Design and in vivo immunogenicity of a polyvalent vaccine based on SIVmac regulatory genes.

Most vaccine modalities for human immunodeficiency virus type 1 (HIV-1) tested for immunogenicity and efficacy in the SIVmac (simian immunodeficiency virus) macaque model do not include the viral regulatory proteins. Because viral regulatory proteins are expressed early during the virus life cycle and represent an additional source of antigens, their inclusion as a vaccine component may increase the overall virus-specific immune response in vaccinees. However, at least two of the early proteins, Tat and Nef, may be immunosuppressive, limiting their usefulness as components of an SIV vaccine. We have constructed a polyvalent chimeric protein in which the open reading frames for Tat and Nef have been reassorted and the nuclear localization sequence for Tat and Rev and the myristoylation site for Nef have been removed. The resulting DNA plasmid (pDNA-SIV-Retanef) (pDNA-SIV-RTN) encodes a protein of 55 kDa (Retanef) that localizes at the steady state in the cytoplasma of transfected cells. Both the DNA-SIV-RTN and the highly attenuated recombinant poxvirus vector NYVAC-SIV-RTN were demonstrated to be immunogenic in SIVmac251-infected macaques treated with ART as well as in naive macaques. An equivalent strategy may be used for the generation of polyvalent antigens encoding the regulatory proteins in a HIV-1 vaccine candidate.

Molecular biology and pathogenesis of the human T-cell leukaemia/lymphotropic virus Type-1 (HTLV-1).

Retroviruses are associated with a variety of diseases, including immunological and neurological disorders, and various forms of cancer. In humans, the Human T-cell Leukaemia/Lymphotropic virus type 1 (HTLV-1), which belongs to the Oncovirus family, is the aetiological agent of two diverse diseases: Adult T-cell leukaemia/lymphoma (ATLL) (Poiesz et al. 1980; Hinuma et al. 1981; Yoshida et al. 1982), as well as the neurological disorder tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM) (Gessain et al. 1985; Rodgers-Johnson et al. 1985; Osame et al. 1986). HTLV-1 is the only human retrovirus known to be the aetiological agent of cancer. A genetically related virus, HTLV-2, has been identified and isolated (Kalyanaraman et al. 1982). However, there has been no demonstration of a definitive aetiological role for HTLV-2 in human disease to date. Simian T-cell lymphotropic viruses types 1 and 2 (STLV-1 and -2) and bovine leukaemia virus (BLV) have also been classified in same group, Oncoviridae, based upon their similarities in genetic sequence and structure to HTLV-1 and -2 (Burny et al. 1988; Dekaban et al. 1995; Slattery et al. 1999). This article will focus on HTLV-1, reviewing its discovery, molecular biology, and its role in disease pathogenesis.

Kinase-inducible domain-like region of HTLV type 1 tax is important for NF-kappaB activation.

Partial proteolysis of HTLV-1 Tax protein has revealed the region surrounding amino acid residues (88)KVL(90) to be highly exposed. The protein sequence surrounding this region ((81)QRTSKTLKVLTPPIT(95)) bears resemblance to the kinase-inducible domain (KID, (129)SRRPSYRKILNE(140)) of CREB and is involved in recruiting transcriptional coactivators, p300 and CBP, for trans-activating the viral long terminal repeat (LTR). Data have also revealed the KID-like region to be important for Tax binding to DNA. Here we report that single (K88A, V89A, L90A) and double alanine substitutions (V89A-L90A) in the (88)KVL(90) motif attenuate the ability of Tax to activate NF-kappaB. Deletions near or spanning this motif also had the same effect. The alanine substitutions affect HTLV-1 LTR activation and NF-kappaB activation differently, with K88A and V89A mutants showing much reduced activities for HTLV LTR activation while retaining attenuated but significant NF-kappaB-activating function. In contrast, although the L90A mutant is similarly attenuated for NF-kappaB activation, it showed significant activity in LTR trans-activation. Incorporation of both V89A and L90A substitutions in a V89A-L90A double mutant further reduced NF-kappaB activation and completely abrogated LTR trans-activation. In aggregate, these results demonstrate the importance of the KID-like domain of Tax and implicate its interaction with cellular factors other than p300/CBP in NF-kappaB activation.

Distinct p300-responsive mechanisms promote caspase-dependent apoptosis by human T-cell lymphotropic virus type 1 Tax protein.

The dysregulation of cellular apoptosis pathways has emerged as a critical early event associated with the development of many types of human cancers. Numerous viral and cellular oncogenes, aside from their inherent transforming properties, are known to induce programmed cell death, consistent with the hypothesis that genetic defects are required to support tumor survival. Here, we report that nuclear expression of the CREB-binding protein (CBP)/p300-binding domain of the human T-cell lymphotropic virus type 1 (HTLV-1) transactivator, Tax, triggers an apoptotic death-inducing signal during short-term clonal analyses, as well as in transient cell death assays. Coexpression of the antiapoptotic factor Bcl-2 increased serum stimulation; incubation with the chemical caspase inhibitor z-Val-Ala-DL-Asp fluoromethylketone antagonized Tax-induced cell death. The CBP/p300-binding defective Tax mutants K88A and V89A exhibited markedly reduced cytotoxic effects compared to the wild-type Tax protein. Importantly, nuclear expression of the minimal CBP/p300-binding peptide of Tax induced apoptosis in the absence of Tax-dependent transcriptional activities, while its K88A counterpart did not cause cell death. Further, Tax-mediated apoptosis was effectively prevented by ectopic expression of the p300 coactivator. We also report that activation of the NF-kappaB transcription pathway by Tax, under growth arrest conditions, results in apoptosis that occurs independent of direct Tax coactivator effects. Our results allude to a novel pivotal role for the transcriptional coactivator p300 in determining cell fate and raise the possibility that dysregulated coactivator usage may pose an early barrier to transformation that must be selectively overcome as a prerequisite for the initiation of neoplasia.

p300 and p300/cAMP-responsive element-binding protein associated factor interact with human T-cell lymphotropic virus type-1 Tax in a multi-histone acetyltransferase/activator-enhancer complex.

The human T-cell lymphotropic virus, type (HTLV)-1 trans-activator, Tax, coordinates with cAMP-responsive element-binding protein (CREB) and the transcriptional co-activators p300/CBP on three 21-base pair repeat elements in the proviral long terminal repeat (LTR) to promote viral mRNA transcription. Recruitment of p300/CBP to the activator-enhancer complex, however, is insufficient to support Tax-dependent LTR trans-activation. Here, we report that the p300/CBP-associated factor (P/CAF) is a critical and integral component of the functional HTLV-1 activator-enhancer complex. The HTLV-1 Tax protein directly binds P/CAF in vitro and co-immunoprecipitates with this co-activator in vivo. The Tax mutants (K88A and V89A) defective for p300/CBP-binding and LTR trans-activation, retained their abilities to interact with P/CAF. The M47 mutant (L319R, L320S) protein, which has previously been shown to interact with p300/CBP, by contrast, failed to form complexes with P/CAF and is impaired in LTR trans-activation. Furthermore, LTR trans-activation by Tax is competitively inhibited by the adenoviral E1A 12S gene product, which displaces P/CAF from p300/CBP and inhibits the histone acetyltransferase activities of both P/CAF and p300/CBP. This inhibition is partially reversed by exogenously added P/CAF. These results imply that simultaneous recruitment of two distinct co-activators (p300/CBP and P/CAF) by Tax is essential for the assembly of a trans-activation competent, nucleoprotein complex.

An extended alpha-helix and specific amino acid residues opposite the DNA-binding surface of the cAMP response element binding protein basic domain are important for human T cell lymphotropic retrovirus type I Tax binding.

The human T cell lymphotropic retrovirus type I (HTLV-I) trans-activator, Tax, interacts specifically with the basic-domain/leucine-zipper (bZip) protein, cAMP response element binding protein (CREB), bound to the viral Tax-responsive element consisting of three imperfect 21-base pair repeats, each with a cAMP response element core flanked by G/C-rich sequences. Here, the minimal CREB-bZip necessary for Tax binding is shown to be composed of amino acid residues 280-341. The Tax-CREB interaction involves an uninterrupted and extended alpha-helix in CREB that spans most of its basic domain to include amino acid residues localized to the NH2 terminus of the DNA binding region. Mutational analyses indicate that three residues, Arg284, Met291, and Glu299 unique to this region of the CREB/activating transcription factor-1 subfamily of bZip proteins, constitute the contact surface for Tax. Amino acid substitutions in these positions had little impact on CREB-bZip binding to DNA but abrogated its binding to Tax. Each of the contact residues for Tax are spaced approximately two helical turns apart on the side of the bZip helix directly opposite to that of the invariant DNA-binding residues. Molecular modeling reveals the Tax-contact residues to be near the minor groove of the G/C-rich DNA in the 21-base pair repeat. They most likely position Tax for minor groove contact with the G/C-rich sequences.

An exposed KID-like domain in human T-cell lymphotropic virus type 1 Tax is responsible for the recruitment of coactivators CBP/p300.

Human T-cell lymphotropic virus type 1 (HTLV-1) transcriptional activation is mediated by the viral transactivator, Tax, and three 21-bp repeats (Tax response element [TxRE]) located in the U3 region of the viral long terminal repeat (LTR). Each TxRE contains a core cyclic AMP response element (CRE) flanked by 5' G-rich and 3' C-rich sequences. The TxRE binds CREB (CRE-binding protein) and Tax to form a ternary complex and confers Tax-dependent transactivation. Recent data indicate that Tax functions as a specific link to connect CREB-binding protein (CBP)/p300 in a phosphorylation-independent manner to CREB/ATF-1 assembled on the viral 21-bp repeats. Glutathione S-transferase pull-down performed with Tax deletion mutants and peptide competition have localized the site in Tax critical for binding CBP/p300 to a highly protease-sensitive region around amino acid residues 81 to 95 (81QRTSKTLKVLTPPIT95) which lies between the domains previously proposed to be important for CREB binding and Tax subunit dimerization. Amino acid residues around the trypsin- and chymotrypsin-sensitive sites (88KVL90) of Tax bear resemblance to those in the kinase-inducible domain of CREB (129SRRPSYRKILNE140) surrounding Ser-133, which undergoes signal-induced phosphorylation to recruit CBP/p300. Site-directed mutagenesis of residues in this domain (R82A, K85A, K88A, and V89A) resulted in proteins which failed to transactivate from the HTLV-1 LTR in vivo. These mutants (K85A, K88A, and V89A) bind CREB with similar affinities as wild-type Tax, yet interaction with CBP/p300 is abrogated in various biochemical assays, indicating that the recruitment of CBP/p300 is crucial for Tax transactivation. A Tax mutant, M47, defective in the COOH-terminal transactivation domain, continued to interact with CBP/p300, suggesting that interactions with additional cellular factors are required for proper Tax function.