DIANE PARDI,I* JONATHAN E. KAPLAN,' JOHN E. COLIGAN,2 THOMAS M. FOLKS,'. AND RENU B. LAL'. Retrovirus Diseases Branch, National Center ...
Vol. 67, No. 12
JOURNAL OF VIROLOGY, Dec. 1993, p. 7663-7667
0022-538X/93/127663-05$02.00/0 Copyright C) 1993, American Society for Microbiology
Identification and Characterization of an Extended Tax Protein in Human T-Cell Lymphotropic Virus Type II Subtype b Isolates DIANE PARDI,I* JONATHAN E. KAPLAN,' JOHN E. COLIGAN,2 THOMAS M. FOLKS,' AND RENU B. LAL'
Retrovirus Diseases Branch, National Center of Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333,1 and Biological Resources Branch, National Institute ofAllergy and Infectious Diseases, Bethesda, Maryland 208942 Received 12 July 1993/Accepted 24 September 1993
The tax gene sequence of human T-cell lymphotropic virus type II isolate G12 (HTLV-11G12) was found to encode an extended Tax protein when compared with that of HTLV-IIMOT. In vitro transcription-translation of the HTLV-11G12 tax gene produced a 40-kDa Tax protein that specifically reacted with serum specimens from HTLV-II-infected individuals. Limited sequence analysis demonstrated that isolates with an extended Tax protein were all HTLV-II subtype b (HTLV-IIb). Therefore, the extended Tax protein appears to be a unique characteristic of most HTLV-IIb isolates and may be useful in designing immunoassays to distinguish between HTLV-IIa and HTLV-IIb.
Human T-cell lymphotropic virus type I (HTLV-I) and type (HTLV-II) are classified as type C retroviruses that share an apparent tropism for T lymphocytes and an ability to transform normal human peripheral blood mononuclear cells upon cocultivation with infected cells (2, 31). Molecularly and serologically, HTLV-I and HTLV-II are easily distinguished from one another by differences in nucleotide sequence and
U.S. blood donors, as well as among patients attending sexually transmitted disease clinics, to be HTLV-II (19, 30). HTLV-II has recently been found to be prevalent in a number of indigenous New World Amerindian populations, including the Navajo and Pueblo tribes in New Mexico, the Seminole Indians in Florida, the Cayapo and Kraho tribes in Brazil, the Wayuu Indians in Colombia, the Tobas and Mata-
II
MoT MAHFPGFGQSLLYGTPVYVFGDCVQADWCPVSGGLCSTRLHRHALLATCPEHQLTWDPID 60 -------------------------------------------. G12.1
GRVVSSPLQYLIPRLPSFPTQRTSRTLKVLTPPITPVSPKVPPAFFQSMRKHTPYRNGCL
120
-----------------------------------------K-
EPTLGDQLPSLAFPEPGLRPQNIYTTWGKTVVCLYLYQLSPPMTWPLIPHVIFCHPRQLG 180
-F_-----------------------
--------
AFLTKVPLKRLEELLYKMFLHTGTVIVLPEDDLPTTMFQPVRAPCIQTAWCTGLLPYHSI 240 -----------------------A--------------------------
LTTPGLIWTFNDGSPMISGPYPKAGQPSLVVQSSLLIFEKFETKAFHPSYLLSHQLIQYS 300 ----------
-c-Q---------Q_----------
SFHNLHLLFDEYTNIPVSILFNKEEADDNGD* 331
----------------RPPEPAAQGESSTQKVRPSHTNNPK*
FIG. 1. Amino acid sequence comparison of the Tax proteins of HTLV-IIMoT and HTLV-II1G2, represented by single-letter codes. Asterisks represent stop codons.
immunodominant epitopes within the Env protein (5, 8, 17, 18, 28). HTLV-I has been associated with adult T-cell leukemia and tropical spastic paraparesis/HTLV-I-associated myelopathy (4, 10, 25, 32). Although HTLV-II infection has not been directly connected with any diseases, the virus has been isolated from two patients with T-cell hairy-cell leukemia (11, 26) and several patients with myelopathies (6, 9). HTLV-II infection appears to be more prevalent than HTLV-I infection within groups of injecting drug users (IDUs) in the United States and Europe (12-14, 20, 33). In addition, seroepidemiologic studies have shown the predominant HTLV type among
*
as
356
previously published (24). Amino acids are
Indians in Argentina, and the Guaymi Indians in Panama (1, 3, 7, 16, 21, 22). The relative isolation of several of these populations (e.g., the Cayapo and Kraho tribes, the Tobas and Matacos Indians, and the Guaymi Indians) provides a unique opportunity to study the natural history, evolution, variation, transmission, and possible disease associations of HTLV-II within these well-defined, endemically infected populations. We have previously reported the complete nucleotide sequence of HTLV-IIG,2, which was originally isolated from an asymptomatic Guaymi Indian (24). Analysis of the predicted amino acid sequence of the HTLV-11,G2 Tax protein revealed that a change of two nucleotides toward the 3' end of the tax sequence resulted in an Arg residue substitution in place of the stop codon normally present at position 332 of the HTLVcos
Corresponding author. 7663
7664
NOTES
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FIG. 2. Analysis of in vitro transcription-translation products. The translation products were analyzed on an SDS-12% polyacrylamide gel. Luciferase is the positive control provided with the TNT Coupled Reticulocyte Lysate System.
IIMo)T Tax protein (Fig. 1). This change resulted in a predicted HTLV-11GI2 Tax protein sequence 25 amino acids longer than the prototype HTLV-IIM,,T Tax protein. This predicted protein would be of approximately the same size as HTLV-IATK Tax (40 kDa), compared with the HTLV-IL,,,.o Tax protein (37 kDa). To determine whether the HTLV-II(,2 tax gene se-
quence would actually produce a protein of larger size, in vitro transcription-translation reactions were performed. The tax genes of HTLV-IIM,,T, HTLV-11GI2, and HTLV-LATK were amplified by polymerase chain reaction under standard conditions (24) and cloned into the pT7Blue T-vector (Novagen, Madison, Wis.) by following the manufacturer's instructions. The resulting clones (MoTtax, G12tax, and MT-2tax) were used in in vitro transcription-translation reactions with a TNT Coupled Reticulocyte Lysate System (Promega, Madison, Wis.) by following the manufacturer's directions, and the products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (15). As predicted, the translation product obtained from G 12tax was larger (40 kDa) than the translation product from MoTtax (37 kDa) and was the same size as the MT-2tax translation product (Fig. 2). A clone with the amplified MT-2 tax product inserted in the reverse orientation (MT-wotax) produced no specific translation products. The G12tax clones consistently produced a second translation product of approximately 18 kDa that was not present in translation reactions with either MoTtax or MT-2tax. The significance of this band is unclear and is still under investigation. We next examined whether the translation products produced by the in vitro transcription-translation reactions represented native proteins, i.e., whether they would react with naturally occurring antibodies in serum from individuals infected with HTLV-I or HTLV-II. The in vitro-expressed translation products were radioimmunoprecipitated with human sera by standard procedures (29) and analyzed by SDSPAGE. Each of the translation products was tested with pooled sera from normal blood donors, HTLV-I-infected donors, HTLV-II-infected individuals (blood donors and IDUs) from the United States, and HTLV-II-infected Guaymi
1ITI.VII (;Guvlli
FIG. 3. Radioimmunoprecipitation of Tax proteins synthesized by in vitro transcription-translation reactions. Tax proteins were analyzed on an SDS-12% polyacrylamide gel. NHS, pooled normal human serum (n = 6); HTLV-I, pooled serum from HTLV-I-infected individuals (n = 6); HTLV-II US, pooled serum from HTLV-II-infected U.S. blood donors and IDUs (n = 6); HTLV-II Guaymi, pooled serum from HTLV-II-infected Guaymi Indians (n = 6).
Indians. The MT-2tax translation product reacted with pooled HTLV-I sera and produced a single distinct band at 40 kDa, while no cross-reactivity with HTLV-II sera was observed. The pooled HTLV-II sera from U.S. individuals and Guaymi Indians reacted with both the MoTtax (37-kDa) and G12tax A
B
Tax22/II312-330
YTNIPVSILFNKEEADDNGD
Tax23 /II327-346
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Tax24/II 337-356
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Tax22/II
Tax23/II
Tax24/II
FIG. 4. (A) Sequence of peptides representing the extended 3' end of the HTLV-II(, 2 Tax protein. (B) Seroreactivity of sera from HTLV-II-infected Guaymi Indians (solid symbols; n = 30) and HTLV1I-infected U.S. individuals (open symbols; n = 30) to synthetic HTLV-II Tax peptides. Horizontal bars represent the negative cutoff points for each Tax peptide established by the mean plus 3 standard deviations of sera from 20 normal blood donors. Numbers in parentheses are the percentage of samples seropositive.
VOL. 67, 1993
NOTES 8190
8141
Subtype
a
Subtype b
MoT US (#184) US (#212) US (H2A) US (H2D) US (H2E) Pueblo (Dsand) Seminole (AI1052)
7665
AATACACCAA CATCCCTUGTC TCTAlw =TAT TTAATAAAGA AGAGGCGGAT
_--__ _--__ __--__ _--__ __--__ _--__ __--__ _--__ _-
G12.1 Guaymi (#128) Guaymi (#133) US (#159) US (*232) Navajo (FH) Pueblo (SC) Mexican (Y01) Seminole (AI1050) 8240 GACAATGGCG ACXACCCTCC CGAGCCAGCC ACCCAGGGCG AGTCATCGAC _ _____ ___--------- T--------- ---------- --------
8191
MoT US (#184) US (#212) US (H2A) US (H2D) US (H2E) Pueblo (Dsand) Seminole (AI1052) G12 1 Guaymi (#128) Guaymi (#133) US (#159) US (#232) Navajo (FH) Pueblo (SC) Mexican (Y01) Seminole (AI1050)
----------
---------
T--------- G---------
----------
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----------
--CG------ T---------
----------
-------------------
----------
--T------- ----------
----------
----------
--C------T----------C------- T----------C------- T----------C------- T----------C------- T---------
G--------G--------G--------G--------G---------
----------------------------
----------
----------
G---------
-------------------------------------
8285 CCAAAAGGTC AGACCGTCTC ACACAAACAA TCCCAAGTAA AGGCTCT
8241
MoT US (#184) US (#212) US (H2A) US (H2D) US (H2E) Pueblo (Dsand) Seminole (AI1052) G12 1
Guaymi (#128) Guaymi (#133) US (#159) US (#232)
Navajo (Fh) Pueblo (SC) Mexican (Y01) Seminole (AI1050)
FIG. 5. Nucleotide sequence of 3' end of tax gene from HTLV-IIM.T aligned with HTLV-Il6, and 15 other HTLV-II tax sequences. Dashes indicate a perfect match to the HTLV-IIM.T sequence. Stop codons used by HTLV-IIM,,T and HTLV-11GI2 are underlined and in bold type. Only 144 bp of the polymerase chain reaction-amplified tax gene fragment are shown.
(40-kDa) translation products and had minimal cross-reactivity with the MT-2tax (40-kDa) translation product (Fig. 3). The G12tax 18-kDa translation product also was immunoprecipitated with HTLV-II sera and may represent a translation product produced by utilization of a different start codon. The difference in size of the HTLV-IIM.T and HTLV-11Gl2 Tax protein translation products was distinct (37 and 40 kDa, respectively) and supported the idea of an extended Tax protein in HTLV-IIG1122 While immunogenicity of the HTLV-IIM,T Tax protein has not been reported, an immunodominant epitope at the C terminus of the HTLV-I Tax protein has been identified (27). To investigate whether the extended Tax protein of HTLVIIG12 contained immunogenic epitopes, overlapping peptides corresponding to the last 45 amino acids of the HTLV-IlG,2 Tax protein (Tax22/II, Tax23/II, and Tax24/II) were synthesized (Fig. 4A). These peptides were used in a synthetic peptide-based enzyme-linked immunosorbent assay (ELISA) by following previously published procedures (18), to screen sera from HTLV-II-infected Guaymi Indians as well as those from HTLV-II-infected U.S. blood donors and IDUs (Fig. 4B). Of 30 serum specimens from Guaymi Indians, 9 (30%) reacted
with peptide Tax22/II, 8 (26.7%) reacted with Tax23/II, and 25 (83.3%) reacted with Tax24/II. In contrast, for 30 U.S. blood donors and IDUs, only 7 specimens (23.3%) reacted with Tax22/II, 2 (6.7%) reacted with Tax23/II, and 1 (3.3%) reacted with Tax24/II. These data strongly suggested that an extended HTLV-II Tax protein was present in a large number of the HTLV-II-infected Guaymi Indians and that this protein elicited a strong humoral response. The HTLV-II12 isolate has been classified as an HTLV-II subtype b isolate on the basis of nucleotide sequence analysis and restriction enzyme patterns within the transmembrane env gene, gp2l (24). Further analysis has identified all the Guaymi Indian isolates tested to be subtype HTLV-IIb, while isolates from other populations were a mixture of HTLV-II subtypes a and b (23). The strong seroreactivity to peptide Tax24/II observed within the HTLV-IIb-infected Guaymi Indian population led us to examine whether an extended Tax protein was a unique feature of the HTLV-IIb isolate found within this population or a characteristic of all HTLV-IIb isolates. To investigate these possibilities a limited sequence analysis of the 3' end of the tax gene was performed. Fifteen DNA samples, including both U.S. (n = 7) and Amerindian (n = 8) isolates,
7666
NOTES
were selected, and a 230-bp fragment was amplified by polymerase chain reaction under previously described conditions (24) and then directly sequenced with an AmpliTaq cycle sequencing kit (Perkin-Elmer Cetus, Norwalk, Conn.) by following the manufacturer's instructions. When the nucleotide sequences from these 15 HTLV-II-infected DNA samples as well as from the MoT and G12.1 cell lines were aligned, they segregated into two distinct groups (Fig. 5). Eight of the isolate sequences (seven HTLV-IIa and one HTLV-Ilb) were identical to or differed by only one base from the prototype HTLV-IIMoT sequence, and all had a stop codon at amino acid position 332. In contrast, seven other isolate sequences (all HTLV-IIb) had either one or two base substitutions at nucleotide position 8203, substituting an Arg or Gln residue in place of the stop codon, which would result in an extended Tax protein similar to that of HTLV-11,12. One Seminole Indian isolate (AII050) was found to be HTLV-II subtype b, although sequence data indicated a stop codon at position 332 similar to the HTLV-IIM,,T sequence. Thus, the extended Tax protein appears to be a characteristic of the majority of HTLV-IIb
isolates. This study showed that the HTLV-IGI2 isolate is capable of producing a Tax protein larger than the prototype HTLVIIMoT Tax protein, as shown by in vitro transcription-translation reactions. The translation product from G12tax was specifically recognized by antibodies from HTLV-II-infected individuals from the United States and from the Guaymi Indian populations, demonstrating that the protein produced in vitro was antigenically similar to virally produced Tax protein. A synthetic peptide-based ELISA was used to screen HTLV-II-infected U.S. blood donors and IDUs and HTLVII-infected Guaymi Indians for seroreactivity to the last 45 amino acids of the HTLV-lc;,12 Tax protein. The major immune response appeared to be directed at the carboxyl end of the Tax protein (peptide Tax24/II), with 83.3% of the Guaymi Indian sera tested found to be seropositive, while only 3.3% of the sera from U.S. individuals were seropositive. These data suggest the presence of an extended Tax protein in a large proportion of the HTLV-II-infected Guaymi Indians and in a much smaller proportion of the HTLV-II-infected populations within the United States. Sequence analysis of 15 DNA samples from HTLV-II-infected individuals demonstrated that all the isolates with an extended Tax protein were HTLV-II subtype b, regardless of geographic origin, and supported the conclusion that an extended Tax protein is a characteristic of most HTLV-II subtype b isolates. These findings might serve as a basis for the design of a synthetic peptide-based immunoassay for serologic differentiation of HTLV-II subtypes. Studies to determine functional differences between HTLV-IIM
