expression in uncloned L cell transformants. L cells transfected with the plasmids pSVTenv(tat+rev+)gpt (abbreviated to tat+rev+) and

—

T3 ¡H

rH

«N

CO

O

|

s

o>

0>

o

a o

Ö

C

o

o

o

s

u

Í 200 kD

O

ö

O

a

o

SO a

—

gpl60 100 kD

69kD 46 kD

the indicated chase intervals, both the cells and chase media were reacted with pooled HIV-positive sera. Immunoprecipitates were then examined by fluorography after SDS-PAGE (Fig. 5). After a 30-min pulse (0 h chase) labeled protein of —160 kD was detected as well as a background band of Mr 200 kD. By 1 h chase period, a band corresponding to gpl20 was apparent, indicating conversion of gp 160 to gp 120/41. Throughout the remaining chase period, free gpl20 was observed to accumulate in the culture medium while the unidentified background band of Mr 200 kD accumulated within the cell lysate, indicating that cells were intact after the 20-h chase period. Interestingly, at the completion of the chase period (20 h), virtually all of the recovered envelope glycoprotein was present in the culture medium. Based upon the intensity of the env bands, a significant proportion of gpl60 could not be accounted for at the end of the chase period, suggesting substantial intracellular degradation. These results, although only semiquantitative, indicate that the kinetics of envelope processing and transport in transfected L cells are similar to those previously described in other cell types,l0'36 except that virtually no env products could be detected in the cellular lysate after a 20-h chase. The HIV-1 envelope glycoprotein is heavily glycosylated; approximately one-half of the apparent molecular weight of the gpl20 protein on SDS-polyacrylamide gels can be attributed to the addition of N-glycans.212'45'46 To evaluate the posttranslational modification of env expressed in transfected L cells, we performed endoglycosidase H and peptide N-glycosidase F digestions of gpl60 and gpl20. The posttranslational modification of gpl60 and gpl20 were studied in separate experiments. Thus gpl60 was immunoprecipitated from cells pulsed with [35S]methionine for only 15 min. Under these conditions gp 120 is not detected. On the other hand, free gpl20 was immunoprecipitated from the culture medium of cells pulsed for 2 h with

—

-

—

pSVTenv(tat—rev+)gpt (abbreviated to tat—rev + ) were pulselabeled for 2 h with 100 p,Ci [35S]methionine. The labeled cells were either collected for preparation of cell lysates, or chased in complete DMEM for 20 h. At the completion of the chase period, the culture medium was collected and clarified by centrifugation. Both culture supernatants and cell lysates were reacted with pooled human sera from HIV-seropositive patients. Immune complexes were adsorbed onto Protein A-Sepharose beads, washed, and separated under reducing conditions by SDS-PAGE in a 7.5% gel. A fluorograph of radiolabelled proteins from the culture medium (M) and cell lysate (C) of each L cell transfection is shown, (b) Env expression in L cell transfectant clones. A number of clones were isolated by limiting-dilution of the pSVTenv(tat—rev + )gpt-transfected L cells. Five clones, as well as the vector-transfected and uncloned Lcell populations, were radiolabeled with 100 p.Ci [35S]methionine for 1 h; under these conditions only gpl60 is detected. Cell lysates were reacted with pooled HIV-seropositive patients sera, adsorbed onto protein A-Sepharose, washed, and analyzed by SDS-PAGE under reducing conditions on a 7.5% gel. Estimates of total protein content were performed on the cell lysates and an equivalent amount of protein was immunoprecipitated.

BIRD ET AL.

2004

Untrensfected

Env-transfected

HIV NHS

NHS

HIV1

O)

CD4-transfected

CD4 -transfected

Fluorescent

+s-gp120

Intensity

FIG. 3. FACS analysis of env expression in cloned env-transfected L cells. L cells were harvested with 50-mm EDTA in the absence of trypsin and incubated with either normal human sera (NHS) or pooled HIV-positive sera (HIV+). After washing, the cells were incubated with FITC-conjugated sheep anti-human immunoglobulin and analyzed by flow cytometry. As a positive control, CD4-.transfected L cells were incubated in medium containing soluble gpl20 then treated with either normal human sera or HIV-positive sera, (a) Untransfected L cells stained with normal human sera or HIV-positive sera, (b) Env-transfected L cells (clone 2) treated with either normal human sera or HIV-positive sera, (c) Human CD4-transfected L cells treated with either normal human sera or HIV-positive sera, (d) Human CD4-transfected L cells incubated with —17 p-g/ml soluble gpl20, then treated with either normal human sera or HIV-positive sera.

[35S]methionine

then chased for 20 h in

complete

DMEM to

optimal conversion of gpl60 to mature shed/secreted gpl20. Glycosidase-digested proteins were analyzed by SDSPAGE followed by fluorography (Fig. 6a and b). As expected, newly synthesized gpl60 was fully endoglycosidase H-sensitive. The bands corresponding to the products of both endoglycosidase H- and peptide N-glycosidase F-treated gpl60 migrated with a molecular weight of —95 kD (Fig. 6a), in agreement with the results of previous studies.210'2 Treatment of secreted gpl20 with peptide N-glycosidase F resulted in a band migrating with an apparent molecular weight of —55 kD, the expected size of the protein core (Fig. 6b). In addition, gpl20 was partially sensitive to endoglycosidase H digestion, indicating that a significant fraction of the N-linked chains in the mature gpl20 glycoprotein were of the high mannose type. allow

for their ability to lyse various L cell targets (Fig. 7). L cells transfected with the vector pSVTgpt construct were not lysed, whereas L cells infected in vitro with VV-gp 160 were lysed even at low effector/ target ratios. Effectors were conventional CD4~ CD8+ CTL (data not shown). Significant cytotoxic activity was also detected against two independent, cloned env-transfectants, clone 4 and clone 1, reflecting the env-specific component of the CTL response in VV-gp 160-infected mice. No such anti-env activity was detected when control rVV (not expressing env) was used to prime mice or to restimulate effectors (data not shown). Thus, although native env was not detected on the cell surface as gp 120/41, env determinants were presented on the membrane for recognition by CTL, presumably as processed peptide fragments complexed to major histocompatibility class I molecules.

Similar results have been reported for endoglycosidase H treatment of gp 120 produced in either transfected or virally infected ceUs 2.12.46.47

Env-specific CTL lyse env-transfected L cells Because of the failure to detect env determinants on the cell membrane of L cell transfectants, and the apparent inefficient processing and export of env proteins, we investigated whether env determinants were being presented on the membrane for recognition by CTL. Env-specific CTL were raised by infecting CBA/H(H-2k) mice with rVV expressing HIV-1 env gpl60. T cells from these mice were restimulated in vitro and then tested

DISCUSSION The expression of HIV-1 envelope glycoprotein is regulated the presence of the rev protein'618 which probably acts to facilitate the transport of unspliced env transcripts from the nucleus to the cytoplasm.19'48 This function of rev is operative in both virally infected cells'8'49 and in expression systems in which env transcription is under the control of the HIV LTR or heterologous promoters.5'21'36'44 Although tat protein is also required when the HIV LTRs are used to direct transcription, the

by

RECOGNITION BY env-SPECIFIC CTL

Untransfected

2005

Env-transfected

B

CD4-transfected

Env-transfected

d

FIG. 4. Cellular distribution of env-protein in env-transfected L cells, (a) Untransfected L cells were fixed, permeabilized, and treated with pooled HIV-positive sera; (b) permeabilized env transfectants (clone 2 cells) were reacted with pooled HIV-positive sera; (c) nonpermeabilized CD4-transfected L cells were reacted with OKT4 monoclonal antibody and then fixed and reacted with FITC-conjugated second antibody; (d) permeabilized, cloned env-transfectant reacted with pooled HIV-positive sera demonstrating an endoplasmic reticulum/vesicular pattern of intracellular env distribution. Nonpermeabilized env-transfectants revealed no detectable surface fluorescence when treated with either HIV-positive or normal human serum.

gene constructs used in this

study were transcribed from a heterologous promoter. Previously, we reported that removal of the tat gene initiation codon greatly enhanced the heterologous expression of the env protein from constructs processing otherwise intact tat, rev, and env genes in transfected COS-1 cells.36 In this study, env proteins were stably expressed in L cells transfected with the pSVTenv(tat—rev+)gpt construct, whereas env determinants were not detected after transfection using the

pSVTenv(tat+rev+)gpt construct. Thus, the negative influence on env expression by the tat initiation codon, previously observed in transiently expressing COS-1 cells, was also evident in the stably transfected L cells. The most likely explanation is that

the to? initiation codon is strong50 and so prevents efficient translational initiation events from down stream start sites.36 It has been suggested that a critical threshold level of rev expression must be achieved before the transition from regulatory to

2006

BIRD ET AL.

20

0 1 t

nr

CMCMCMCMCM 200 kD

100 kD

—

^gpl60 -«gpl20 —

Pulse-chase metabolic labeling of env proteins in transfected L cells. Cloned env-transfectants (clone 2) were 100 p,Ci [35S]methionine and then chased in complete DMEM. At the end of each chase interval (0, 1,3,6, and 20 h), chase medium was collected and the cells lysed in 0.5 ml Nonidet P-40 lysis buffer as described.36 Nuclei and cell debris were removed by centrifugation at 10,000 g for 5 min and both cell extracts and chase media were reacted with pooled HIV-positive sera. The immune complexes were adsorbed onto protein A-Sepharose, washed, and separated by SDS-PAGE in a 7.5% gel, under reducing conditions. The fluorograph of radiolabeled proteins from the chase medium (M) and cell extract (C) is shown. Bands corresponding in Mr to gp!60 and gp!20 are arrowed.

FIG. 5.

pulse-labeled for 30 min with

structural gene expression can occur in virally infected cells. Thus it seems likely that the failure to detect env expression in pSVTenv(tat+rev + )gpt-transfected L cells was due to insufficient rev expression. Although env could be immunoprecipitated from radiolabeled cell extracts and from the culture supernatants of transfected cells (Figs. 2, 5, and 6), surface determinants could not be detected by indirect immunofluorescence and FACS analysis of these cells (Figs. 3 and 4). By contrast, surface env expression could be demonstrated in COS-1 and CV-1 cells transfected with plasmids similar to the ones used in this study36'51 as well as in cells expressing env proteins from rVV systems.52'53 Presumably, the exaggerated replication and transcription of the genes in these systems results in the production of such large amounts of protein that a detectable level of env remains cell surface associated. Despite the lack of detectable surface env expression

in the L cell transfectants these cells were able to form syncytia with human CD4-positive HUT 78 cells. This finding is consistent with the lack of detectable cell surface envelope in retroviral vector-transduced mouse fibroblasts stably expressing env glycoprotein and capable of forming syncytia.54 Taken together these findings suggest that syncytia formation is a more sensitive assay for surface env expression than conventional antibody

staining techniques.

As in the case of COS-1 transfectants,36 indirect immunofluorescent staining of fixed and permeabilized pSVTenv(tat—rev+)gpt-transfected L cells revealed that most of the env proteins were located intracellularly. In most cases, intracellular env was distributed in a perinuclear Golgi-like distribution, however, in some cells the pattern of staining resembled that of the endoplasmic reticulum. Surprisingly, the pattern of env-staining in the transfected cell population was

Culture Medium

Cell Extract EndoH

N-glycanase

r~"-1

EndoH

N-glycanase

~

C 200 kD

—100 kD

—

-«gpl60 —

100 kD 69 kD

69 kD

—

—

46kD

—

46 kD

-

B

FIG. 6. Endoglycosidase H and peptide N-glycosidase F digestion of env proteins from transfected L cell clones, (a) Clone 2 cells were labeled with [35S]methionine for 15 min such that only env gpl60 would be detectable. Radiolabeled env proteins from cell extracts were then reacted with HIV-positive sera and immunoprecipitates were incubated in buffer containing ( + ) or lacking ( ) endoglycosidase H (Endo H), or peptide -N glycosidase F (N-glycanase). In each case untreated immunoprecipitates were included as a control (C). (b) Clone 2 cells were labeled with [35S]mefhionine for 2 h followed by a 20-h chase in complete medium. Radiolabeled env proteins from culture medium were then reacted with antisera and glycosidases as described above. —

RECOGNITION BY env-SPECIFIC CTL

2007

90-,

Vv-gp160

80

70

u

50

| 40o,

M

30 20

ion

pSVTgpt

0 10

0

30

E:T Ratio

Env-transfected L cell clones are lysed by envCBA/H mice were infected with rVV expressing env gp 160, and 3 wk later splenic T cells were restimulated for 6 days in vitro. Cytolytic activity of effector T cells was measured against env-transfected clones (clone 1 and clone 4), pSVTgpttransfected L cells (pSVTgpt) and L cells infected with rVV

FIG. 7.

specific CTL.

expressing env-gpl60 (VV-gp 160).

heterogeneous, despite several rounds of single-cell cloning. The heterogeneity in env expression was not obviously related to cell cycle (data not shown). Heterogeneity of env expression might arise from selective growth advantage of spontaneously

transformants expressing low levels of env. However, when clone 2 was recloned after an 8-mo period in continuous culture (—80 passages) six randomly selected subclones were found to produce similar amounts of [35S]methionine labeled env protein, despite persistent heterogeneity in their intracellular env staining (data not shown). We assume that heterogeneity of the env-expression in L cell transfectant population is not due to polyclonality, but represents an artefact of the immunofluorescence detection assay or some variable affecting env expression. The lack of detectable surface env expression by antibody staining of L cell populations expressing high levels of intracellular env determinants is of particular interest. We suggest that this apparent discrepancy is due in part to the generally ineffi-

arising

cient processing of the envelope glycoprotein. Other pulsechase studies have shown that* only a fraction of newly synthesized gpl60 is processed by proteolytic cleavage to gp 120/41 (