Serological cross-reactivity between envelope gene products of type I ...

Proc. Nati. Acad. Sci. USA Vol. 81, pp. 7579-7583, December 1984 Medical Sciences

Serological cross-reactivity between envelope gene products of type I and type II human T-cell leukemia virus (gp6l/gp67/radiosequence analysis/human T-cell leukemia virus-associated cell membrane antigen)

T. H. LEE*, J. E. COLIGANt, M. F. MCLANE*, J. G. SODROSKI§, M. Popovict, F. WONG-STAALt, R. C. GALLO*, W. HASELTINE*§, AND M. ESSEX*¶ *Department of Cancer Biology, Harvard University School of Public Health, Boston, MA 02115; tLaboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, and tLaboratory of Tumor Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20205; and §Laboratory of Biochemical Pharmacology, Dana-Farber Cancer Institute, Department of Pathology, Harvard Medical School, Boston, MA 02115

Communicated by Elkan R. Blout, July 16, 1984

ABSTRACT People exposed to type I human T-cell leukemia virus (HTLV-I) develop antibodies to an antigen at the surface of virus-infected cells, designated human T-cell leukemia virus membrane antigen (HTLV-MA). In an earlier study, we demonstrated that the major component of HTLV-MA is gp6l, a glycoprotein encoded by the HTLV env gene. In the current study, we found that human antibodies that react with HTLV-MA on cells infected with HTLV-I react equally well with HTLV-MA on C3-44/MO, a target cell infected with type II HTLV. A glycoprotein with an approximate size of 67 kDa, gp67, was identified in C3-44/MO using immunoprecipitation and NaDodSO4/PAGE analysis. The positions of serine and cysteine residues were determined in the amino terminus of gp67 by radiolabel sequencing analysis. Comparison with the amino acid sequence deduced from the primary nucleotide sequence of HTLV-IIMo virus reveals that gp67 is also encoded, at least in part, by the env gene. The gp67 of HTLV-IIMo, like the env gene product of HTLV-ICR, gp6l, is recognized both by antibodies from a HTLV-IIMO-infected patient with a variant form of hairy cell leukemia, and by antibodies from patients with HTLV-I-associated adult T-cell leukemia/lymphoma. These results indicate that, despite the divergence between HTLV-I and HTLV-II, the major env gene products of the two types of HTLV are conserved to the degree that they are serologically cross-reactive. Human T-cell leukemia virus (HTLV) is a retrovirus of humans and Old World primates that is distinct from previously

known retroviruses of lower species (1-5). Type I HTLV (HTLV-I) is closely associated with, if not the cause of, an aggressive type of adult T-cell malignancy whose clinical manifestations are very similar to those of adult T-cell leukemia/lymphoma (ATLL) (6). The majority of HTLV isolates from the United States, England, and Japan, including those originally designated as adult T-cell leukemia virus, belong to the HTLV-I class (7-10). A second distinct class of HTLV (HTLV-II) was described in 1982 (11). Common features shared by the HTLV-I isolates are the extensive homology of the gag gene-encoded antigens and the homology of the primary nucleotide sequence, as indicated by the conservation of several restriction enzyme sites and by the DNA sequence of selected regions of proviral DNA (12-15). Based on nucleic acid hybridization analysis (16, 17), heteroduplex mapping (18), direct comparison of primary nucleotide sequence of long terminal repeats (19, 20), and the degree of cross-reactivity between gag gene-encoded products (11), it has been concluded that HTLV-I and HTLV-II, although related, are clearly distinguishable. We previously identified a glycoprotein, gp6l, expressed

on the cytoplasmic membrane of a HTLV-I-infected prototype tumor cell line, HUT 102 (clone B2) (21-23). This antigen is the most immunogenic species in HTLV-exposed humans (21). It can be precipitated by serum samples from ATLL patients (21), healthy carriers living in endemic areas (21), and with most acquired immune deficiency syndrome (AIDS) and hemophiliac patient antisera that are positive for antibody to HTLV-associated cell membrane antigen (HTLV-MA) as measured by membrane immunofluorescence (21-23). Using radiolabel sequencing analysis, we have concluded that gp61 is an env gene-encoded product of HTLV-ICR (21). The env gene-encoded products of HTLV-IIMo have not been identified, nor has the extent of serological relatedness between env gene products of HTLV-I and HTLV-II been determined. We report here that human antiserum samples known to have antibody reactivity to gp6l of HTLV-ICR cross-react with HTLV-MA expressed by HTLV-IIMO-infected cells in the membrane immunofluorescence assay and recognize at least one env gene-encoded product of HTLVIlMo. This serological cross-reactivity can be explained by the conserved protein sequence in the env gene regions of these two types of HTLV.

MATERIALS AND METHODS Cells. The HTLV-ICR-infected Hut 102 clone B2 (Hut 102) has been described (1, 21, 24). Uninfected cell lines 8402, Jurkat, and NC37 have also been described (21). C3-44/MO is a HTLV-lIMO-infected cell line (25). All the cells were cultured as described (21, 25). Sera. Serum samples from healthy blood donors and from ATLL patients were from Miyazaki Medical School and were standardized earlier (21, 26). Sources of reference serum samples from AIDS patients and asymptomatic hemophiliacs have been described (22, 23, 26). Serum from a Taiwanese ATLL patient was from the College of Medicine, National Taiwan University. All the sera were initially tested by us under double blind code and were scored positive for HTLV-specific antigens by analysis with the live cell membrane immunofluorescence assay and by radioimmunoprecipitation. The specificity of the goat antiserum to p24 and the monoclonal antibody to p19 was described (3, 4). Serology and Protein Analysis. Detailed procedures for these analyses have been described (21, 22). To enrich glycoprotein fractions, soluble cell lysate first reacted with lentil lectin-Sepharose 4B (Pharmacia) at a ratio of 30 x 106 cells to 0.5 ml of lentil lectin-Sepharose 4B at 40C for 3-6 hr. RIPA buffer in the presence of 0.2 M methyl-a-D-mannoAbbreviations: ATLL, adult T-cell leukemia/lymphoma; HTLV, human T-cell leukemia virus; HTLV-MA, HTLV-associated cell membrane antigen; AIDS, acquired immune deficiency syndrome. $To whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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chance alone is 4.0 x 10-10. These results strongly indicate that both tests are highly related in detecting specific antibody to HTLV-related antigens and suggest that the HTLVMA detected on both cells may be serologically cross-reactive. All 66 antisera were negative for reactivity with uninfected T- and B-lymphoid cell lines. Recognition of HTLV-I-Encoded gp6l by a HTLV-II-Infected Patient. Two env gene-encoded products of HTLV-ICR, gp6l and gp45, were shown by us to be the major antigens recognized by antiserum samples that are positive for antibody to HTLV-MA on Hut 102 cells (21). Because antiserum collected from patient MO, from whom the first isolate of HTLV-II was obtained, also scored as positive for HTLVMA on Hut 102, we examined the possibility that gp6l and gp45 of HTLV-ICR might be precipitated by antiserum from patient MO. As shown in Fig. 1A, both gp6l and gp45 were precipitated from lentil-lectin-enriched glycoproteins of [35S]cysteine-labeled Hut 102 cells by antiserum from patient MO as well as by a reference antiserum from a Japanese ATLL patient, a healthy carrier living in the same endemic area as the ATLL patient, and a Taiwanese ATLL patient. These antigens were not precipitated by a reference negative serum that was collected from a healthy Japanese adult also living in the same endemic area as the ATLL patients. To determine whether the gp6l recognized by antiserum from patient MO and that recognized by other patients were identical, [35S]cysteine-labeled antigens prepared from Hut 102 cells that were treated with a glycosylation inhibitor, tunicamycin, then underwent reaction with the antiserum from the MO patient. As shown in Fig. 1B, the MO patient antiserum, like antiserum from the two ATLL patients, and antiserum from a reference healthy carrier, all precipitated the unglycosylated p50, which was previously shown to be the protein backbone of gp6l (21) from tunicamycin-treated Hut 102 cells. All these antisera precipitate gp6l from the untreated Hut 102 cells. A representative reference negative serum did not precipitate either gp6l or p50. The unglycosylated protein backbone of gp45 was shown to have an approximate size of 34 kDa (21). Antisera from people exposed to HTLV-I and from patient MO also precipitate this species. In addition, all these antisera were shown to precipitate gp45 from the Hut 102 cells that were not treated with tunicamycin. Recognition of an HTLV-IIMO-Specific Glycoprotein by Antiserum from HTLV-I-Exposed People. Precipitation of gp6l by antiserum from the HTLV-II-infected patient MO suggests that env gene products of HTLV-I and HTLV-II are serologically cross-reactive. To identify the cross-reactive

Table 1. Reactivity of antibodies from ATLL patients and healthy blood donors from Miyazaki, Japan, for HTLV-MA on cells infected with HTLV-I vs. cells infected with HTLV-II

Antibody reactivity to Hut 102 Antibody reactivity Total Positive Negative to C3-44/MO 43 7 36 Positive 23 1 22 Negative 66 37 29 Total X2 = 37.75 with 1 degree of freedom. P = 4 x 10-10.

side, but in the absence of deoxycholate, was used to elute glycoprotein fractions. Procedures for tunicamycin treatment and amino acid sequence analysis have been described (21, 27). The amounts of [35S]cysteine (specific activity, >988 Ci/nmol; 1 Ci = 37 GBq) and [3H]serine (specific activity, 29 Ci/nmol) used were 5 mCi and 10 mCi, respectively. DNA Sequence Analysis. Subclones of XMO15A, an apparently complete HTLV-II proviral clone (16), were constructed in pBR322 derivatives. The plasmids were 5' and 3' endlabeled at a BamHI site immediately 5' to the env gene-coding region and were sequenced according to the procedure of Maxam and Gilbert (28).

RESULTS Membrane Immunofluorescence. In a previous study, we reported that specific antibody to HTLV-MA expressed by HTLV-I-infected cells could be detected in antiserum from all 8 Japanese ATLL patients tested and in -10% of the healthy blood donors living in the same endemic area as the ATLL patients (21). Of 37 antisera samples that were previously positive for antibody to HTLV-MA on Hut 102 cells, which are infected with HTLV-I, 36 were also positive for HTLV-MA on C3-44/MO, a line that is persistently infected with HTLV-II. The results are shown in Table 1. The 36 positive antiserum samples include 6 from ATLL patients living in the Miyazaki prefecture of Japan, 29 from healthy carriers in the same endemic area as the ATLL patients, and 1 from patient MO. Twenty-nine antisera samples that were negative for HTLV-MA on Hut 102 were also tested on C344/MO cells. Twenty-two of the 29 samples, which were all from healthy donors from Miyazaki, Japan, were also negative on C3-44/MO cells, while 7 were positive. Using x2 analysis, the probability of having such a concordance by

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FIG. 1. Analysis of HTLV-ICR-encoded glycoprotein detected by antiserum of the patient infected with HTLV-IIMo, using NaDodSO4/ 4 61 PAGE. (A) [35S]Cysteine-labeled glycoproteins bound to lentil lectin-Sepharose 4B reacted 50 with 5 ,ul of reference negative serum (23) from a healthy adult living in the Miyazaki area (lane 1), 5 ,ul of antiserum from patient MO (lane 2), 5 4 34 ,ul of a reference antiserum from an ATLL patient (23) living in the Miyazaki area (lane 3), 3 ,ul of a reference antiserum from a HTLV-exposed healthy adult (23) living in the Miyazaki area (lane 4), and 8 ,ul of antiserum from a Taiwanese ATLL patient (lane 5). (B) Same serum samples numbered the same way as indicated in A reacted with [35S]cysteine-labeled cell lysate from tunicamycin-treated Hut 102 cells (lanes b) or from untreated Hut 102 cells (lanes a).

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env gene product(s) encoded by HTLV-IIMo, we used several antiserum samples known to have antibodies to gp6l of HTLV-ICR (21-23) to precipitate [35S]cysteine-labeled antigens from the HTLV-IIMo immortalized cell line C3-44/MO. One of the glycoproteins bound to the lentil lectin-Sepharose 4B had an approximate size of 67 kDa (gp67) and was detectable with antisera from ATLL patients, patient MO, a reference healthy carrier living in the HTLV-I endemic area, a reference AIDS patient, and a reference asymptomatic hemophiliac patient (Fig. 2A). At least two other antigens with approximate sizes of 52 and 21 kDa could be detected by the same antisera. None of these three species was detected in the cells not infected with HTLV, such as Jurkat, 8402, and NC37, by the reference positive antisera (data not shown), or by the reference negative sera (Fig. 2A). The gp67 species was not detected when C3-44/MO cells were treated with tunicamycin. Instead, both the MO antiserum and the antisera of ATLL patients and a reference healthy carrier precipitate a species with an approximate size of 53 kDa (Fig. 2B). This result provides additional evidence for the glycosylated nature of gp67 and indicates that the unglycosylated portion of gp67 is s53 kDa. Similar results were obtained with endoglycosidase-H digestion of gp67 (data not shown). The 53-kDa species, like gp67, was not precipitated by the reference negative control serum. Two other HTLV-specific antigens coprecipitated by antisera positive for antibody to gp67 are a 52-kDa and a 21-kDa species (Fig. 2). NH2-Terminal Amino Acid Sequence Analysis. To address the question of whether gp67 of HTLV-IIMo, like gp6l of HTLV-ICR, is encoded by the env gene of HTLV-IIMo, the NH2-terminal sequence of the [35S]cysteine- and [3H]serinelabeled gp67 was determined. As shown in Fig. 3, 35S peaks were detected at residues 3, 17, and 24, and 3H peaks were detected at 1, 10, 11, 14, 15, and 18, indicating the presence of cysteine and serine at these positions in the protein sequence. The repetitive yield of 35S peaks was 96.5%, indicating that all the residues are in the same sequence. The amount of radioactivity detected for the serine at position 1 A

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is lower than expected based on the yield of radioactivity for subsequent serine residues. Although the reason for this is unknown, this peak is not artifactually caused by "wash out" (27), because only a background level of 35S count was detected. Comparison of the NH2-terminal amino acid sequence of [35S]cysteine- and [3H]serine-labeled gp67 with the protein sequence of the NH2 terminus of the env gene deduced by DNA sequence analysis of a HTLV-IlMo proviral DNA reveals that at least the NH2-terminal part of gp67 is encoded by. the env gene (Table 2). The first [3H]serine peak of gp67 corresponds to the 21st amino acid deduced from the primary nucleotide sequence of the env gene of HTLV-IIMo, and the rest of the [PH]serine and [35S]cysteine peaks match exactly the rest of the deduced protein sequence. This indicates that the first 20 amino acids deduced from the primary nucleotide sequence of the env gene, which include several hydrophobic residues, represent the leader sequence of gp67 that is post-translationally cleaved from the gp67. Deduced NH2 Terminus Sequence Homology Between env Gene Products of HTLV-I and HTLV-II. We have previously shown that NH2-terminal cysteine residues of gp6l are at positions 6, 7, 21, and 28, thus establishing that the first 20 amino acids deduced from the env gene DNA sequence of HTLV-I (29) represent the leader sequence. By aligning the 1st serine immediately following the leader sequence of HTLV-IIMo with the 5th amino acid beyond the leader sequence of HTLV-ICR, and then comparing the next 39 amino acid residues of HTLV-IIMo and HTLV-ICR, it is revealed that there are 25 (including the fifth amino acid) identical residues (Table 2). Of 15 nonidentical residues, 10 result from the change of a single nucleotide in the triplet codon. The strong degree of the conservation in the deduced amino acid sequence is compatible with our finding the gp67 and gp61 are serologically cross-reactive. Contrary to the extensive homology described above, when the leader sequence of HTLV-I and HTLV-II are compared, there are only 5 identical residues in the leader sequence.

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FIG. 2. Analysis of a HTLV-IIMO-specific glycoprotein detected by human antibodies positive for gp6l, using NaDodSO4/PAGE. (A) 10 of two reference negative sera (23) from two patients [35S]Cysteine-labeled glycoproteins bound to lentil lectin-Sepharose 4B reacted with MA with non-ATLL tumors living in the Miyazaki area (lanes 1 and 2), 5 ,A of four reference positive antisera (23) from four ATLL patients living in the Miyazaki area (lanes 3-6), 5 Al1 of antiserum from patient MO (lane 7), 3 ,ul of a reference positive antiserum (23) from a healthy adult living in the Miyazaki area (lane 8), 10 ,l of a reference negative serum (23) from a healthy adult living in the Miyazaki area (lane 9), 10 Al of a reference positive antiserum (22) from an AIDS patient (lane 10), and 8 Al1 of a reference positive antiserum (23) from an asymptomatic hemophiliac (lane 11). (B) Soluble cell lysate from tunicamycin-treated C3-44/MO (lanes 2, 4, 6, 8, and 10) and from untreated C3-44/MO (lanes 1, 3, 5, 7, and 9) reacted with 10 ,u1 of a reference negative serum (23) from a healthy adult living in the Miyazaki area (lanes 1 and 2), 5 ,ul of antiserum from patient MO (lanes 3 and 4), 5 ,ul of a reference positive antiserum (23) from an ATLL patient living in the Miyazaki area (lanes 5 and 6), 10 ,u1 of antiserum from a Taiwanese ATLL patient (lanes 7 and 8), and 3 ,l of a reference positive antiserum (23) from a healthy adult living in the Miyazaki area (lanes 9 and 10).

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Sequence step FIG. 3. NH2-terminal amino acid sequence analysis of [35S]cysteine and [3H]serine-labeled gp67. Automated Edman degradation was performed and the radioactivity in the fraction obtained from each cycle is listed on the vertical axis in disintegrations per minute (dpm). Both cysteine and serine were determined from the same sequence analysis, but for clarity, cysteine peaks and serine peaks were plotted separately. Radioactive cysteine (A) was detected at cycles 3, 17, and 24. Radioactive serine (B) was detected at cycles 1, 10, 11, 14, 15, 18, and 32.

DISCUSSION Using antiserum samples known to have antibodies to gp6l, which is encoded by the env gene of HTLV-ICR, we identified at least one serologically cross-reactive glycoprotein, gp67, from a HTLV-IIMo immortalized cell line, C3-44/MO. To delineate the coding origin of gp67, radiolabel sequencing analysis was used to determine where in the NH2 terminus of gp67 cysteine and serine residues are located. This approach reveals that there are three 35S and six 3H peaks (seven 3H peaks if residue 32 is included) in the first 35 NH2terminal amino acids analyzed. Comparing this sequence with the deduced protein sequence from the primary nucleotide sequence of a molecularly cloned HTLV-IIMo, we can

conclude with almost complete certainty that gp67 is encoded, at least in part, by the env gene of HTLV-IIMo. The probability of finding such a radiolabeled amino acid sequence in any given HTLV-specific protein by chance alone is