Characterization of an HIV-2-related virus with a smaller sized extracellular envelope glycoprotein

VIROLOGY

173,258-267

(1989)

Characterization

MARIE-ANNE

Unite’ d’Oncologie

of an HIV-2-Related Extracellular Envelope

Virus with a Smaller Sized Glycoprotein

REY, BERNARD KRUST, ANNE G. LAURENT, DENISE GUiTARD, LUC MONTAGNIER, AND ARA G. HOVANESSIAN’ Virale (URA CNRS

153), lnstitut

Received

May

Pasteur,

25, rue du Dr Roux,

1, 1989; accepted

75724

Paris Ckdex

15, France

July 27, 1989

A new isolate of the human immunodeficiency virus (HIV) related to the HIV-2 strain was isolated from peripheral blood lymphocytes of an Ivory Coast patient with AIDS. This isolate referred to as HIV-2 EHO could be differentiated by its envelope precursor and external glycoprotein which are PO-kDa smaller than those of HIV-2 ROD isolate. Furthermore, the apparent molecular weight of the major core protein of HIV-2 EHO is 27 kDa instead of 26 kDa as in HIV-2 ROD isolate. In addition, the product of the vpx gene which is a characteristic feature of the HIV-2 strain, is 14 kDa in HIV-2 EHO compared with 16 kDa in HIV-2 ROD. In contrast to these, the envelope precursor of HIV-2 EHO forms a transient dimer during its maturation as is the case for HIV-2 ROD. In both cases the transmembrane proteins are 36 kDa and exist as homodimers of 80 kDa. Endoglycosidase H digestion experiments indicated that the 20-kDa difference between the two HIV-2 isolates is not due to a difference in the number of N-linked oligosaccharide chains per polypeptide, since deglycosylated envelope precursors of HIV-2 ROD and EHO have an apparent molecular weight of 80 and 60 kDa, respectively. Partial proteolysis of the envelope precursors from the two isolates with Staphy/ococcus aureus V8 protease gave a distinct pattern of polypeptides. These results suggest that the differences between the external envelope proteins of the two HIV-2 isolates are due to their amino acid composition. Accordingly, polyclonal antibodies raised against HIV-2 ROD envelope do not recognize the corresponding envelope proteins of HIV-2 EHO by immunoblotting and immunoprecipitation assays. These data illustrate that analysis of viral proteins could be useful for a rapid characterization of new viral isolates and show the heterogeneity of HIV-2 isolates in West Africa. o 1989Academic Press, Inc.

INTRODUCTION

to all retroviruses, HIV-l, HIV-2, and SIV encode genes that regulate virus replication as well as genes that encode proteins of yet unknown function. The only notable difference in the genetic organizations of HIV-l, HIV-2, and SIV resides in the open reading frames referred to as vpx and vpu:vpx is present in HIV-2 and SIV but is absent in HIV-1 whereas vpu is present in HIV-1 but not in HIV-2 and SIV (Guyader et al., 1987; Cohen et a/., 1988). These viruses are both tropic and cytopathic for CD4-positive T lymphocytes (Klatzmann et al., 1984; Dalgleish et al., 1984; Daniel et al., 1985; Clavel et al., 1986a). A great number of studies have indicated that CD4 functions as the cellular receptor of HIV (Weiss, 1988). HIV-1 was probably originated in Central Africa whereas HIV-2 was shown to be widespread in several West African countries such as Guinea Bissau, Cape Verde Islands, Senegal, Ivory Coast, Ghana, The Gambia, and Mali (Clavel, 1987). Besides cytopathic isolates of HIV-2 (referred to as ROD, SBL6669, and GHl), two noncytopathic HIV-2 isolates (referred to as UCI and ST) have been reported (Clavel et al,, 1986a; Guyader et a/., 1987; Albert et al., 1987; Arya et al., 1987; Ishikawa et al., 1988; Evans et al., 1988; Kong et al.,

Human immunodeficiency virus (HIV) is considered to be the etiologic agent of the acquired immunodeficiency syndrome (AIDS) (Montagnier er a/., 1984). To date, two related but distinct types, HIV-1 and HIV-2, have been identified (Barre-Sinoussi et al., 1983; Popovic et al., 1984; Ratner et a/., 1985; Wain-Hobson et a/., 1985; Clavel e2 al., 1986a,b; Brun-Vezinet et al., 1987). HIV-2 is closely related to simian immunodeficiency virus (SIVmac), which causes an AIDS-like disease in macaques (Daniel et a/., 1985; Fultz et al., 1986; Chakrabarti et a/., 1987). Alignments of the nucleotide sequences of HIV-l, HIV-2, and SIV reveal a considerable homology between HIV-2 and SIVmac. These two viruses share about 75% overall nucleotide sequence homology; both of them are only distantly related to HIV-l with about 40% overall homology(Guyader et a/., 1987; Chakrabarti ef a/., 1987). In addition to the genes that encode structural proteins (the virion capsid and envelope proteins) and the enzymes required for proviral synthesis and integration common ’ To whom

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258

CHARACTERIZATION

1988). Here, we report the isolation and characterization of a new cytopathic HIV-Z-related virus from a patient in the ivory Coast. This isolate designated as HIV2 EHO was studied parallel with the first HIV-2 isolate, HIV-2 ROD. HIV-2-positive patient sera cross-react with viral proteins from HIV-2 ROD and HIV-2 EHO. The envelope precursor of HIV-2 EHO forms a dimer during its processing and the transmembrane glycoproteins exist as homodimers as is the case for HIV-2 ROD (Rey et al,, 1989a,b). In addition, HIV-2 EHO produces a 14kDa protein related to the product of the vpx gene of HIV-2 ROD. These results indicate that this new HIV isolate belongs to the HIV-2 family. However, HIV-2 EHO was differentiated from HIV-2 ROD by its envelope precursor and the extracellular glycoprotein which are 20 kDa smaller than those of HIV-2 ROD. This difference is probably due to major differences in the amino acid composition of the envelope glycoprotein precursor of HIV-2 EHO compared to HIV-2 ROD since different polypeptide profiles were obtained as a result of partial proteolysis of the purified precursors. By the use of murine polyclonal and monoclonal antibodies against the envelope glycoproteins of HIV-2 ROD, we were able to demonstrate that the envelope glycoprotein precursor of HIV-2 EHO has a distinct polypeptide structure compared to that of HIV-2 ROD. Furthermore, the extracellular glycoprotein of HIV-2 EHO is 20 kDa smaller, which should be accounted for by a significant deletion in the envelope gene of the classical HIV-2. MATERIALS

AND METHODS

Materials L-[35S]Methionine (sp act > 1000 &i/mmol) and D[6-3H]glucosamine (sp act: 20-40 ~Ci/mmol) were purchased from Amersham (Amersham, UK). Endo-/3-Nacetylglucosaminidase H and Staphylococcus V8 protease were from Calbiochem (San Diego, CA). Poly(A). poly(U) was the generous gift of M. Michelson (Institut Curie, Paris). The monoclonal antibody specific for the transmembrane glycoprotein was obtained from Dr. Jan McClure (Oncogen, Seattle, WA). The preparation of this monoclonal antibody (referred to as mAb 1 H8) will be described elsewhere (Rey et a/., 1989b). The monoclonal antibody specific for the major core protein of HIV-2 ROD was provided by Genetic Systems. Rabbit serum against the vpx protein was provided by Dr. Moustafa Bahraoui. This latter was prepared by immunizing rabbits with a synthetic peptide deduced from the nucleotide sequence of the N-terminal region of HIV-2 ROD vpx gene corresponding to amino acids 1 to 35.

259

OF HIV-2 EHO

Virus and ceils HIV-1 BRUisolate of the human immunodeficiency virus type 1 (Montagnier et al., 1984) HlV-2RoD isolate of the human immunodeficiencyvirus type 2 (Clavel eta/., 1986a), and simian immunodeficiencyvirus, SlVmac,,, (Daniel eta/., 1985) were used in this study. The different cell lines and human lymphocytes were cultured in suspension medium RPMI 1640 (GIBCOBRL, Cergy-Pontoise, France) containing 10% (v/v) fetal calf serum; 2 pg/ml Polybrene (Sigma) was added for HIV-infected cell cultures. CEM clone 13 cells are derived from the human lymphoid cell line CEM (ATCCCCL1 19) and express the T4 antigen to a high level. Five days after infection with HIV-1 BRUor HIV-2Ror-, isolates, about 80-90% of the cells produce viral particles and can be identified by a cytopathic effect corresponding to vacuolization of cells and appearance of small syncitia. The HUT-78 cell line is another human T4-positive lymphoid cell line that is highly permissive for the replication of SIVmac,,, (Daniel eta/., 1985). Peripheral blood lymphocytes from healthy blood donors were stimulated for 3 days with 0.2% (w/v) phytohemagglutinin (PHA)fraction P (Difco, Detroit, MI) in RPMI 1640 medium supplemented with 10% fetal calf serum. Cells were then cultured in RPMI 1640 medium containing 10% (v/v) T-cell growth factor (TCGF, Biotest). After infection with HIV-2, lymphocytes were cultured in the presence of 10% (v/v) TCGF, and 2 pg/ml Polybrene and sheep anti-human a-interferon antibodies (25 units/ml). The HIV-2 EHO isolate was obtained from blood lymphocytes of Ivory Coast patient (EHO) with AIDS. Blood lymphocytes were stimulated with PHAand cocultured with PHA-stimulated, normal human lymphocytes. These cultures were maintained in the presence of TCGF. The production of virus was monitored by cytopathic effects and by the reverse transcriptase activity in the supernatant (Rey et a/., 1984). The virus (HIV-2 EHO) was then cultured on CEM clone 13 cells. Four to 5 days after infection with HIV-2 EHO, more than 80% of the cells produced viral particles. Metabolic

labeling

of cells

For metabolic labeling of proteins, infected cells were incubated for 16 hr at 37” in MEM culture without L-methionine and serum but supplemented with 200 &i/ml [35Slmethionine. For metabolic labeling of glycoproteins, infected cells were incubated for 16 hr at 37” in MEM culture medium lacking serum and glucose but supplemented with 200 &i/ml [3H]glucosamine.

260

REY ET AL.

Cells and viral extracts Cell pellets corresponding to lo7 cells were resuspended in 100 ~1 of buffer: 10 rnM Tris-HCI, pH 7.6, 150 mM NaCI, 1 mM EDTA, 0.2 mM PMSF, 100 units/ ml aprotinin (Iniprol, Choay) before addition of 100 ~1 of the same buffer containing 2% (v/v) Triton X-l 00. Cells extracts were centrifuged at 12,000 g for 10 min, and the supernatant was stored at -80” until used. For viral extract preparations, 100 PI of 1 OX lysis buffer (100 mMTris-HCI, pH 7.6, 1.5 h/l NaCI, 10 mM EDTA, 10% (v/v) Triton X-l 00, 1000 units/ml aprotinin) was added per milliliter of clarified supernatant from infected CEM cells and processed as above. For the preparation of extracts from virus pellets, culture medium from infected cells was first centrifuged at 12,000 gfor 10 min before high speed centrifugation at 100,000 g for 15 min in a Beckman TLl 00 centrifuge. Virus pellets (material from 1O7 cells) were then solubilized in 200 ~1 of lysis buffer.

Preparative

electrophoresis

HIV-2 glycoproteins purified by immunoprecipitation were resolved by polyacrylamide gel electrophoresis as previously described (Rey et al., 1989a), and the regions of the gel containing the viral glycoproteins were cut out by reference to the position of prestained molecular weight protein markers (BRL). Glycoproteins were eluted by incubation for 16 hr at 4’ in elution buffer (0.1 M NaHCO,, 0.5 mM EDTA, 0.05% (w/v) SDS, 0.2 mM PMSF). The glycoprotein fractions thus obtained were lyophilized and kept refrigerated until used.

Preparation antibodies,

of murine polyclonal anti-gp300

HIV-2 ROD envelope glycoprotein gp300 was purified from extracts of infected CEM cells (3 X 1 O* cells) by immunoaffinity chromatography on the HIV-2 serum-Sepharose and followed by preparative gel electrophoresis (Rey eta/., 1989a). The purified preparation of gp300 was dissolved in 10 ml of 150 mM NaCl containing 0.5 M urea and 1 mg/ml of mouse serum proteins and dialyzed for 24 hr against the solution containing 150 mM NaCl and 0.5 Murea. The dialyzed material was then centrifuged and 2-ml aliquots were stored at -80”. Five mice (6 weeks old) were injected intraperitoneally, five times at 12-day intervals with 350 ~1 of the gp300 preparation (about 0.1 pg of gp300). Poly(A). poly(U) (200 pg; 1 mg/ml in 150 ml\/l NaCI) was used as an adjuvant which was administered intravenously during each immunization. Five days before the

last injection mice were injected intraperitoneally with a suspension of 1O6 sarcoma 180/TG cells to prepare hyperimmune ascitic fluid (Hovanessian et a/., 1988). A week following the booster, mice were sacrificed and the ascitic fluids were collected. Ascitic cells were removed by centrifugation (200 g, 5 min) and the peritoneal fluid was collected.

Radioimmunoprecipitation

assay (RIPA)

Cell or viral extracts (20 ~1) (material corresponding to 1 X lo6 infected cells) were first diluted in 2 vol of RIPA buffer 110 mM Tris-HCI, pH 7.6, 150 mM NaCI, 1 mM EDTA, 1% Triton X-l 00 (v/v), 0.2% sodium deoxycholate (wUV), 0.1% SDS (wt/v), 7 mlVI 2-P-mercaptoethanol, 0.2 mM PMSF, 100 U/ml of aprotinin (Iniprol, Choay)]. Diluted extracts were incubated (45 min, 4’) with sera or with murine polyclonal or monoclonal antibodies (2-5 ~1). Protein A-Sepharose was then added and the samples were further incubated for 3 hr at 4”. These samples were washed in the RIPA buffer. Proteins recovered by immunoprecipitation were eluted by heating (95”, 5 min) in the electrophoresis sample buffer [125 ml\/lTris-HCI, pH 6.8, 19/o SDS (wt/v), 20% glycerol (v/v), 1% 2-P-mercaptoethanol]. Eluted proteins were resolved by electrophoresis on polyacrylamide-SDS gels containing 0.1% bis-acrylamide instead of 0.2% (wt/v).

Electrophoretic Western blot

transfer immunoblot

analysis:

Proteins were subjected to analysis by polyacrylamide gel electrophoresis before being electrophoretitally transferred to 0.45-pm nitrocellulose sheets (Schleicherand Schtill, Dassel, FRG) in electrode buffer (20 mM Tris base, 150 mM glycine, 20% methanol, v/ v) as described (Burnette, 1981). The electrophoretic blots were saturated with 5% (w/v) nonfat dry milk in PBS (Johnson et al., 1984). They were then incubated in a sealed bag (overnight 4”) either with HIV-1 - or HIV2-positive sera (at 1: 100 dilution) or with mouse polyclonal or monoclonal antibodies (at 1:200 dilution) in PBS containing 10% FCS. The sheets were subsequently washed in PBS and in PBS containing 5% Nonidet-P40 and then resaturated in PBS containing nonfat milk (59/o). The washed sheets were then incubated (2 hr, room temperature) in a sealed bag either with a preparation of ‘251-labeled protein A (Amersham, >30 mCi/mg) to reveal the human polyclonal antibodies from the HIV-1 or HIV-2 sera or with a preparation of ‘251-labeled goat anti-mouse immunoglobulins (Amersham; 2-10 &ilpg). The sheets were removed from

CHARACTERIZATION

OF

HIV-2

the bags and washed again, dried, and autoradiographed (Kodak RP Royal, X-ray films) for 24-48 hr.

RESULTS Isolation

261

EHO

1

gp125--

ROD 2

*L --SW

3

1

EHO 2

3

gplOO-

of HIV-2 EHO

HIV-2 EHO was isolated from the peripheral blood lymphocytes of an AIDS patient (EHO) from Ivory Coast. Virus was isolated by coculturing PHA-stimulated lymphocytes from the patient EHO and PHA-stimulated normal human lymphocytes in the presence of TCGF. Reverse transcriptase activity in the culture supernatant was detectable at 15 days associated with a typical cytopathic effect. The new virus was then propagated on CEM cells by coculturing with infected lymphocytes. Supernatants of such cultures were then used to infect CEM cells and prepare the stock of virus used in the experiments discussed here. Initially this virus was designated as HIV-2 since in a dot-blot assay its genomic RNA hybridized with a probe from HIV-2 ROD but not from HIV-1 BRU (data not shown). The stock preparation of HIV-2 EHO infects CEM cells and causes a cytopathic effect. Four to 5 days after infection of CEM cells with HIV-2 EHO, about 80 to 90% of cells produced viral proteins detectable by immunofluorescence studies using an HIV-2-positive serum. Four days after infection most of the cells showed a clear cytopathic effect corresponding to vacuolization of cells and appearance of small syncitia. In order to characterize HIV-2 EHO virus by its proteins, infected cells were metabolically labeled with [35S]methionine and the viral pellet in the culture supernatant was assayed by immunoprecipitation using different sera: (1) the serum of the patient infected by HIV-2 EHO; (2) a typical HIV-2-positive serum; and (3) a typical HIV-1 -positive serum. These assays were carried out parallel with extracts of [35S]methionine-labeled HIV-2 ROD virus. The HIV-2-positive serum and the serum of the patient EHO both recognized the extracellular glycoprotein gp125 of HIV-2 ROD. However, only the HIV-2-positive serum recognized the major core protein p26 of HIV-2 ROD (Fig. 1, lanes 1 and 2, ROD). The HIV-l-positive serum did not recognize gpl25 but it could immunoprecipitate p26 (Fig. 1, lane 3, ROD). This is in accord with previous results indicating that the gag proteins of HIV-1 and HIV-2 are antigenitally cross-reactive whereas the env proteins are not (Clavel et al., 1986a). The reactivity of these three sera with the viral proteins of HIV-2 EHO was similar to that of HIV-2 ROD. The serum of the patient EHO and the HIV-2-positive serum immunoprecipitated a 1 OOkDa protein which was presumed to be the extracellular envelope glycoprotein of HIV-2 EHO (Fig. 1, lanes 1

p26--

p27-

FIG. 1. Identification of HIV-2 EHO proteins by immunoprecipitation. HIV-2 ROD- or EHO-infected CEM cells were labeled with [35S]methionine and the culture supernatant was used in an immunoprecipitation assay using serum from the patient EHO (lanes l), a typical HIV-2-positive serum (lanes 2). and a typical HIV-l-positive serum (lanes 3). Samples were analyzed by polyacrylamide gel (12.5%) electrophoresis. A fluorograph is shown. gp125 and p26 refer to the extracellular envelope glycoprotein and the major core protein of HIV-2 ROD, respectively. gplO0 and p27 refer to the extracellular envelope glycoprotein and the major core protein of HIV-2 EHO, respectively.

and 2 EHO). This 1 00-kDa protein was specific to HIV2 since it was not recognized by the HIV-l -positive serum. Both HIV-l- and HIV-2-positive sera precipitated a 27-kDa protein which was probably the major core protein of HIV-2 EHO. However, this latter protein was not recognized by the serum from patient EHO (Fig. 1, EHO). These observations indicate the absence of antigag antibodies in the serum of patient EHO. Such a phenomenon has been reported in HIV-1 -infected individuals and it is associated with the development of AIDS. The late stages of HIV-1 infection are characterized by an increased production of antigen and a decreased level of antibodies directed against the major core protein. In fact this observation is routinely used as a marker to monitor the switch from latent to active HIV infection (Lange et a/., 1986; Pedersen et al., 1987).

Identification of env, gag, and vpx proteins of HIV-2 EHO In these experiments, HIV-2 ROD and EHO infected CEM cells were metabolically labeled with [35S]methionine and the labeled extracts were assayed by immunoprecipitation using different types of antibodies: a typical HIV-2-positive serum which identifies envelope proteins of HIV-2 ROD (Rey et al., 1989a), a monoclonal antibody specific for the transmembrane glycoprotein of HIV-2 ROD, a monoclonal antibody specific for the major core protein p26 of HIV-2 ROD, and rabbit polyclonal antibodies specific for the vpx protein.

262

REY ET AL.

a

b ROD

gp300--,

gpl40--, gp125-

MO

1

+gP27@

ROD

EHO

gp300-D

~~~270

gp140--,

cgp120 cgpf+O

gpsO+

,-gPl20 *I+gplOO

FIG. 2. Characterization of HIV-2 EHO envelope glycoproteins. (a) HIV-2 ROD- and EHO-infected cells were labeled with [?S]methionine and extracts were then used in an immunoprecipitation assay using a typical HIV-2-positive serum. The samples were analyzed on a 5% polyacrylamide gel containing 0.1% bis-acrylamide. (b) Extracts from HIV-2-infected cells labeled with [3H]glucosamine were assayed by immunoprecipitation using a monoclonal antibody specific for the transmembrane glycoprotein of HIV-2 ROD. The samples were analyzed by polyactylamide gel (10%) electrophoresis. Fluorographs are shown. Envelope precursors: gp140 ROD, gp120 EHO; dimeric forms of the precursor: gp300 ROD, gp270 EHO; extracellular glycoprotein: gp125 ROD, gplO0 EHO; transmembrane glycoprotein: gp80 for both ROD and EHO.

In the case of HIV-2 ROD envelope glycoproteins, we have previously shown that the envelope precursor (gp140) forms a dimer (gp300) during its processing required for the production of mature envelope proteins: the extracellular glycoprotein gp125 and the dimeric form of the transmembrane glycoprotein gp80. The monomer of the transmembrane glycoprotein is rarely detectable under the experimental conditions used in our experiments, In order to compare the molecular weights of the envelope glycoproteins we performed an electrophoresis in a 5% polyacrylamide gel containing 0.1% bis-acrylamide instead of 0.2%. Figure 2a shows the results of this experiment indicating that the envelope precursor, the dimeric form of the envelope precursor, and the extracellular envelope glycoprotein are 20-30 kDa smaller in HIV-2 EHO compared to those of HIV-2 ROD. In order to identify the transmembrane glycoprotein of HIV-2 EHO we used a monoclonal antibody against the transmembrane glycoprotein of HIV-2 ROD. As expected, this monoclonal antibody also identifies the envelope precursor and its dimeric form. Accordingly, the monoclonal antibody immunoprecipitated gp120, gp270, and gp80 from HIV-2 EHO-infected cells compared to gpl40, gp300, and gp80 from HIV-2 ROD-infected cells (Fig. 2b). The electrophoretic mobility of the transmembrane glycoprotein dimer of HIV-2 EHO was slightly slower than that of HIV-2 ROD. Since this difference is not signifi-

cant, for convenience we will refer to it as gp80. Pulsechase experiments and studies on the kinetics of accumulation of envelope proteins of HIV-2 EHO indicated that gpl20 was the first glycoprotein synthesized before the formation of the precursor dimer gp270 and the production of mature envelope proteins: extracellular glycoprotein gplO0 and the transmembrane glycoprotein dimer gp80. Glycosylation of all envelope proteins was blocked by tunicamycin, an antibiotic which inhibits N-linked glycosylation of proteins (Schwartz et al., 1976; Kornfeld and Kornfeld, 1985). Among these glycoproteins of the envelope, only gplO0 and gp80 were found to be associated with virus particles (data not shown, similar to previously reported results on HIV-2 ROD) (Rey et al., 1989a). The gag precursor polyprotein and the major core protein of HIV-2 EHO were identified by a monoclonal antibody raised against the major core protein p26 of HIV-2 ROD. lmmunoprecipitation with this antibody indicated that the gag precursor and the major core protein of HIV-2 EHO have a slightly higher molecular weight compared to those of HIV-2 ROD: 56 and 27 kDa in HIV-2 EHO compared to 55 and 26 kDa in HIV2 ROD (Fig. 3a). Despite this slight difference, the gag products of these two viruses should be quite similar because of the presence of common epitopes recognized by the monoclonal and polyclonal (patients sera) antibodies (Figs. 1 and 3). One of the specific features of the HIV-2 is the open reading frame called vpx which encodes for a 16-kDa protein (Guyader et a/., 1987; Franchini et al., 1988; Yu eta/., 1988). This protein is associated with the mature virus and appears to play an important role in the patho-

a

b

ROD

ROD EHO

Pr55-c

EHO

--Pr56 p16-w cp14

~264

cp27

FIG. 3. Identification of HIV-2 EHO gag and vpx proteins by specific antibodies. [35S]Methionine-labeled extracts from HIV-2 EHO- and ROD-infected CEM cells were assayed by immunoprecipitation using a monoclonal antibody specific for the major core protein p26 of HIV-2 ROD (a) or rabbit polyclonal antibodies specific for the vpx protein of HIV-2 ROD (b). All the samples were analyzed by polyactylamide gel (12.5%) electrophoresis. Fluorographs are shown.

CHARACTERIZATION

ROD EndoH:

-

EHO +

-

+

gpl40-,

-

gp’*O-

FIG. 4. Electrophoretic mobilities of deglycosylated envelope precursors of HIV-2 ROD and EHO. [?S]Methionine-labeled gp300 (ROD) and gp270 (EHO) were purified by immunoprecipitation followed by preparative gel electrophoresis (Materials and Methods). The lyophilized samples were suspended in the endo H buffer (which results in the dissociation of dimers) containing 150 mM sodium citrate, pH 5.5, 0.1% SDS (w/v), and 0.5 mM PMSF. These samples were incubated (30”, 2 hr) in the absence (lanes -) or presence (lanes +) of 10 units of endo H. Reactions were stopped by the addition of twofold concentrated electrophoresis sample buffer. Samples were analyzed by polyacrylamide gel (7.5%) electrophoresis. Fluorographs are presented. On the right the arrows indicate the position of the digested products, 80 and 60 kDa for ROD and EHO, respectively.

genesis of HIV-2. (Guyader et al., 1989). The vpx protein was identified by a specific rabbit serum raised against a synthetic peptide deduced from the N-terminal region of HIV-2 ROD vpx gene. Figure 3b shows the vpx proteins purified by immunoprecipitation using [35S]methionine-labeled viral extracts from HIV-2 ROD and EHO. The vpx protein in HIV-2 EHO is 14 kDa (~14) compared to that of HIV-2 ROD which is 16 kDa (~16). These same viral extracts were also immunoprecipitated using rabbit serum raised against a peptide deduced from the C-terminal region of the vpx gene of HIV-2 ROD. This latter serum identified pl6 of HIV-2 ROD and p14 of HIV-2 EHO (data not shown). Thus, these results indicated that despite the difference in their size, the ~16 and p14 have some common sequences at least at their N-and C-terminal regions.

OF

HIV-2

EHO

263

fl-N-acetylglucosaminase H (endo H), which cleaves high-mannose-type oligosaccharide chains (Tarentino et a/., 1974). HIV-2 EHO- or ROD-infected cells were labeled with [35S]methionine and cell extracts were used for the purification of the dimeric forms of envelope precursors by immunoprecipitation and preparative gel electrophoresis. The purified dimer precursors were then dissociated in the presence of SDS and acidic pH (Rey et al., 1989a) before digestion with endo H. The apparent molecular weights of the envelope precursors gp140 (ROD) and gpl20 (EHO) were reduced to proteins of 80 and 60 kDa, respectively (Fig. 4). These results therefore indicated that the 20-kDa difference between the envelope precursors of the two HIV-2 isolates was not due to a difference in the number of N-linked oligosaccharide chains. It should be noted that under the experimental conditions of these experiments, digestion with endo H resulted in a complete cleavage of all oligosaccharide chains (data not shown). In order to compare the polypeptide pattern of the envelope precursors of HIV-2 ROD and EHO, we carried out partial proteolysis experiments with Staphylococcus aureus V8 protease, which manifests specificity for glutamoyl bonds (Houmard and Drapeau, 1972). The purified envelope dimer precursors were first dissociated, before partial proteolysis with different concentrations of the V8 protease. The gpl40 arising from the dissociation of gp300 of HIV-2 ROD was converted to three major polypeptides of 130, 110, and 80 kDa whereas the gp120 arising from the dissociation of gp270 of HIV-2 EHO was converted to a major 60-kDa polypeptide and two faint 90- and 75-kDa polypeptides (data not shown). Thus digestion of gpl40 and gpl20 with the V8 protease resulted in the production of distinct polypeptide patterns which probably reflected

HIV-1 Sl Ab 160-W 126+

ROD Sz Ab

EHO SZ Ab

300-b

270-r

I:%= 60*

:'"oz lo--

SIVMAC

SZ Ab

Characterization of HIV-2 ROD and HIV-2 EHO envelope precursors

There are at least 30 potential N-linked glycosylation sites on the envelope precursor of HIV-2 ROD. In view of this, it remained possible that the 20-kDa difference between the envelope precursor of HIV-2 EHO and ROD might be due to a reduced number of oligosaccharide chains in the envelope precursor of HIV-2 EHO. For this reason, we analyzed the electrophoretic mobilities of these precursors after deglycosylation by

FIG. 5. lmmunoprecipitation assay using polyclonal antibodies raised against gp300 of HIV-2 ROD. [3H]Glucosamine-labeled extracts from HIV-1 BRU, HIV-2 ROD, HIV-2 EHO, and SIVmac-infected cells were assayed by immunoprecipitation using a typical human HIV-l -positive serum (lane Sl), a typical human HIV-2-positive serum (lanes S2), and murine polyclonal antibodies raised against gp300 of HIV-2 ROD (lanes Ab). Samples were analyzed by polyacrylamide gel (12.5%) electrophoresis. Fluorographs are shown.

264

differences in the amino acid composition these two envelope precursors.

REY ET AL.

between

Serum

HIV-2

a gp300 30 8 Lu

r:

HIV-2 EHO envelope glycoproteins are not recognized by murine polyclonal antibodies against the envelope glycoproteins of HIV-2 ROD

w

gp300 d

,!3P’40

Polyclonal antibodies were prepared by immunizing mice with a purified preparation of gp300 from cells infected with HIV-2 ROD (Materials and Methods). These antibodies were referred to as anti-gp300 polyclonal antibodies. The reactivity of these antibodies was tested in an immunoprecipitation assay using extracts from HIV-1 BRU-, HIV-2 ROD-, HIV-2 EHO-, and SIVmat-infected cells labeled with [3H]glucosamine (Fig. 5). These assays were carried out parallel with typical human sera specific for HIV-l and HIV-2 (corresponding to the ROD isolate). Anti-gp300 polyclonal antibodies immunoprecipitated gp300, gpl40, gpl25, and gp80 of HIV-2 ROD with an affinity similar to that of the HIV-2-positive human serum. In contrast, anti-gp300 antibodies did not recognize any of the envelope glycoproteins of HIV-2 EHO (Fig. 5, sections ROD and EHO). However, these latter (gp270, gpl20, gp100, and gp80) were immunoprecipitated by the typical HIV-2positive human serum. These observations indicate the presence of similar and some distinct epitopes on both the extracellular and the transmembrane envelope glycoproteins precursor of HIV-2 ROD and EHO. Anti-gp300 antibodies did not recognize HIV-1 envelope glycoproteins, the precursor gpl60, and the extracellular glycoprotein gpl20, which, as expected, were immunoprecipitated by the HIV-l specific human serum (Fig. 5, section HIV-l). Interestingly, anti-gp300 antibodies immunoprecipitated SlVmac envelope glycoproteins: the envelope precursor gpl40, the precursor dimer gp300, and the extracellular glycoprotein gpl30. These SlVmac glycoproteins were also immunoprecipitated by the HIV-2-specific human serum (Fig. 5 section SIVmac). The reactivity of anti-gp300 antibodies with envelope glycoproteins of both HIV-2 ROD and SIVmac, but not with those of HIV-2 EHO, confirms the important homology between HIV-2 ROD and SlVmac and also suggests that HIV-2 EHO belongs to a subtype of the HIV-2 that could be differentiated by its lower homology to both HIV-2 ROD and SIVmac. Anti-gp300 antibodies along with HIV-2- and HIV-lspecific human sera were used in an immunoblot assay with extracts from HIV-l BRU-, HIV-2 ROD-, and HIV-2 EHO-infected cells (Fig. 6). The HIV-2-positive serum strongly identified gp300, gp140, and gp80 of HIV-2 ROD, and gp270, gp120, and gp80 of HIV-2

xgp125

FIG. 6. Envelope glycoproteins of HIV-2 EHO are not recognized by anti-gp300 antibodies. Extracts from CEM cells infected with HIV1 or HIV-2 ROD or HIV-2 EHO were analyzed by immunoblotting using the serum of the patient EHO (section Serum HIV-2) or the murine anti-gp300 antibodies (section anti-gp300); Polyacrylamide gel electrophoresis was carried out on 10% (in sections Serum HIV-2) or 7.5% polyacrylamide (sections anti-gp300) gels. Autoradiographs are presented.

EHO, but it showed no reactivity with envelope glycoproteins of HIV-1 (Fig. 6, section Serum HIV-2; lOq/o polyacrylamide gel). The poor affinity of this serum with the extracellular glycoprotein of HIV-2 was probably due to low reactivity with the denatured protein since this same serum could identify gp125 (ROD) and gp100 (EHO) under native conditions of the immunoprecipitation assay(Fig. 5). Anti-gp300 antibodies identified strongly all the envelope glycoprotein of HIV-2 ROD (gp300, gp140, gpl25, and gp80) but it showed no reactivity with the corresponding envelope glycoproteins of HIV-2 EHO nor with proteins of HIV-1 (Fig. 6, section anti-gp300; 7.5% polyactylamide gel). The reactivity of anti-gp300 antibodies with the 60-kDa protein was found to be not specific because it was observed in cell extracts independent of virus infection. These results indicate that anti-gp300 antibodies do not react with envelope glycoproteins of HIV-2 EHO, either native or denatured form. Comparison of the results obtained by immunoblot analysis and the immunoprecipitation assays (Figs. 5 and 6) show that patient serum and anti-gp300 polyclonal antibodies recognize the denatured form of gp80 better than its native form. The native form of this transmembrane glycoprotein dimer probably has a conformation which masks the epitopes identified by these different antibodies.

DISCUSSION Recently, we reported that HIV-2 and SlVmac can be differentiated from HIV-l by the processing pathway of

CHARACTERIZATION

their envelope glycoprotein precursors (Rey and Hovanessian, 1989). HIV-2 and SIV envelope precursors form an homologous dimer during their processing into the mature products, the extracellular and the transmembrane glycoproteins (Rey et a/., 1989a). Furthermore, transmembrane glycoproteins of HIV-2 and SIV exist as homodimers (Rey et a/., 198913). Under similar experimental conditions, dimerization of the envelope precursor and dimeric forms of the transmembrane glycoprotein are not observed in the case of HIV-l. Dimerization of the envelope glycoproteins, therefore, might be a specific property of HIV-2 and SIV envelope gene expression. Accordingly, this property could be used as a convenient marker to differentiate between different isolates of HIV and SIV. Here we describe a new cytopathic isolate of HIV-2 from an Ivory Coast patient with AIDS. The unusual feature of this new HIV-2 isolate (referred to as HIV-2 EHO) is that its envelope precursor and the extracellular glycoproteins are 20 kDa smaller than those of HIV-2 ROD. This difference is not due to the number of N-linked oligosaccharide chains per polypeptide. In fact, deglycosylated envelope precursors of HIV-2 ROD and EHO have an apparent molecular weight of 80 and 60 kDa, respectively. Several other HIV isolates apparently related to HIV2 EHO have been described recently (Jan McClure, personal communication). So far, the different isolates of HIV-2 fall into two major prototypes: isolates related to HIV-2 ROD or those related to HIV-2 EHO. These two prototypes can be differentiated simply by polyacrylamide gel electrophoresis of their envelope glycoproteins. Table 1 gives the molecular weights of envelope glycoproteins of HIV-2 ROD and EHO estimated in relation to the molecular weights of the HIV-1 envelope precursor (gpl60), the extracellular glycoprotein (gpl20), and the transmembrane glycoprotein (gp41). The envelope precursor and the extracellular protein in the case of HIV-2 ROD are 140 and 125 kDa, in contrast to 120 and 100 kDa in the case of HIV-2 EHO. Despite this difference between HIV-2 ROD and EHO, envelope precursors of both viruses form dimers (gp300 for ROD, gp270 for EHO) during processing into the mature envelope products. In both viruses, the transmembrane proteins are 36 kDa and exist as homodimers with comparable electrophoretic mobilities. HIV-2 EHO can also be distinguished by its protein vpx (14 kDa), its gag precursor (56 kDa), and the major core protein (27 kDa) which are slightly smaller or bigger than the corresponding proteins of HIV-2 ROD. In regard to the size of the vpx and the gag products, HIV-2 EHO seems to be closer to SlVmac than to HIV-2 ROD. On the other hand, envelope precursor and the extracellular envelope glycoproteins of SlVmac seem to be

OF

HIV-2

EHO

265 TABLE

1

COMPARATIVE MOLECULAR WEIGHT (kDa) OF VIRAL PROTEINS: HIV-l BRU, HIV-2 ROD, HIV-2 EHO, AND SlVmac env Pr

TM cm

Virus

Monomer

Dimer

ECgp

HIV-1 BRU

160 140 120 140

-

120

300 270 300

125

HIV-2RoD HIV-2EHo

SIVm.c

100 130

Monomer

gag Dimer

Pr

MCP

41

-

36 36 32

80 80 65

55 55 56 56

25 26 27 28

“Px X 16 14 14

Note. The apparent molecular weights of viral proteins were estimated in relation to the molecular weights of HIV-l BRU proteins (Montagnier et al., 1985; Rey et al., 1989a,b; Veronese et al., 1989; Figs. 2 and 3). The extracellular glycoprotein and the major core protein of HIV-1 are also referred to as gpl 10 and ~24, respectively. It should be noted that under nondenaturing conditions (i.e., in the absence of SDS), oligomeric forms of the transmembrane glycoprotein of HIV-1 have been reported (Pinter et a/., 1989). Pr, precursor; EC gp, extracellular glycoprotein; TM gp, transmembrane glycoprotein; MCP, major core protein.

closer to those of HIV-2 ROD compared to HIV-2 EHO (Table 1). The transmembrane glycoproteins of SlVmac also exist as homodimers of 65 kDa. Under experimental conditions of our assays, the monomeric forms of the transmembrane glycoproteins of HIV-2 ROD, EHO, and SlVmac are seldomly detectable. In vitro, these transmembrane dimers could be dissociated at low pH in the presence of SDS to give a 36-kDa protein in the case of HIV-2 ROD and EHO or a 32-kDa protein in the case of SIVmac. Previously, we have postulated that conformational modifications brought about by the dimerization of the envelope precursor might be necessary for transport of the glycoprotein precursor to the Golgi apparatus and its processing into the mature envelope products (Rey et a/., 1989a). Dimeric forms of transmembrane glycoproteins might be essential for optimal structure of the virion and thus its infectivity. Several observations suggested that differences between the envelope precursors of HIV-2 ROD and EHO are due to their amino acid composition. For example, partial proteolysis of the envelope precursors from the two HIV isolates with Staphylococcus aureus V8 protease resulted in the production of distinct polypeptide patterns. In accord with this difference, polyclonal antibodies raised against HIV-2 ROD gp300 recognized all the envelope glycoprotein products of HIV-2 ROD but did not at all recognize the corresponding envelope glycoproteins of HIV-2 EHO. These results therefore, confirm the existence of distinct epitopes on the envelope glycoproteins of the two viral isolates. The 20-kDa

266

REY ET AL

difference between the envelope precursors of HIV-2 ROD and EHO indicate an important deletion in the env gene of HIV-2 EHO. The observation that the transmembrane glycoprotein of HIV-2 EHO has a comparable molecular size to that of HIV-2 ROD whereas the extracellular glycoproteins are 20-25 kDa different in size indicates that the deletion is in the region of the env gene encoding the extracellular glycoprotein. Future work, therefore, will be concentrated on the molecular cloning of the HIV-2 EHO RNA and determination of its nucleotide sequence. These studies are essential to find the variability among HIV-2-related viruses and also to characterize the homology at the nucleotide level between HIV-2 EHO compared to HIV1 and to different SIV isolates. REFERENCES J., BREDBERG, U., CHIODI, F., B~~TTIGER, B., FENYB, E. M., NORRBY, E., and BIBERFELD, G. (1987). A new human retrovirus isolate of West African origin (SBL-6669) and its relationship to HTLVIV, LAV-II and HTLV-IIIB. AlDSRes. Hum. Refroviruses 3,3-10. ARYA, S. K., BEAVER, B., JAGODZINSKI, L., ENSOLI, B., KANKI, P. I., ALBERT, J., FENYB, E. M., BIBERFELD, G., ZAGURY, J. F., LAURE, F., ESSEX, M., NORRBY, E., WONG-STAAL, F., and GALLO, R. C. (1987). New human and simian HIV-related retroviruses possess functional transactivator (tat) gene. Nature (LondonJ 328, 548-550. BARR~~INOUSSI, F., CHERMANN, J. C., REY. F., NUGEYRE, M. T., CHAT MARET, S., GRUEST, J., DAUGUET, C., AXLER-BLIN, C., BRUN V~ZINET, F., ROUZIOUX, C., ROZEMBAUM, W., and MONTAGNIER, L. (1983). Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 220, 868871. BRUN-V~ZINET, F., REY, M. A., KATLAMA, C., GIRARD, P. H., ROULOT, D., YENI, P., LENOBLE, L., CLAVEL, F., ALIZON, M., GADELLE. S., MADJAR, J. J., and HARZIC, M. (1987). Lymphadenopathy associated virus type 2 in AIDS-related complex. Lancet i, 128-l 32. BURNEITE, W. N. (1981). “Western blotting”: Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioionidated protein A. Anal. Biochem. 112, 195-230. CHAKRABARTI. L., GUYADER, M., ALIZON, M., DANIEL, M. D., DESROSIERS, R. C., TIOLLAIS, P., and SONIGO, P. (1987). Sequence of simian immunodeficiency virus from macaque and its relationship to other human and simian retroviruses. Nature (London) 328, 543547. CLAVEL, F. (1987). Editorial review: HIV-2, The West African AIDS virus. AlDS 1, 135-l 40. CLAVEL, F., GU~TARD. D., BRUN-V~ZINET, F., CHAMARET, S., REY, M. A., SANTOS-FERREIRA, M. O., LAURENT, A. G., DAUGUET, C., KATLAMA, C., ROUZIOUX, C., KLATZMANN, D., CHAMPALIMAUD, J. L., and MONTAGNIER, L. (1986a). Isolation of a new retrovirus from West African patients with AIDS. Science 233,343-346. CLAVEL, F., GUYADER, M., GU~TARD, D.. SALLY, M., MONTAGNIER, L.. and ALIZON, M. (1986b). Molecular cloning and polymorphism of the immune deficiency virus type 2. Nature (London) 324, 691694. COHEN, E. A., TERWILLIGER, J. G., SODROSKI, 1. G., and HASELTINE. W. A. (1988). Identification of a protein encoded by the vpu of HIV1. Nature (London) 334, 532-534.

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