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WO2006133795A2 - Nouveau virus t-lymphotropique humain (htlv-3), acides nucleiques et sequences peptidiques de ce virus, et methode de detection de htlv-3 - Google Patents

Nouveau virus t-lymphotropique humain (htlv-3), acides nucleiques et sequences peptidiques de ce virus, et methode de detection de htlv-3 Download PDF

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WO2006133795A2
WO2006133795A2 PCT/EP2006/004951 EP2006004951W WO2006133795A2 WO 2006133795 A2 WO2006133795 A2 WO 2006133795A2 EP 2006004951 W EP2006004951 W EP 2006004951W WO 2006133795 A2 WO2006133795 A2 WO 2006133795A2
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htlv
purified
seq
polypeptide
nucleic acid
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PCT/EP2006/004951
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WO2006133795A3 (fr
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Antoine Gessain
Renaud Mahieux
Sara Calattini
Sébastien CHEVALIER
Renan Duprez
Alain Froment
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Institut Pasteur
Centre National De La Recherche Scientifique (Cnrs)
Institut National De La Sante Et De La Recherche Medicale (Inserm)
Universite Paris Vii Denis Diderot
Institut De Recherche Pour Le Developpement (Ird)
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Publication of WO2006133795A2 publication Critical patent/WO2006133795A2/fr
Publication of WO2006133795A3 publication Critical patent/WO2006133795A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/14011—Deltaretrovirus, e.g. bovine leukeamia virus
    • C12N2740/14021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/14011—Deltaretrovirus, e.g. bovine leukeamia virus
    • C12N2740/14022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • HTLV-3 NUCLEIC ACID AND PEPTIDIC SEQUENCES THEREOF AND METHOD FOR DETECTING HTLV-3
  • This invention relates to human T-cell leukemia viruses, specifically HTLV-3.
  • the invention is directed to purified and isolated nucleic acids of the HTLV-3 genome and to polypeptides encoded by the nucleic acids of the HTLV-3 genome.
  • the invention is directed to processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides, and the use of such nucleic acids and polypeptides in diagnostic methods, kits, vaccines, or antiviral therapy.
  • PTLVs Primate T-cell Lymphotropic viruses
  • PTLV-1 , PTLV-2 and PTLV-3 include human (HTLV-1 , HTLV-2, HTLV-3) and simian (STLV-1 , STLV-2, STLV-3) viruses
  • HTLV-4 the fourth type (HTLV-4) consists only, so far, of a unique human isolate (Calattini et al., 2005; Wolfe et al., 2005).
  • STLV-3 formerly named PTLV-L, was isolated shortly before STLV-2 (Goubau et al., 1994). STLV-3 belongs to a third group of lymphotropic viruses that infect numerous African monkeys species. Sequence comparisons of STLV-3 proviruses indicate that these strains are highly divergent from HTLV-1 (60% nucleotide similarity), HTLV-2 (62%), or STLV-2 (62%) prototype sequences.
  • STLV-3 viruses cluster in a highly supported group, indicating an evolutionary lineage independent from PTLV-1 and PTLV-2. Nevertheless, the STLV-3 lineage is composed of at least three subtypes that correspond more or less to the geographical origin of the virus (East, West or Central Africa) (Goubau et al.
  • the overall STLV-3 genomic organization is similar to that of HTLV-1 and HTLV-2, with the presence of gag, pro, pol, env, tax and rex genes (Meertens and Gacin, 2003; Meertens et al., 2002; Van Brussel et al., 1996; Van Brussel et al., 1997; Van Dooren et al., 2004).
  • the invention is based on the discovery of HTLV-3, a virus that infects humans.
  • the invention relates to this new human retrovirus, HTLV-3 virus, and provides its nucleic acid and polypeptide sequences.
  • the invention provides means for molecular and/or serological detection of the virus, assays for the detection of the virus, and expression vectors that utilize the genetic sequences of the virus.
  • HTLV-3p y ⁇ 43 The complete sequence of HTLV-3p y ⁇ 43 was determined in order to gain information from its genomic organization and to characterize its Tax protein. The genome sequence can be found in Genbank under accession number DQ 462191. As expected, HTLV-3p y i 43 contains Open Reading Frames (ORFs) corresponding to gag, pol, env, tax and rex genes.
  • ORFs Open Reading Frames
  • Tax3p y i 43 displays strong similarities with HTLV-1 Tax including the sequence of a putative PDZ binding motif. In transient transfection assays, Tax3pyi 43 activates the transcription from the HTLV-3, HTLV-1 , and HTLV-2 LTRs. Mutational analysis indicates that two functional domains (M22 and M47) important for transactivation through the CREB/ATF or NF- ⁇ B pathway are similar but
  • Tax3p y i 43 transactivates the human
  • interleukin-8 and BCI-XL promoters through the induction of the NF- ⁇ B pathway.
  • Tax3p y i 43 represses the transcriptional activity of the p53 tumor suppressor protein as well as the c-Myb promoter.
  • HTLV-3 genome organization is similar to that of HTLV-1 and HTLV-2 and is closely related to central African STLV-3 strains.
  • STLV-3 and STLV-2 HTLV-3 LTRs contain only two Tax responsive elements.
  • a 366 bp sequence is lacking in the HTLV-3 pX proximal region. This sequence corresponds in part to the Rorfll sequence that was previously described for STLV-3PH 969 , but also to the 3 1 end of a putative antisense transcript.
  • HTLV-3 Tax protein is also expressed in vivo (Chevalier et al. 2006).
  • the invention encompasses a purified nucleic acid molecule comprising the DNA sequence of the HTLV-3 genome and fragments thereof.
  • the invention also encompasses nucleic acid molecules complementary to these sequences.
  • the invention also encompasses purified polypeptides encoded by these nucleic acid molecules.
  • Both single-stranded and double-stranded RNA and DNA nucleic acid molecules are encompassed by the invention. These molecules can be used to detect both single-stranded and double-stranded RNA and DNA variants of encoding polypeptides encompassed by the invention.
  • a double-stranded DNA probe allows the detection of nucleic acid molecules equivalent to either strand of the nucleic acid molecule.
  • nucleic acid molecules that hybridize to a denatured, double- stranded DNA comprising the nucleic acids of the invention under conditions of moderate stringency in 50% formamide and 6XSSC, at 42 0 C with washing conditions of 60 0 C, 0.5XSSC, 0.1 % SDS are encompassed by the invention.
  • the invention further encompasses purified nucleic acid molecules derived by in vitro mutagenesis from the genome of HTLV-3.
  • In vitro mutagenesis includes numerous techniques known in the art including, but not limited to, site-directed mutagenesis, random mutagenesis, and in vitro nucleic acid synthesis.
  • the invention also encompasses purified nucleic acid molecules degenerate from the nucleic acids of the HTLV-3 genome, as a result of the genetic code, purified nucleic acid molecules, which are allelic variants of the HTLV-3 genome or a species homolog of HTLV-3 genome nucleic acids.
  • the invention also encompasses recombinant vectors that direct the expression of these nucleic acid molecules and host cells transformed or transfected with these vectors.
  • the invention further encompasses methods for the production of polypeptides encoded by the HTLV-3 genome, including culturing a host cell under conditions promoting expression, and recovering the polypeptide from the culture medium.
  • the polypeptides encoded by the HTLV-3 genome in bacteria, yeast, plant, and animal cells is encompassed by the invention.
  • This invention also provides labeled polypeptides encoded by the HTLV-3 genome.
  • the labeled polypeptides are in purified form. It is also preferred that the unlabeled or labeled polypeptide is capable of being immunologically recognized by human body fluid containing antibodies to HTLV-3.
  • the polypeptides can be labeled, for example, with an immunoassay label selected from the group consisting of radioactive, enzymatic, fluorescent, chemiluminescent labels, and chromophores.
  • Immunological complexes between the polypeptides encoded by the HTLV-3 genome of the invention and antibodies recognizing the polypeptides are also provided.
  • the immunological complexes can be labeled with an immunoassay label selected from the group consisting of radioactive, enzymatic, fluorescent, chemiluminescent labels, and chromophores.
  • this invention provides a method for detecting infection by HTLV-3.
  • the method comprises providing a composition comprising a biological material suspected of being infected with HTLV-3, and assaying for the presence of polypeptides encoded by the HTLV-3 genome.
  • the polypeptides are typically assayed by electrophoresis or by immunoassay with antibodies that are immunologically reactive with polypeptides encoded by the HTLV-3 genome.
  • This invention also provides an in vitro diagnostic method for the detection of the presence or absence of antibodies, which bind to an antigen comprising polypeptides encoded by the HTLV-3 genome or mixtures of the polypeptides.
  • the method comprises contacting the antigen with a biological fluid for a time and under conditions sufficient for the antigen and antibodies in the biological fluid to form an antigen-antibody complex, and then detecting the formation of the complex.
  • the detecting step can further comprising measuring the formation of the antigen- antibody complex.
  • the formation of the antigen-antibody complex is preferably measured by immunoassay based on Western blot technique, ELISA (enzyme linked immunosorbent assay), indirect immunofluorescent assay, or immunoprecipitation assay.
  • a diagnostic kit for the detection of the presence or absence of antibodies which bind to a polypeptide encoded by the HTLV-3 genome or mixtures of polypeptides encoded by the HTLV-3 genome, contains antigen comprising polypeptides encoded by the HTLV-3 genome, or mixtures thereof, and means for detecting the formation of immune complex between the antigen and antibodies.
  • the antigens and the means are present in an amount sufficient to perform the detection.
  • This invention also provides an immunogenic composition
  • an immunogenic composition comprising a polypeptide encoded by the HTLV-3 genome or a mixture thereof in an amount sufficient to induce an immunogenic or protective response in vivo, in association with a pharmaceutically acceptable carrier therefor.
  • polypeptides of this invention are thus useful as a portion of a diagnostic composition for detecting the presence of antibodies to antigenic proteins associated with HTLV-3.
  • polypeptides encoded by the HTLV-3 genome can be used to raise antibodies for detecting the presence of antigenic proteins associated with HTLV-3.
  • the polypeptides of the invention can be also employed to raise neutralizing antibodies that either inactivate the virus, reduce the viability of the virus in vivo, or inhibit or prevent viral replication. The ability to elicit virus-neutralizing antibodies is especially important when the proteins and polypeptides of the invention are used in immunizing or vaccinating compositions to activate the B-cell arm of the immune response or induce a cytotoxic T lymphocyte response (CTL) in the recipient host.
  • CTL cytotoxic T lymphocyte response
  • this invention provides a method for detecting the presence or absence of HTLV-3 comprising: (1 ) contacting a sample suspected of containing viral genetic material of HTLV-3 with at least one nucleotide probe, and (2) detecting hybridization between the nucleotide probe and the viral genetic material in the sample, wherein said nucleotide probe is complementary to the full-length sequence of purified HTLV-3 nucleic acids of the invention.
  • Figure 1 depicts the close relationship of HTLV-3 to STLV-3.
  • An unrooted phylogenetic tree was generated with the neighbor-joining method, performed in the PAUP program (v4.0b10), on a 180 bp fragment of the tax gene using all full length PTLV-1/2 available sequences and all published STLV-3 tax sequences.
  • the final alignment was submitted to the Modeltest program (version 3.6) to select, according to the Akaike Information Criterion (AIC), the best model to apply to phylogenetic analyses.
  • the selected model was the TrN+G one. Bootstrap support (1 ,000 replicates) is noted on the branches of the tree. The branch lengths are drawn to scale, with the bar indicating 0.1 nucleotide replacement per site.
  • Figure 2 depicts the serological pattern of a person infected by the HTLV- 3 Py143 strain.
  • A Western blot from Diagnostic Biotechnology (HTLV blot 2.4 version) and
  • B line immunoassay (INNO-LIA HTLV confirmation Immunogenetics).
  • Lane 1 HTLV-1 positive control
  • lane 2 HTLV-2 positive control
  • lane 3 STL V-3 positive control (STLV-3604 strain)
  • lane 4 STLV-3 positive control (STLV-3 F3)
  • lane 5 HTLV-1/2 negative control
  • lane 6 plasma from a person infected by HTLV-3 (Pyl43 strain).
  • Figure 3 depicts the nucleotide sequence of the fragment of 1519 bp of the Pol gene of the novel HTLV-3 virus (strain PYL 43) (SEQ ID NO: 1).
  • Figure 4 depicts the amino acid sequence of the fragment of 1519 bp of the Pol gene of the novel HTLV-3 virus (strain PYL 43) (SEQ ID NO: 2).
  • Figure 5 depicts the sequence comparison of the fragment of 1519 bp of the Pol gene of the novel HTLV-3 virus (strain PYL 43) with 4 other STLV-3 viruses of simian origin CTO 604 (SEQ ID NO: 28), CTO NG 409 (SEQ ID NO: 29), F3 (SEQ ID NO: 30), and PH969 (SEQ ID NO: 3).
  • a consensus sequence is also provided (SEQ ID NO: 52).
  • Figure 6 depicts the nucleotide sequence of a fragment of 279 bp of the tax gene of the novel HTLV-3 virus (strain PYL 43) (SEQ ID NO: 4).
  • Figure 7 depicts the amino acid sequence of the fragment of 279 bp of the tax gene of the novel HTLV-3 virus (strain PYL 43) (SEQ ID NO: 5).
  • Figure 8 depicts the sequence comparison of the fragment of 279 bp of the fax gene of the novel HTLV-3 virus (strain PYL 43) (SEQ ID NO: 4) with 4 other STLV-3 viruses of simian origin CTO 604 (SEQ ID NO: 6), CTO NG 409 (SEQ ID NO: 7), F3 (SEQ ID NO: 31), PH969 (SEQ ID NO: 8).
  • a consensus sequence is also provided (SEQ ID NO: 53).
  • Figure 9 depicts the positions according to CTO 604 of the different primers used for amplifying and cloning the Tax and Pol PCR products from HTLV-3 PYL 43. The size of the PCR product is also indicated.
  • FIG. 10 depicts the HTLV-3 Py i 43 genome.
  • A PCR strategy for amplifying the complete HTLV-3p y ⁇ 43 provirus. Nineteen PCR fragments are shown.
  • B Schematic representation of the HTLV-3p y i 43 genome.
  • C The HTLV-3 pX proximal sequence is shorter than that of the prototypical STLV-3 strain. PCR amplification using high molecular weight DNA extracted either from HTLV-3p y ⁇ 43 (lane 1 , 4) or from STLV3PH 969 (lanes 3, 6). DNA was not added to the PCR mix in lanes 2 and 5.
  • Lane 3 and 6 expected size: 1027 bp and 1097 bp for STLV-3 PH969 - Lane 1 and 6 observed size 660 bp and 730 bp for HTLV-3p y ⁇ 43 (D) Proximal pX organization. The deleted region, which encompasses the ORFII, is shown.
  • Figure 11 depicts data showing that HTLV-3p y ⁇ 43 belongs to the central African STLV-3 group. Unrooted phylogenetic tree generated with the Neighbor-joining method, performed in the PAUP program (v4.0b10), on the PTLVs complete sequences available in GenBank. Bootstrap support (1 ,000 replicates) is noted on the branches of the tree. The branch lengths are drawn to scale, with the bar indicating 0.1 nucleotide substitutions per site. [044] Figure 12 depicts a schematic display of different Tax3 and Taxi
  • HTLV-1 A ⁇ is used as a prototypical sequence.
  • A HTLV-1 Tax CBP/p300 binding sequence as reported in (Harrod et al., 1998) is compared to that of Tax3pyi 43 .
  • B and (C), HTLV-1 Tax M22 , Tax M 47 and Tax CR2 binding domain as reported in (Smith and Greene, 1990; Scoggin et al., 2001) are compared to Tax3p y ⁇ 4 3-
  • D As Taxi (Rousset et al., 1998), but not Tax2 (Endo et al., 2002), Tax3 Py ⁇ 43 contains a PDZ binding motif.
  • FIG. 13A-B demonstrate that Tax3p y ⁇ 43 intracellular localization is similar to that of Taxi .
  • HeLa cells were transiently transfected with GFP (a), GFP-Tax1 (b), GFP-Tax2 (c) various GFP-Tax3p y i43 (d, e, f) or GFP-Tax3 60 2 (g) plasmids. Images of cells that are representative of the entire population are shown.
  • FIG 14 demonstrates that Tax3p y ⁇ 43 is a viral transactivator of the HTLV- 1 , HTLV-2 and HTLV-3 promoters.
  • Jurkat cells were transiently transfected with: (A) 250 ng of HTLV-3 LTR-luc, (B) of HTLV-1 LTR-luc, (C) of HTLV-2 LTR-luc, or (D) 200
  • HeIa cells were transiently transfected with 100 ng of PG13pyLuc, 200 ng of the different Tax3p y ⁇ 43 constructs, or of empty pSG5M vector and 100 ng of a p53-expressing plasmid as previously reported
  • Tax3 p y ⁇ 43 transactivates the Bcl-X L promoter through the
  • Jurkat cells were transfected with 500 ng of the Pr-
  • Tax3 PyI43 transactivates the IL-8 promoter through the NF- ⁇ B
  • Figure 16A-16B depicts the full length sequence of the genome of the HTLV-3 Py143 virus (SEQ ID NO: 47).
  • Figure 17A-17AB depicts the alignment of the genomic sequence of HTLV-3 Py143 (SEQ ID NO: 47) with the following sequences: K96 (SEQ ID NO: 54), RP329 (SEQ ID NO: 55), SPW (SEQ ID NO: 56), AF41231 Hsa (SEQ ID NO: 57), G12 (SEQ ID NO: 58), GAB (SEQ ID NO: 59), EFE2 (SEQ ID NO: 60), PanP (SEQ ID NO: 61), PP1664 (SEQ ID NO: 62), AF139170Hsa (SEQ ID NO: 63), RK13 (SEQ ID NO: 64), BRRP438 (SEQ ID NO: 65), BOI (SEQ ID NO: 66), K30 (SEQ ID NO: 67), ATK (SEQ ID NO: 68), Y
  • HTLV-1 and STLV-2 are pathogenic retroviruses that infect humans.
  • HTLV-1 causes severe hematological disease, while both HTLV-1 and HTLV-2 cause neurological disease. Both viruses have simian counterparts (STLV-1 and STLV-2).
  • STLV-3 belongs to a third group of lymphotropic viruses that infect numerous African monkeys species.
  • Cameroon has a remarkable diversity of retroviruses. All the subtypes of HIV-1 group M (A to H) are present, subtype-recombinant strains co-circulate, and HIV- 1 groups O and N have been reported. HTLV-1 subtypes B and D, as well as HTLV-2 type A and B, are also present in Cameroonian individuals, while STLV-1 and STLV-3 strains have been isolated from several non-human primates (NHPs) species living in this region (Goubau et al., 1994; Courgnaud et al., 2004; Van Brussel et al., 1996).
  • NHS non-human primates
  • HTLV-3 a new human retrovirus in the peripheral blood cells of a Central African native, called HTLV-3. This virus is closely related to STLV-3.
  • HTLV-3 infection is reflected by an HTLV indeterminate serological WB pattern.
  • Emerging viral pathogens have either invaded a new host species or expanded into new geographic populations of host species. As represented by the human immunodeficiency virus epidemy, or by the Spanish influenza outbreak in the early 20's, viruses can be highly transmissible and virulent. These episodes appear to be unpredictable. A viral pathogen may be benign while residing within a reservoir species, yet on entering a new host, increases in virulence (Slattery et al., 1999).
  • HTL V- 1 known as the etiological agent of ATLL, which is one of the worst leukemia
  • HTLV- 2 which, although very similar in sequence to HTLV-1 , is barely leukemogenic.
  • a third member of the HTLV group was identified (Calattini et al., 2005; Wolfe et al., 2005). It is important to determine whether HTLV-3 is more similar to HTLV-1 or to HTL V-2 in terms of pathogenicity.
  • STLV-3 infected animals were reported almost a decade ago (Goubau et al., 1994), but these animals were never studied longitudinally. For this reason, it is not possible to determine whether, as some STLV-1 infected animals, they can suffer of ATLL-like diseases (Debacq et al., 2005).
  • HTLV-3 The complete sequence of the HTLV-3 provirus has been determined.
  • HTLV-3 is very similar to that of Central African STLV-3.
  • the viral promoter the LTR found at nucleotides 1 to 695, contains only two Tax responsive elements, a trait that was also described earlier for STLV-3 (Meertens and Gappeln, 2003; Meertens et al., 2002; VanBrussel et al., 1997). This confirms that, as it is the case for HTLV-1 (Slattery et al., 1999; Liu et al., 1996), HTLV-3 arose in humans from interspecies transmissions.
  • STLV-3 minus strand contains an ORF that can encode a 221 amino acid long protein. Similar to HTLV-1 HBZ, this protein can possess several Nuclear Localization Signals (NLS). The NLS sequences that are present in HTLV-1 HBZ, are critical for its localization, and likely for its functions (Hivin et al., 2005). On the other hand, the putative HTLV-3 antisense-encoded protein is only 103 amino acid long due to the 366 bp deletion in the proximal pX region. This sequence is located at nucleotides 6701 -7067 of the HTLV-3 genome. The protein lacks the NLS domains and is functionally impaired. Because an HTL V-3 or an STLV-3 infected cell line is not available for analysis, the existence and role of such a protein cannot be investigated.
  • NLS Nuclear Localization Signals
  • Tax is one of the key players in HTLV-1 pathogenicity
  • HTLV-3 Tax was examined. Tax is located at nucleotides 5068-5071 and 6882-7931 of the HTLV-3 genome. Sequence analysis first revealed that, like Taxi but not Tax2, Tax3p y i 43 contains a PDZ binding motif. The PDZ binding motif is located at amino acids 348-350 of the Tax3p y143 protein. Such a domain, which is absent from Tax2, is critical for the ability of the viral protein to induce the T-cell proliferation and the viral persistence in vivo (Xie et al., 2005). Therefore, HTLV-3 possesses some of the HTLV- 1 properties. [062] In addition, Tax3p y ⁇ 43 is capable of inducing the CREB/ATF and the NF- ⁇ B
  • Tax3p y ⁇ 43 transactivates both the HTLV-1 and HTLV-2 promoters. This is important because HTLV-3 Py ⁇ 4 3 was discovered in a population that is endemic for both HTLV-1 and HTLV-2. Interestingly, animals that are co-infected by STLV-1 and STLV-3 were described earlier (Courgnaud et al., 2004). Co-infection can accelerate the occurrence of a PTLV-associated disease. So far, though, both cases of HTL V-3 infection were reported in individuals that are not co-infected with other HTLV viruses.
  • Tax3p y ⁇ 43 like Taxi , transrepresses some cellular genes (p53, c-Myb) and transactivates others (IL-8, BCI-XL), the mechanisms that are involved are slightly different. In particular, the activation of NF- ⁇ B by Tax3p y ⁇ 43 is not
  • Both an HTLV-3 and an STLV-3 molecular clone can be used to investigate the viral expression in the context of a provirus. These clones will allow determination of whether the 366 bp deletion has an impact on the HTLV-3 viral life cycle. Indeed, earlier analyses show that not only Tax but other pX-encoded proteins (p12, p13, p30, HBZ) are involved in the viral pathogenesis. Due to the deletion in the putative HTLV-3 antisense encoded protein (AEP) sequence, HTLV-3 encodes a nonfunctional protein as compared to its simian STLV-3 counterpart.
  • AEP antisense encoded protein
  • the invention encompasses the genetic sequence of the HTLV-3p y i43 genome (SEQ ID NO: 47), which can be found in Genbank under accession number DQ462191.
  • An embodiment of the invention includes the genetic sequence of the tax (located at nucleotides 5068-5071 and 6882-7931 of the HTL V-3 genome) (SEQ ID NO: 48) and pol genes (located at nucleotides 239-5075 of the HTLV-3 genome) (SEQ ID NO: 49), and fragments thereof, and the amino acid sequences of the full length Tax (SEQ ID NO: 50) and Pol (SEQ ID NO: 51) proteins of HTLV-3, strain Py143. Fragments of the genetic sequences are found in GenBank under accession numbers DQ020492 (SEQ ID NO: 4) for fax and DQ020493 (SEQ ID NO: 1) for pol.
  • Yet another embodiment includes the protein or polypeptide sequences or fragments of the fax and pol genes, and fragments thereof, of HTLV-3, strain Py143, (SEQ ID NO: 5) and (SEQ ID NO: 2), respectively.
  • HTLV-3 PYL43 POL1 comprising a polynucleotide corresponding to fragment 1 (SEQ ID NO: 41) of Figure 9
  • I-3420 for HTLV-3 PYL43 POL2 comprising a polynucleotide corresponding to fragment 2 (SEQ ID NO: 42) of Figure 9
  • 1-3421 for HTLV-3 PYL43 POL3 comprising a polynucleotide corresponding to fragment 3 (SEQ ID NO: 46) of Figure 9).
  • Recombinant viral polypeptides can be prepared, for instance, by using such polynucleotides in an expression host.
  • An embodiment also encompasses the fragments depicted as fragments 1 (nt 3119-3510) (SEQ ID NO: 43), 2 (nt 3348-4128) (SEQ ID NO: 44), and 3 (nt 3348- 3839) (SEQ ID NO: 45) of Figure 9.
  • Nucleic acids within the scope of the invention include isolated DNA and RNA that hybridize to the nucleic acids of the invention, especially as disclosed in Figures 3, 5, 6, and 8, and to the nucleic acids of under conditions of moderate or severe stringency.
  • kits capable of diagnosing HTLV infections including HTLV-1 and -2 co-infection with HTLV-3, is described.
  • This kit in one embodiment, contains the DNA or RNA sequences of this invention, which are capable of hybridizing to viral RNA or analogous DNA sequences to indicate the presence of an HTLV infection.
  • Different diagnostic techniques can be used which include, but are not limited to: (I) Southern blot procedures to identify cellular DNA, which may or may not be digested with restriction enzymes; (2) Northern blot techniques to identify RNA extracted from cells; and (3) dot blot techniques, i.e., direct filtration of the sample through an ad hoc membrane, such as nitrocellulose or nylon, without previous separation on agarose gel.
  • Suitable material for dot blot technique could be obtained from body fluids including, but not limited to, serum and plasma, supernatants from culture cells, or cytoplasmic extracts obtained after cell lysis and removal of membranes and nuclei of the cells by centrifugation.
  • the invention encompasses Tax3 Py i 43 mutants, including, but not
  • Tax3 M 22 (Ai3o-Mi 31 ->A 13 o->Si3i) and Tax3 M 47 (L 3 i9L 32 o->R3i9S 3 2o).
  • HTLV-3 The genome of HTLV-3 has been disclosed in SEQ ID NO: 47.
  • This discovery of the HTLV-3 genome enables construction of expression vectors comprising nucleic acid sequences encoding HTLV-3 polypeptides; host cells transfected or transformed with the expression vectors; biologically active HTLV-3 polypeptides and HTLV-3 polypeptides as isolated or purified proteins; and antibodies immunoreactive with polypeptides encoded by the HTLV-3 genome.
  • understanding of the mechanism by which HTLV-3 polypeptides function enables the design of assays to detect inhibitors of HTLV-3 activity.
  • polypeptides encoded by the HTLV-3 genome or "HTLV-3 polypeptide” refers to a genus of polypeptides that further encompasses proteins encoded by SEQ ID NO: 47, as well as those proteins and polypeptides having a high degree of similarity (at least 90% homology) with such amino acid sequences and which proteins and polypeptides are immunoreactive.
  • polypeptides encoded by the HTLV-3 genome refers to the gene products of the nucleotides of SEQ ID NO: 47.
  • purified means that the polypeptides encoded by the HTLV-3 genome are essentially free of association with other proteins or polypeptides, for example, as a purification product of recombinant host cell culture or as a purified product from a non-recombinant source.
  • substantially purified refers to a mixture that contains polypeptides encoded by the HTLV-3 genome and is essentially free of association with other proteins or polypeptides, but for the presence of known proteins that can be removed using a specific antibody, and which substantially purified polypeptides encoded by the HTLV-3 genome can be used as antigens.
  • an HTLV-3 polypeptide "variant" as referred to herein means a polypeptide substantially homologous to native HTLV-3 polypeptides, but which has an amino acid sequence different from that of native HTLV-3 polypeptides because of one or more deletions, insertions, or substitutions.
  • the variant amino acid sequence preferably is at least 80% identical to a native HTLV-3 polypeptide amino acid sequence, most preferably at least 90% identical.
  • the percent identity can be determined, for example by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. ⁇ Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG).
  • the GAP program utilizes the alignment method of Needleman and Wunsch (J. MoI. Biol. 48:443, 1970), as revised by Smith and Waterman ⁇ Adv. Appl. Math 2:482, 1981).
  • the preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
  • Variants can comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics.
  • conservative substitutions include substitution of one aliphatic residue for another, such as lie, VaI, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; GIu and Asp; or GIn and Asn.
  • Other such conservative substitutions for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known.
  • Naturally occurring HTLV-3 polypeptide variants are also encompassed by the invention. Examples of such variants are proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the HTLV-3 polypeptides.
  • Variations attributable to proteolysis include, for example, differences in the termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the HTLV-3 polypeptides.
  • Variations attributable to frameshifting include, for example, differences in the termini upon expression in different types of host cells due to different amino acids of the polypeptides of HTLV-3.
  • the invention provides isolated and purified, or homogeneous, HTLV-3 polypeptides, both recombinant and non-recombinant.
  • Variants and derivatives of native HTLV-3 polypeptides that can be used as antigens can be obtained by mutations of nucleotide sequences coding for native HTLV-3 polypeptides. Alterations of the native amino acid sequence can be accomplished by any of a number of conventional methods. Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence.
  • oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered gene wherein predetermined codons can be altered by substitution, deletion, or insertion.
  • Exemplary methods of making the alterations set forth above are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); Kunkel (Proc. Natl. Acad. Sci.
  • HTLV-3 polypeptides can be utilized to prepare antibodies that specifically bind to HTLV-3 polypeptides.
  • the term "antibodies” is meant to include polyclonal antibodies, monoclonal antibodies, fragments thereof such as F(ab')2 and Fab fragments, as well as any recombinantly produced binding partners. Antibodies are defined to be specifically binding if they bind HTLV-3 polypeptides with a K 3 of greater than or equal to about 10 7 M '1 . Affinities of binding partners or antibodies can be readily determined using conventional techniques, for example, those described by Scatchard et al., Ann. N. YAcad. Sci., 51:660 (1949). Polyclonal antibodies can be readily generated from a variety of sources, for example, horses, cows, goats, sheep, dogs, chickens, monkeys, rabbits, mice, or rats, using procedures that are well known in the art.
  • the invention further encompasses isolated fragments and oligonucleotides derived from the nucleotide sequence of SEQ ID NO: 47.
  • the invention also encompasses polypeptides encoded by these fragments and oligonucleotides.
  • Nucleic acid sequences within the scope of the invention include isolated DNA and RNA sequences that hybridize to the native HTLV-3 nucleic acids disclosed herein under conditions of moderate or severe stringency, and which encode HTLV-3 polypeptides.
  • conditions of moderate stringency as known to those having ordinary skill in the art, and as defined by Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1 , pp.
  • One embodiment of the invention encompasses amplification-based or hybridization-based assays, which use DNA or RNA primers to amplify or hybridize to genetic sequences of HTLV-3.
  • the primers can be derived from regions of the HTLV-3 genome that have appropriate levels of specificity. (See Table 1.)
  • primer Mac2se (5'-accaggtcacctgggaccccatcgatggacg-3' (SEQ ID NO: 11 )) was used.
  • Amplification-based assays include PCR assays.
  • Hybridization-based assays rely on probes that specifically recognize DNA or RNA from HTLV-3 (annealing temperature: 53 0 C to 57 0 C, Mg 2+ 2mM).
  • a DNA sequence can vary from that shown in SEQ ID NO: 47 and still encode an HTLV-3 polypeptide having the amino acid sequence of SEQ ID NO: 47.
  • Such variant DNA sequences can result from silent mutations (e.g., occurring during PCR amplification), or can be the product of deliberate mutagenesis of a native sequence.
  • the invention thus provides equivalent isolated DNA sequences, encoding HTLV-3 polypeptides, selected from: (a) DNA derived from the coding region of a native
  • HTLV-3 gene (b) cDNA comprising the nucleotide sequence of SEQ ID NO: 47; (c) DNA capable of hybridization to a DNA of (a) under conditions of moderate stringency and which encode HTLV-3 polypeptides; and (d) DNA which is degenerate as a result of the genetic code to a DNA defined in (a), (b) or (c) and which encodes HTLV-3 polypeptides.
  • HTLV-3 polypeptides encoded by such DNA equivalent sequences are encompassed by the invention.
  • DNA that is equivalent to the DNA sequence of SEQ ID NO: 47 will hybridize under moderately stringent conditions to the double-stranded native DNA sequence that encode polypeptides comprising amino acid sequences of SEQ ID NO: 47.
  • Examples of HTLV-3 polypeptides encoded by such DNA include, but are not limited to, HTLV-3 polypeptide fragments and HTLV-3 polypeptides comprising inactivated N-glycosylation site(s), inactivated protease processing site(s), or conservative amino acid substitution(s), as described above.
  • HTLV-3 polypeptides encoded by DNA derived from other species, wherein the DNA will hybridize to the complement of the DNA of SEQ ID NO: 47 are also encompassed.
  • Recombinant expression vectors containing a nucleic acid sequence encoding HTLV-3 polypeptides can be prepared using well known methods.
  • the expression vectors include a HTLV-3 DNA sequence operably linked to suitable transcriptional or translational regulatory nucleotide sequences, such as those derived from a mammalian, microbial, viral, or insect gene.
  • suitable transcriptional or translational regulatory nucleotide sequences such as those derived from a mammalian, microbial, viral, or insect gene.
  • regulatory sequences include transcriptional promoters, operators, or enhancers, an mRNA ribosomal binding site, and appropriate sequences which control transcription and translation initiation and termination.
  • Nucleotide sequences are "operably linked" when the regulatory sequence functionally relates to the HTLV-3 DNA sequence.
  • a promoter nucleotide sequence is operably linked to a HTL V-3 DNA sequence if the promoter nucleotide sequence controls the transcription of the HTL V-3 DNA sequence.
  • the ability to replicate in the desired host cells, usually conferred by an origin of replication, and a selection gene by which transformants are identified can additionally be incorporated into the expression vector.
  • sequences encoding appropriate signal peptides that are not naturally associated with HTLV-3 polypeptides can be incorporated into expression vectors.
  • a DNA sequence for a signal peptide secretory leader
  • a signal peptide that is functional in the intended host cells enhances extracellular secretion of the HTLV-3 polypeptide.
  • the signal peptide can be cleaved from the HTLV-3 polypeptide upon secretion of HTLV-3 polypeptide from the cell.
  • Expression vectors for use in prokaryotic host cells generally comprise one or more phenotypic selectable marker genes.
  • a phenotypic selectable marker gene is, for example, a gene encoding a protein that confers antibiotic resistance or that supplies an autotrophic requirement.
  • useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids.
  • Commercially available vectors include those that are specifically designed for the expression of proteins. These include, but are not limited to, pMAL-p2 and pMAL-c2 vectors, which are used for the expression of proteins fused to maltose binding protein (New England Biolabs, Beverly, MA, USA).
  • Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include, but are not limited to, ⁇ -lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 5:4057, 1980; and EP-A-36776), and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412, 1982).
  • ⁇ -lactamase penicillinase
  • lactose promoter system Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544, 1979
  • tryptophan (trp) promoter system Goeddel et al., Nucl.
  • Suitable host cells for expression of HTLV-3 polypeptides include prokaryotes, yeast, or higher eukaryotic cells.
  • Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described, for example, in Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, New York, (1985). Cell-free translation systems could also be employed to produce HTLV-3 polypeptides using RNAs derived from DNA constructs disclosed herein.
  • the present invention is intended to encompass the previously described proteins in isolated or purified form, whether obtained using the techniques described herein or other methods.
  • the HTLV-3 polypeptides are substantially free of human tissue and human tissue components, nucleic acids, extraneous proteins and lipids, and adventitious microorganisms, such as bacteria and viruses.
  • the invention encompasses equivalent proteins having substantially the same biological and immunogenic properties.
  • this invention is intended to cover serotypic variants of the proteins of the invention.
  • suitable labels are radioactive labels, enzymatic labels, fluorescent labels, chemiluminescent labels, and chromophores.
  • the methods for labeling proteins and glycoproteins of the invention do not differ in essence from those widely used for labeling immunoglobulin.
  • the need to label may be avoided by using labeled antibody to the antigen of the invention or antiimmunoglobulin to the antibodies to the antigen as an indirect marker.
  • HTLV-3 polypeptides of the invention can be used to produce polyclonal and monoclonal antibodies reactive therewith.
  • a protein or polypeptide of the invention can be used to immunize an animal host by techniques known in the art. Such techniques usually involve inoculation, but they may involve other modes of administration. A sufficient amount of the protein or the polypeptide is administered to create an immunogenic response in the animal host. Any host that produces antibodies to the antigen of the invention can be used. Once the animal has been immunized and sufficient time has passed for it to begin producing antibodies to the antigen, polyclonal antibodies can be recovered.
  • the general method comprises removing blood from the animal and separating the serum from the blood.
  • the serum which contains antibodies to the antigen, can be used as an antiserum to the antigen.
  • the antibodies can be recovered from the serum.
  • Affinity purification is a preferred technique for recovering purified polyclonal antibodies to the antigen, from the serum.
  • Monoclonal antibodies to the antigens of the invention can also be prepared.
  • One method for producing monoclonal antibodies reactive with the antigens comprises the steps of immunizing a host with the antigen; recovering antibody producing cells from the spleen of the host; fusing the antibody producing cells with myeloma cells deficient in the enzyme hypoxanthine-guanine phosphoribosyl transferase to form hybridomas; select at least one of the hybridomas by growth in a medium comprising hypoxanthine, aminopterin, and thymidine; identifying at least one of the hybridomas that produces an antibody to the antigen, culturing the identified hybridoma to produce antibody in a recoverable quantity; and recovering the antibodies produced by the cultured hybridoma.
  • polyclonal or monoclonal antibodies can be used in a variety of applications. Among these is the neutralization of corresponding proteins. They can also be used to detect viral antigens in biological preparations or in purifying corresponding proteins, glycoproteins, or mixtures thereof, for example when used in a affinity chromatographic columns.
  • the HTLV-3 polypeptides can be used as antigens to identify antibodies to HTLV-3 in materials and to determine the concentration of the antibodies in those materials.
  • the antigens can be used for qualitative or quantitative determination of the virus in a material.
  • materials include human tissue and human cells, as well as biological fluids, such as human body fluids, including human sera.
  • the antigens of the present invention provide an assay that is convenient, rapid, sensitive, and specific.
  • the antigens of the invention can be employed for the detection of HTLV-3 by means of immunoassays that are well known for use in detecting or quantifying humoral components in fluids.
  • immunoassays that are well known for use in detecting or quantifying humoral components in fluids.
  • antigen-antibody interactions can be directly observed or determined by secondary reactions, such as precipitation or agglutination.
  • immunoelectrophoresis techniques can also be employed. For example, the classic combination of electrophoresis in agar followed by reaction with anti-serum can be utilized, as well as two-dimensional electrophoresis, rocket electrophoresis, and immunolabeling of polyacrylamide gel patterns (Western Blot or immunoblot).
  • immunoassays in which the antigens of the present invention can be employed include, but are not limited to, radioimmunoassay, competitive immunoprecipitation assay, enzyme immunoassay, and immunofluorescence assay. It will be understood that turbidimetric, colorimetric, and nephelometric techniques can be employed. An immunoassay based on Western Blot technique is preferred.
  • Immunoassays can be carried out by immobilizing one of the immunoreagents, either an antigen of the invention or an antibody of the invention to the antigen, on a carrier surface while retaining immunoreactivity of the reagent.
  • the reciprocal immunoreagent can be unlabeled or labeled in such a manner that immunoreactivity is also retained.
  • enzyme immunoassays such as enzyme linked immunosorbent assay (ELISA) and competitive inhibition enzyme immunoassay (CIEIA).
  • the support is usually a glass or plastic material.
  • Plastic materials molded in the form of plates, tubes, beads, or disks are preferred. Examples of suitable plastic materials are polystyrene and polyvinyl chloride.
  • a carrier material can be interposed between the reagent and the support. Examples of suitable carrier materials are proteins, such as bovine serum albumin, or chemical reagents, such as gluteraldehyde or urea. Coating of the solid phase can be carried out using conventional techniques.
  • kits capable of diagnosing an HTLV-3 infection contains the DNA sequences of this invention, which are capable of hybridizing to viral RNA or analogous DNA sequences to indicate the presence of an HTLV-3 infection.
  • Different diagnostic techniques can be used which include, but are not limited to: (1 ) Southern blot procedures to identify cellular DNA which may or may not be digested with restriction enzymes; (2) Northern blot techniques to identify RNA extracted from cells; and (3) dot blot techniques, i.e., direct filtration of the sample through an ad hoc membrane, such as nitrocellulose or nylon, without previous separation on agarose gel.
  • Suitable material for dot blot technique could be obtained from body fluids including, but not limited to, serum and plasma, supernatants from culture cells, or cytoplasmic extracts obtained after cell lysis and removal of membranes and nuclei of the cells by centhfugation.
  • the 240 plasma samples were tested at a 1/40 dilution for the presence of HTLV-1/2 antibodies with a highly sensitive immunofluorescence test (IF) that uses MT2 and C19 as HTLV-1 and HTLV-2 viral antigen producing cells, respectively.
  • IF immunofluorescence test
  • This test also allows the detection of STLV-3 positive samples (Meertens et al., 2002; Meertens and Gussin, 2003).
  • the 48 plasma samples that were IF reactive on MT2, C19, or both were further tested by western blot (WB) HTLV-I/II Blot 2.4 (Diagnostic Biotechnology, Singapore).
  • the tax primers were: SCTaxoutse: ⁇ '-CTHTAYGGRTACCCHGTCTACGT-S' (SEQ ID NO: 9) and SCTaxoutas: ⁇ '-AGGGGAGBCGAGGGATAAGG-S' (SEQ ID NO: 21) corresponding to nucleotides 7279 to 7301 and 7455 to 7474, respectively, of the prototype STLV-3 PH A 969 sequence (GenBank accession number Y07616).
  • the pol primers were SCPOL1 outse: 5'- TTAAACCDGARCGCCTCCAGGC-3' (SEQ ID NO: 13) (nt 2485 to 2506) SCPOUoutas: 5'- GGDGTDCCYTTRGAGACCCA-3' (SEQ ID NO: 12) (nt 3201 to 3220) and SCPOU inse: 5'- TAYHHAGGRCCAGGMAATAACCC-3' (SEQ ID NO: 15) (nt 2556 to 2578).
  • HTLV-1 and HTLV-2 tax sequences were obtained for 4 and 11 samples that exhibited complete HTLV-1 and HTLV-2 WB profiles, respectively, but no PCR signals were obtained for any of the WB indeterminate samples. Consistent results were obtained for these HTLV-1 and HTLV-2 strains with the pol hemi-nested PCR. However, a faint band (665 bp) was also obtained for one sample (Py 143), which was previously found to be tax PCR negative. Sequencing of this fragment indicated the presence of an HTLV pol sequence that is highly related to STLV-3 strains (86.6% to 99.2% nucleotide identity).
  • the HTLV-3 sample originated from a 62 year old Bakola Pygmy living in a remote settlement in the ocean department of Southern Cameroon. His plasma was reactive on MT2 cells (titer: 1/320) but was negative on C19 cells.
  • the HTLV-I/II Blot 2.4 WB exhibited a strong reactivity to p19 and a faint reactivity to MTA-1 (Figure 2A).
  • On the INNO-LIA strip Innogenetics, Ghent, Belgium), it reacted faintly (+/-) with the generic p19 (l/ll), but strongly to the generic env gp46 (l/ll) and to the specific HTLV-2 gp46 ( Figure 2B).
  • Jurkat cells were grown in RPMI 1640 medium
  • the reaction mixture contained 1 ⁇ g of DNA, 10 mM Tris-HCI pH8.3, 50
  • Thermo-start DNA polymerase (Abgene). Table 1 shows the primer sequences and the corresponding annealing temperatures.
  • the nested PCR products were purified on a 1.5% agarose gel using the Qiaquick gel extraction kit (Qiagen) and cloned into the pCR2.1 vector (TA cloning kit, Invitrogen). For each PCR fragment, 6-10 clones were sequenced using the BigDye terminator cycle kit and an ABI 3100 automated sequencer (Applied Biosystem).
  • Phylogenetic analyses Multiple nucleotide sequences alignment was performed with the DAMBE program on the PTLVs complete sequences available in GenBank. The final alignment was submitted to the Modeltest program to select the best phylogenetical model to apply for the phylogenetical analyses. The model has been chosen accordingly to the Akaike Information Criterion (AIC). In this analysis, the selected model was the GTR+I+G one, with a shape of 2.0497 and a pinvar of 0.3239. The phylogeny was derived by the Neighbour-Joining method (with a bootstrap value of 1000), performed in PAUP program (Posada and Crandall, 1998; Xia and Xie, 2001).
  • HTLV-3 LTR To amplify and clone the HTLV-3 LTR, we used a previously described PCR technique (Meertens et al., 2004a), on three partially overlapping HTLV-3p y i 43 clones corresponding to nt 1 ->137 (part of clone 18), 44->213 (clone 19) and 111->446 (part of the clone 1 ) nucleotides. The following primers were used:
  • LTR-pyl43-Mlu1 -sens CCCCCCACGCGTTGTCAGTGATGATGAGCCTCG (SEQ ID NO: 22)
  • Tax8511as GGGTAGGGAGAGACGTCAGAGCC (SEQ ID NO: 23)
  • LTR8268se GGAGGACAAATAGCTGAATCATCCG
  • LTR8411 as: CAAA I I I I I AGGGTTATCGTCAGAGCC (SEQ ID NO: 25)
  • LTR111se CCAAGGCTCTGACGTCTCTCCCTAC (SEQ ID NO: 26)
  • LTR-pyl43-Bgl2-antisense AAAAAAAGATCTGGAGTGATGGCCTAGCTCGAC (SEQ ID NO: 27)
  • the 446 bp long LTR fragment encompassing the U3 (i.e. with the TREs) and part of the R sequence was then digested and inserted into the PGL2 basic plasmid as previously described (Chevalier et al., 2006).
  • the nucleotide sequence of the construct was determined as described above.
  • GFP-Tax3 and pSG5M-Tax plasmids Because human HTLV-3 viral RNA is not available, the tax3 cDNA sequence from STLV-3 CTO 6 o 2 , which is highly related to Pyl43, was used as a matrix for in vitro site-directed mutagenesis. Simian Tax3 sequence was therefore converted into human Tax3 sequence (S 3 22->N 322 ; L 33 3-> P33 3 , D 3 35->G 335 ). The nucleotide sequence of the both human pSG5M-Tax3 and human GFP-Tax3 constructs was determined as described above.
  • the pSG5M-Tax3 and GFP-Tax3 M22 (Ai 3 oM 13 i-> A 13 oSi3i) and M47 (L 3 i 9 L 32 o-> R3i ⁇ S 3 2o) were also constructed using the QuikChange Site-Directed mutagenesis kit (Stratagene) as previously described (Meertens et al., 2004a).
  • Green Fluorescent Protein analyses For microscopic analyses, HeLa cells were seeded on an eight-well chamber glass slide at a concentration of 3.5 x10 4
  • Luciferase assays Jurkat cells (3x10 6 ) were transfected with HTLV-3,
  • HTLV-1 and HTLV-2-LTR-luc NF-KB-IUC, Pr-Bcl-X L , MRE-luc, IL-8 536 -luc plasmids
  • RT-PCR analyses Twenty-four hours after transfection of Jurkat cells, total RNA was extracted with the Rneasy Mini Kit (QIAGEN) and treated with DNAsel RNAse free DNA set (Qiagen) to avoid any carry-over of the Tax containing plasmid. 0.5 ⁇ g of total RNA was then used as a matrix for reverse transcriptase PCR (RT-PCR) according to the instructions with the OneStep RT-PCR kit (QIAGEN).
  • PCR was performed using the following Tax primers: 602TaxEcoR1 and 602TaxBamH1 primers, which allow the amplification of a 1050 bp PCR product, which was then resolved on a 1 % agarose gel as previously described (Chevalier et al., 2006).
  • Tax primers 602TaxEcoR1 and 602TaxBamH1 primers, which allow the amplification of a 1050 bp PCR product, which was then resolved on a 1 % agarose gel as previously described (Chevalier et al., 2006).
  • HTLV-3 LTR is 695 bp long and possesses only two 21 -bp repeat sequences while HTLV-1 and HTLV-2 LTR have three of these TREs. This is due to a deletion of the TATA-distal 21 -bp repeat in the U3 region, a characteristic that has previously been reported for STLV-3 strains (VanBrussel et al., 1996).
  • HTLV-1 pX region encodes at least 5 proteins (Tax, Rex, p12, p13 and p30) whose functions are now well characterized (for a review see Nicot, et al, 2005). Apart from Tax and Rex, the existence of a doubly spliced viral mRNA that would be generated by alternative splicing, was reported for the STLV-3PH 969 prototypical strain. This transcript is predicted to encode a 84 amino acid long, highly hydrophobic and leucine rich protein designated Rorfll.
  • Rorfll transcript was not detected when investigators analyzed two other STLV-3 strains transcripts by RT-PCR, due either to mutations in the Rorfll splice acceptor sequence and/or to stop codon in the Rorfll coding sequence.
  • HTLV-3 Py ⁇ 4 3 Rorfll could not be amplified when using primers positioned within this sequence.
  • Sequence analysis subsequently revealed a 366 bp deletion in the HTLV-3 pX proximal region, leading to the absence of the putative Rorfll DNA sequence ( Figure 10D).
  • HTL V- 1 strand encodes a protein, HTLV-1 b-ZIP factor (HBZ), whose role might be to support the proliferation of adult T cell leukemia cells (Arnold et al., 2006; Satou, et al, 2006) and to enhance infectivity and persistence in inoculated animals (Xie et al., 2005).
  • HBZ HTLV-1 b-ZIP factor
  • AEP antisense encoded protein
  • HTLV-3p y ⁇ 4 3 belongs to the PTLV-3 Central African group. It displays 99.12% homology with the STLV-3 CTO 6 M strain if excluding the 366 bp deletion for the analysis, or 95.06% if considering the deletion in the sequence comparison ( Figure 11).
  • HTLV-3 Py i43 presents only 87% and 88% homology with the East African STLV-3PH 969 and the West African STLV-3 C TONG 409 isolates, respectively.
  • Tax3p y i 43 sequence analysis [0125] Tax3p y i 43 is expressed in vivo and elicits a humoral response in the HTLV- 3p y i 43 infected individual (Chevalier et al., 2006). Sequence comparisons reveal that the Tax3p y i4 3 protein displays 25% and 21% divergence at the amino acid level with HTLV-1 and HLTV-2 Tax respectively. Nevertheless, most of the previously reported Taxi domains are conserved in the Tax3p y i 43 sequence.
  • Tax3p y ⁇ 43 putative KID-like domain (aa 81 to 95), which is critical for the binding to CBP/p300 (Harrod et al., 1998), is similar to that of Taxi .
  • the 2 amino acids corresponding to the "M22 domain" (Smith and Greene, 1990) are also different in the Taxi vs. Tax3p y ⁇ 43 protein (T 130 L 131 -> A 130 M 131 ) ( Figure 12B).
  • Tax3p y i 43 CR2 binding sequence (aa 312 to 319) ( Figure 12C).
  • This domain has been shown to be important for the ability of Taxi to bind CBP/p300 (Scoggin et al., 2001).
  • Tax3p y ⁇ 43 "M47 domain” is identical to that of Taxi .
  • Tax3p y ⁇ 43 contains a PDZ binding motif at its C-terminus ( Figure 12D).
  • Such a domain has been repeatedly shown to be required for the ability of Taxi to transform rat fibroblasts or to induce the proliferation of human PBMCs (Xie et al., 2005; Endo, et al, 2002).
  • Taxi is a shuttling protein that is predominantly found in the cell nucleus where it forms speckled structures (Semmes and Jeang, 1996). Taxi contains a non- canonical nuclear localization signal (NLS) and a nuclear export signal (NES). In contrast, HTLV-2 Tax (Tax2) is governed by a minimal domain encompassing amino acids 90-100 and is mainly located in the cytoplasm of the HTLV-2 immortalized or transformed T-cells (Chevalier et al., 2005; Meertens et al., 2004a).
  • Tax3M22 (Ai3o-Mi 3 r>Ai 3 o->S 1 3i) and Tax3M47 (L 319 L 3 2o->R 3 i 9 8 3 2 0 )-
  • the localization of both mutants is similar to that of Tax3 with, however, in both cases fewer cytoplasmic speckles ( Figure 13A, panels d vs. e and f).
  • Western-blot analysis demonstrated that all GFP-fusion proteins were expressed to similar levels (Figure 13B upper panel).
  • the membrane loading As a control for protein loading, the membrane
  • Tax3p y i43 is a viral transactivator
  • Taxi protein activates both the CREB/ATF and the NF- ⁇ B signaling
  • HTLV-3 LTR constructs were preformed with different HTLV LTR constructs. First, whether Tax3p y ⁇ 43 could mediate the transcription from the HTLV-3 LTR in lymphocytes was determined. This cell population is likely to be the natural target of HTLV-3 infection in vivo, as it is the case for HTLV-1 and HTLV-2 (Manel et al., 2005). As stated above, HTLV-3 LTR sequence lacks the distal TRE.
  • Tax3p y ⁇ 43 activates efficiently the transcription from both LTRs in transiently transfected lymphocytes.
  • Tax3 sequence contains another domain that can, to some extent, compensate for the M22 mutation.
  • Taxi or Tax2 specific antibodies do not allow the detection of Tax3. Therefore, Tax3 expression was monitored by RT-PCR.
  • a Tax3 specific signal was present in the Tax3p y ⁇ 43 transfected Jurkat ( Figure 14E, lanes 4, 6, 8). This band was absent in mock transfected cells ( Figure 14E, lane 2), or when the reverse transcriptase was not added to the PCR mix ( Figure 14E, lanes 1 , 3, 5, 7).
  • Tax3p y ⁇ 43 represses or activates some cellular promoters was investigated. Regulation of these promoters has previously been shown to be
  • Tax3 Py ⁇ 43 or the different Tax3p y ⁇ 43 mutants inhibit p53 transcriptional activity was investigated ( Figure 15A). As it is the case with Taxi (Meertens et al., 2004b; Mulloy et al., 1998; Pise-Masison et al., 2001), Tax3 Py ⁇ 43 and Tax3 M22 , repressed p53 activity in HeIa cells. Thus, in these cells, the activation of NF-
  • KB is not needed for inhibiting p53 functions.
  • a series of western blots was performed using cellular extracts obtained from the transfected HeIa cells. Consistent with the ability of Tax3p y i4 3 and of Tax3M22 to inhibit p53 activity better that Tax3M47, the p53 protein was slightly more efficiently stabilized in these cells ( Figure 15A, lower panel). This observation was also reported when repressing p53 functions with HTLV-1 Tax.
  • c-Myb is essential for a controlled balance between cell growth and differentiation and aberrant c-Myb activity has been reported for numerous human cancers.
  • Tax3p y i 43 is functionally related to Taxi .
  • MRE c-Myb promoter construct
  • Jurkat cells express c-Myb and therefore do not require the addition of ectopically expressed c-Myb.
  • Tax3pyi 43 and Tax3 M 47 but not Tax3 M22 transrepress the MRE construct ( Figure 15B).
  • Tax3p y ⁇ 43 transactivates two cellular promoters that were previously reported to be upregulated by Taxi through the
  • Taxi partially activates the NF- ⁇ B pathway
  • Tax3p y i43 but also of Tax3 M 22 (Figure 15C). This result confirms that NF- ⁇ B is involved in the Bcl-X L transactivation. Also, Tax3M 47 , even if it activates this pathway, has another defect that prevents it from transactivating the Bcl-X L promoter.
  • Tax3p y i 43 dependent lnterleukin-8 transactivation [0136]
  • an interleukin-8 promoter construct that is also known to be
  • Taxi upregulated by Taxi was tested for activation of the NF- ⁇ B pathway (Mori et al., 1998).
  • Tax3 is necessary but is not sufficient to transactivate the BCI-XL and the IL-8 promoters. Thus, Taxi and Tax3, although functionally highly related in vitro, are not entirely equivalent.
  • Retrovirology 2 70. Courgnaud V, Van Dooren S, Stanfordois F, Pourrut X, Abela B, Loul S, Mpoudi-Ngole E,
  • Vandamme AM (2001) J Virol. 75: 11939-41. Van Dooren S, Shanmugam V, Bhullar V, Parekh B, Vandamme AM, Heneine W,

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Abstract

L'invention concerne la séquence génétique de HTLV-3, un virus T-lymphotropique humain différent de HTLV-1 et de HTLV-2. En outre, elle concerne les séquences des polypeptides codés par le génome de HTLV-3. Ces polypeptides permettent la production de formes recombinées de ces polypeptides et d'anticorps dirigés contre ces polypeptides, ainsi que l'utilisation de ces acides nucléiques et de ces polypeptides dans des méthodes diagnostiques, des trousses, une composition immunogène ou un traitement antiviral. Les méthodes diagnostiques comprennent également des méthodes basées sur l'amplification et sur l'hybridation faisant appel à des amorces ou des sondes issues de la séquence génétique du génome de HTLV-3.
PCT/EP2006/004951 2005-05-02 2006-04-28 Nouveau virus t-lymphotropique humain (htlv-3), acides nucleiques et sequences peptidiques de ce virus, et methode de detection de htlv-3 WO2006133795A2 (fr)

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US67669905P 2005-05-02 2005-05-02
US60/676,699 2005-05-02
US74105805P 2005-12-01 2005-12-01
US60/741,058 2005-12-01

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WO2006133795A2 true WO2006133795A2 (fr) 2006-12-21
WO2006133795A3 WO2006133795A3 (fr) 2007-04-26

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AU2063795A (en) * 1994-03-25 1995-10-17 Stichting Rega Vzw T-lymphotropic primate virus l (ptlv-l) and its applications
EP2194127B1 (fr) * 2005-02-21 2011-12-28 The Government of the United States of America as represented by the Secretary of the Department of Health and Human Services Virus lymphotropes T de primates

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