Mar 28, 1988 - the rhesus monkey (Maccaca mulatta), sooty mangabey. (Cercocebus atys), African green monkey (Cercopitecus aethiops), and mandrill ...
JOURNAL OF VIROLOGY, Dec. 1988, p. 4782-4785
Vol. 62, No. 12
0022-538X/88/124782-04$02.00/0 Copyright © 1988, American Society for Microbiology
Production and Characterization of Monoclonal Antibodies Specific for the Transmembrane Protein of Simian Immunodeficiency Virus from the African Green Monkey TOSHIAKI KODAMA,' YOSHIHIRO OHTA,' TAKAO MASUDA,l KOH-ICHI ISHIKAWA,' HAJIME TSUJIMOTO,' MOHAMED ISAHAKIA,2 AND MASANORI HAYAMI1* Department of Animal Pathology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minatoku,
Tokyo 108, Japan,1 and Institute of Primate Research, National Museums of Kenya, Karen, Kenya2 Received 28 March 1988/Accepted 12 August 1988
Mouse monoclonal antibodies were produced against simian immunodeficiency virus (SIV) from the African monkey (SIVAGM). The antibodies reacted with the transmembrane protein of all five SIVAGM isolates but not with those of SIVs from the rhesus macaque and mandrill or of human immunodeficiency virus type 1 or type 2, indicating that they recognize a species-specific epitope strongly conserved in SIVAGM. The transmembrane proteins of several SIVAGM isolates were found to vary in molecular size, even in the deglycosylated form after N-glycanase treatment, indicating heterogeneity of the SIVAGM isolates. green
The human immunodeficiency virus type 1 (HIV-1) was first isolated as an etiological agent of acquired immunodeficiency syndrome (AIDS) in humans (1, 20, 24). Later, simian retroviruses closely related to HIV-1, referred to as simian immunodeficiency viruses (SIVs), were isolated from several species of nonhuman primates: those isolated from the rhesus monkey (Maccaca mulatta), sooty mangabey (Cercocebus atys), African green monkey (Cercopitecus aethiops), and mandrill (Papio sphinx) were designated
SIVMAC (7, 15), SIVSMM (9), SIVAGM (23), and SIVMND (28a), respectively. SIVs have several properties similar to those of HIV-1: tropism and cytopathic effects to CD4bearing human T lymphocytes in culture, Mg2l preference of reverse transcriptase, characteristic virion morphology, antigenicity (7, 23), and genetic organization (3, 8, 13). Recently, some AIDS patients in West Africa were shown to be infected with a second AIDS virus designated HIV-2 (2, 5), which was shown to be antigenically (5) and genetically closer to SIVMAC than to HIV-1 (3, 6, 11). It is important to compare and differentiate these HIV and SIV group retroviruses to clarify their origin, transmission, and evolution. To investigate the antigenetic relationships of these viruses, we prepared monoclonal antibodies (MAbs) against SIVAGM, which was isolated in our laboratory (23) and differs from STLV-IIIAGM (14), which is now considered to be derived from SVMAC (17). Five-week-old female BALB/c mice were immunized intraperitoneally with virion protein (50 jig) purified from the culture supernatant of MOLT-4 cells infected with SIVAGM[TYO-l] (previously referred to as SIV[AGM-1]) (23) by two-step sucrose gradient centrifugation. The virion protein was emulsified in complete Freund adjuvant for the first immunization and in incomplete adjuvant for two subsequent boosters. In Western blotting (immunoblotting) analyses (28), the immune mouse serum reacted with all the major SIVAGM viral proteins of 24, 18, 66/55, 145, and broad-banded 32 to 34 kilodaltons (kDa), which are analogous to the gag, pol, and env gene products of HIV-1 (Fig. 1A, lane P). Immune splenic lymphocytes were fused with the X63*
Corresponding author. 4782
Ag8.653 myeloma line (16) with polyethylene glycol, and hybridomas were selected in HAT medium (10). The hybridoma supernatants were screened by enzyme-linked immunosorbent assay with 1% Triton X-100-disrupted SIVAGM[TYO-1] virions (30), and antibody-secreting hybridomas were cloned twice by limiting dilution. In independent fusions, 4 of 400 hybridomas, named TM105, TM211, TM219, and TM304, were found to secrete antibodies specific for a broad band of protein of 32 to 34 kDa, which was shown to be the transmembrane protein (TMP) of SIVAGM[TYO-lI (23) (Fig. 1A, lanes 1 to 4). TM304 and TM105 are of the immunoglobulin G2a (IgG2a) subclass, and TM211 and TM219 are of the IgGl subclass, and all four have a K light chain. A competitive binding assay was carried out to determine the epitope recognized by these MAbs (21). TM105, TM211, and TM219 significantly inhibited the binding of biotinated TM304, indicating that they had a closely related epitope (Fig. 1B). In immunofluorescence assays, TM304 caused positive staining of the cytoplasm of acetonefixed SIVAGM-infected MOLT-4 cells and the plasma membrane of unfixed cells. Therefore, the epitope recognized by the MAb is expressed on the surface of virus-infected cells. The TMPs of all five SIVAGM strains, including the strain isolated in Kenya, were detected as broad bands with TM304. The Kenyan strain SIVAGM[KE-1I was isolated in Kenya from the native seropositive African green monkey by cocultivation of its concanavalin A-stimulated peripheral blood lymphocytes with MOLT-4 clone 8 cells (18). These TMPs of SVAGM varied in size, being 32 to 34 kDa in SIVAGM[TYO-l] and a Kenyan strain (KE-1), 36 to 38 kDa in TYO-5, and 34 to 36 kDa in TYO-2 and TYO-7 (Fig. 2). The molecules recognized by TM304 were thought to be TMPs by analogy with gp4l of HIV-1 (29), but the proteins of SIVAGM and HIV-1 showed very weak cross-reactivity with env-related proteins (23). Although TM304 did not react with gp4l of prototype HTLV-IIIB (24) (Fig. 3), the putative TMPs of HIV-2 (5), SVMAC, and SIVMND (data not shown) were highly reactive with SIVAGM-infected sera (Fig. 3, lane S). SVMND was isolated from a mandrill in Gabon with antibodies to SIVAGM[TYO-1 by the same procedure. Therefore, they might have a common epitope in TMP; however, these TMPs did not react with TM304 (Fig. 3, lane
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NOTES 2 3 4
FIG. 2. Reactivities with TM304 and heterogeneities of the TMPs of several SIVAGM isolates. Purified viral proteins of five SIVAGM isolates were treated with simian positive sera (A) and TM304 culture supernatants (B). Lane 1, TYO-1; lane 2, KE-1; lane 3, TYO-5; lane 4, TYO-2; lane 5, TYO-7. Lane M, Molecular size standards (in kilodaltons).
w Z : Co
0.4 - -
6 0. 3---
0.2 - 0.7
25 50 100
mAb (pg/ml) FIG. 1. (A) Western blotting analysis of reactivities of four MAbs with SIVAGM[TYO-1I. SIVAGM[TYO-1] viral proteins (1 Rg per lane) were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose sheets. Strips were treated with 1:100-diluted immune mouse sera (lane P) and culture supernatants from hybridomas TM304 (lane 1), TM105 (lane 2), TM211 (lane 3), and TM219 (lane 4). (B) Competitive binding assays with MAbs to SIVAGM. Volumes (100 ,±l) of serially diluted MAb TM105 (A), TM211 (A), TM219 (0), and TM304 (0) were incubated overnight at 4°C in wells coated with detergentdisrupted SIVAGM[TYO-1I. Then the wells were washed, and 100 Rl of biotin-conjugated TM304 was added. The binding of biotinconjugated TM304 was detected with biotinylated peroxidase-streptoavidin complex and substrate.
M). These results suggest that the antigenic epitope of the TMP recognized by TM304 is species-specifically conserved in SIVAGM isolates. The reactivity of TM304 with an extract of [3H]leucine-
labeled cells producing SIVAGM was mainly directed against a protein of 140 kDa, but it was weak against the protein of 32 to 34 kDa (Fig. 4A). On the other hand, with labeled virions the protein of 32 to 34 kDa was the predominant antigen detected by TM304 (Fig. 4B). Therefore, the protein of 32 to 34 kDa should be derived from the 140-kDa protein present in the cell extract, and the processing and maturation of the env gene product in SIVAGM may occur mainly after budding from the plasma membrane, as is the case in HIV-1 (22). To examine the glycosylation of the TMP of SIVAGM, we treated the virions with N-glycanase (Genzyme, Su Hulk, England), which hydrolyzes asparagine-linked oligosaccharides of glycoproteins (27). After enzymatic treatment, TM304 reacted with a distinct band of 23 kDa instead of the glycosylated form (32 to 34 kDa) in SIVAGM[TYO-l] (Fig. 5), indicating that the TMP of TYO-1 is glycosylated and that TM304 recognizes its protein backbone. The glycosylated and deglycosylated forms differ in size by approximately 10 kDa, indicating that the TMP contains three or four asparagine-linked oligosaccharides. From our nucleotide sequence data on SIVAGM[TYO-1] (8), the TMP-encoding region has three potential glycosylation sites and is predicted to encode a protein of 26 kDa in the unglycosylated form. As shown in Fig. 2, there was a variation in the molecular size of the TMPs among several SIVAGM isolates. After N-glycanase treatment, the TMP of SIVAGM[TYO-5] was reduced in size H
5-IV siVui5 t
_a! I _so
FIG. 3. Reactivities of TM304 MAb with HIV-1, HIV-2, and SIVMAC proteins. Prototype HTLV-IIIB was treated with HIV-1 positive sera (H) and TM304 culture supernatants (M). Viral proteins of HIV-2ROD and SIVMAC were treated with SIVAGM positive sera (S) and TM304 culture supernatants (M). The TMP of each virus is indicated.
J. VIROL. B
protein (23), like that of HIV-1 (25), but its functional role is not yet clear. The MAbs produced in this study will be useful for functional analysis of the TMP of SIVAGM.
200 ..14 b K
32-3s4K 29 24_
FIG. 4. Electrophoretic analysis (10% acrylamide gel) of metabolically labeled SIVAGM protein immunoprecipitated with TM304. The lysate of SIVAGM[TYO-1]-infected MOLT-4 cells, labeled for 24 h with [3H]leucine (80 pCi/ml) (A), and of labeled virions concentrated from the culture supernatant (B) were immunoprecipitated with immune mouse sera (lanes 1), monoclonal antibody TM304 (lanes 2), and normal mouse sera (lane 3). M, Molecular size standards (in kilodaltons).
from 36 to 38 kDa to 26 kDa, and there was a difference in size of 3 kDa between the deglycosylated forms of TMP from SIVAGM strains TYO-1 and TYO-5 (Fig. 5). Therefore, the size heterogeneity of the TMPs was probably due to differences in the molecular weight of their protein backbones. Genetic heterogeneity of HIV-1 has been demonstrated (31), especially that of the exterior env gene (12), but the TMPencoding portion is relatively well conserved (26). Conceivably, SIVAGM has genetic diversity in the TMP-encoding gene, resulting in the variations in the molecular weight of the products. SIVs have several features similar to HIV-1 and HIV-2, suggesting that the HIV/SIV retrovirus group arose from a common ancestor virus. But the present study indicated that the TMP of SIVAGM has a species-specific antigenic epitope that is strongly conserved in SIVAGM but is not detected in HIV-1, HIV-2, and SIVs from other nonhuman primates. Nucleotide sequence analyses also indicate that SIVAGM is quite distinct from both HIV-1 and HIV-2 (8). Therefore, the human AIDS-related viruses cannot have originated from African green monkeys in recent times. The TMP of HIV-1 has a neutralizing epitope (4) and has potential roles in replication and cytopathology (19). The TMP of SIVAGM is known to be the most immunogenic a YOC-1
36-38K 26 K
FIG. 5. Effect of N-glycanase on putative TMPs of SIVAGM strains. Denatured viral proteins of SIVAGM strains TYO-1 and TYO-5 were incubated in the absence (A) or presence (B) of 250 mU of N-glycanase. The products were blotted onto membranes and treated with TM304 culture supernatants.
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