Human T cell leukaemia virus type 2 tax protein mediates CC-chemokine expression in peripheral blood mononuclear cells via the nuclear factor kappa B canonical pathway.

Retroviral co-infections with human immunodeficiency virus type-1 (HIV-1) and human T cell leukaemia virus type 1 (HTLV-1) or type 2 (HTLV-2) are prevalent in many areas worldwide. It has been observed that HIV-1/HTLV-2 co-infections are associated with slower rates of CD4(+) T cell decline and delayed progression to AIDS. This immunological benefit has been linked to the ability of Tax2, the transcriptional activating protein of HTLV-2, to induce the expression of macrophage inflammatory protein (MIP)-1α/CCL3, MIP-1ß/CCL4 and regulated upon activation normal T cell expressed and secreted (RANTES)/CCL5 and to down-regulate the expression of the CCR5 co-receptor in peripheral blood mononuclear cells (PBMCs). This study aimed to assess the role of Tax2-mediated activation of the nuclear factor kappa B (NF-κB) signalling pathway on the production of the anti-viral CC-chemokines MIP-1α, MIP-1ß and RANTES. Recombinant Tax1 and Tax2 proteins, or proteins expressed via adenoviral vectors used to infect cells, were tested for their ability to activate the NF-κB pathway in cultured PBMCs in the presence or absence of NF-κB pathway inhibitors. Results showed a significant release of MIP-1α, MIP-1ß and RANTES by PBMCs after the activation of p65/RelA and p50. The secretion of these CC-chemokines was significantly reduced (P < 0·05) by canonical NF-κB signalling inhibitors. In conclusion, Tax2 protein may promote innate anti-viral immune responses through the activation of the canonical NF-κB pathway.

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.

In vitro selection of DNA elements highly responsive to the human T-cell lymphotropic virus type I transcriptional activator, Tax.

The human T-cell lymphotropic virus type I (HTLV-I) transactivator, Tax, the ubiquitous transcriptional factor cyclic AMP (cAMP) response element-binding protein (CREB protein), and the 21-bp repeats in the HTLV-I transcriptional enhancer form a ternary nucleoprotein complex (L. J. Zhao and C. Z. Giam, Proc. Natl. Acad. Sci. USA 89:7070-7074, 1992). Using an antibody directed against the COOH-terminal region of Tax along with purified Tax and CREB proteins, we selected DNA elements bound specifically by the Tax-CREB complex in vitro. Two distinct but related groups of sequences containing the cAMP response element (CRE) flanked by long runs of G and C residues in the 5' and 3' regions, respectively, were preferentially recognized by Tax-CREB. In contrast, CREB alone binds only to CRE motifs (GNTGACG[T/C]) without neighboring G- or C-rich sequences. The Tax-CREB-selected sequences bear a striking resemblance to the 5' or 3' two-thirds of the HTLV-I 21-bp repeats and are highly inducible by Tax. Gel electrophoretic mobility shift assays, DNA transfection, and DNase I footprinting analyses indicated that the G- and C-rich sequences flanking the CRE motif are crucial for Tax-CREB-DNA ternary complex assembly and Tax transactivation but are not in direct contact with the Tax-CREB complex. These data show that Tax recruits CREB to form a multiprotein complex that specifically recognizes the viral 21-bp repeats. The expanded DNA binding specificity of Tax-CREB and the obligatory role the ternary Tax-CREB-DNA complex plays in transactivation reveal a novel mechanism for regulating the transcriptional activity of leucine zipper proteins like CREB.

KSHV vCyclin counters the senescence/G1 arrest response triggered by NF-κB hyperactivation.

Many oncogenic viruses activate nuclear factor-κB (NF-κB) as a part of their replicative cycles. We have shown recently that persistent and potentially oncogenic activation of NF-κB by the human T-lymphotropic virus 1 (HTLV-1) oncoprotein Tax immediately triggers a host senescence response mediated by cyclin-dependent kinase inhibitors: p21(CIP1/WAF1) (p21) and p27(Kip1) (p27) Here we demonstrate that RelA/NF-κB activation by Kaposi sarcoma herpesvirus (KSHV) latency protein vFLIP also leads to p21/p27 upregulation and G1 cell cycle arrest. Remarkably, KSHV vCyclin, another latency protein coexpressed with vFLIP from a bicistronic latency-specific mRNA, was found to prevent the senescence and G1 arrest induced by HTLV-1 Tax and vFLIP, respectively. This is because of the known ability of vCyclin/cyclin-dependent kinase 6 complex to resist p21 and p27 inhibition and cause p27 degradation. In KSHV-transformed BCBL-1 cells, sustained vFLIP expression with small hairpin RNAs-mediated vCyclin depletion resulted in G1 arrest. The functional interdependence of vFLIP and vCyclin explains why they are cotranslated from the same viral mRNA. Importantly, deregulation of the G1 cyclin-dependent kinase can facilitate chronic I-κB kinases/NF-κB activation.

No evidence of KSHV/HHV-8 in mycosis fungoides or associated disorders.

The recently discovered human virus known as Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus-8 (HHV-8) has been associated with body-cavity-based lymphomas in AIDS patients. It is most closely related to two other herpesviruses, the Epstein-Barr virus and herpesvirus saimiri, which are known to be associated with lymphomas in humans and nonhuman primates, respectively. To determine whether KSHV/HHV-8 is involved in the pathogenesis of mycosis fungoides (MF) and related disorders, we used a genomic PCR assay followed by confirmatory Southern blot analysis with a nested oligonucleotide probe to analyze cases for the presence of this virus. The specimens studied included fresh-frozen lesional tissues obtained from 16 patients with MF, seven with lymphomatoid papulosis, seven with primary cutaneous CD30+ large cell lymphoma of T-cell lineage, and five with Hodgkin's disease. Two T-cell tumor lines were also studied: MT4 (derived from a patient with adult T-cell leukemia/lymphoma) and Jurkat (derived from a patient with T-cell acute lymphoblastic leukemia). All cases were uniformly negative for KSHV/HHV-8, whereas Kaposi's sarcoma-positive controls and human beta-globin DNA integrity controls were appropriately positive. These findings provide strong evidence against a role for KSHV/HHV-8 in the pathogenesis of MF or associated lymphoproliferative disorders.

Mycobacterium tuberculosis and its purified protein derivative activate expression of the human immunodeficiency virus.

To examine the effects of Mycobacterium tuberculosis on human immunodeficiency virus type 1 (HIV-1) expression, the monocytoid cell line U1 containing integrated provirus was incubated with the H37Ra strain of M. tuberculosis. This resulted in heightened expression of virus in supernatant that was partially inhibited by antibody to tumor necrosis factor-alpha (TNF-alpha). Purified protein derivative (PPD) prepared from M. tuberculosis also could activate HIV expression, and this was less affected by anti-TNF antibody. PPD could activate the HIV promoter in both U937, the monocytoid cell line from which U1 was derived, and Jurkat, a CD4+ lymphoid line. Activation was abolished by mutations in the nuclear factor (NF)-kB binding domains. Jurkat cells transfected with a plasmid construct linking 8 NF-kB binding domains to the chloramphenicol acetyltransferase (CAT) gene showed increased activity of the reporter gene after activation with PPD. Transcriptional activation of HIV expression by mycobacteria and mycobacterial products may enhance propagation of HIV in monocytoid and lymphoid cells. This may result in accelerated HIV disease progression in persons coinfected with M. tuberculosis.

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.

HTLV-I tax gene product activates transcription via pre-existing cellular factors and cAMP responsive element.

Human T-cell leukemia virus type I (HTLV-I) is the etiological agent of adult T-cell leukemia. The 3' end of HTLV-I proviral DNA encodes the synthesis of two regulatory proteins, tax and rex. The 40-kDa tax protein is a nuclear protein which positively stimulates transcription from the U3 region of the viral long terminal repeat sequence. Three 21-base pair sequences in the U3 region have been found to serve as the cis-element for tax-mediated trans-activation. We now report that the tax protein can trans-activate HTLV-I LTR in the absence of de novo cellular protein synthesis. Saturated mutagenesis of the 21-base pair repeat sequence showed that specific mutations clustered in sequences homologous to the cAMP responsive element (TGACGTCA) abolish trans-activation by tax. Furthermore, although the TGACGTCN element is nearly palindromic, the mutations that abolish trans-activation are localized exclusively in the 5' 6 bases, suggesting the orientation of this element may play a role in transcription. That the purified tax protein does not bind the 21-base pair repeats or nonspecific DNA lends further support to the notion that tax protein does not directly interact with the 21-base pair repeats to activate transcription. Instead, tax most likely acts via cellular transcriptional factor(s) to bring about trans-activation.

In vivo and in vitro autoprocessing of human immunodeficiency virus protease expressed in Escherichia coli.

The viral-specific protease of human immunodeficiency virus (HIV) has been expressed as a lacZ-protease fusion protein. This fusion protein contains protease cleavage sites at the gag/protease and protease/reverse transcriptase junctions and undergoes autoprocessing in vivo when expressed in Escherichia coli. The purified lacZ-protease fusion protein precursors also exhibit autoproteolytic activity in vitro. One cleavage product of the autoprocessing reactions is a 10-kDa protein that cross-reacts with peptide antisera prepared against the putative protease sequence. Consistent with the notion that HIV protease is an acid protease, its autoproteolytic activity is inhibited in alkaline buffers and by pepstatin A. The in vivo and in vitro autocleavage assays for HIV protease together with the overproduction of the protease should facilitate design and testing of therapeutic agents that inhibit gag-pol polyprotein processing and HIV virion maturation.

Human T-cell lymphotropic virus type I (HTLV-I) transcriptional activator, Tax, enhances CREB binding to HTLV-I 21-base-pair repeats by protein-protein interaction.

HTLV-I Tax protein activates transcription from three 21-base-pair (bp) repeat sequences in the viral enhancer. The HTLV-I 21-bp repeat contains a TGACGT motif that is homologous to the cAMP-responsive element (CRE) and crucial for tax transactivation. Tax exhibits marginal affinity for DNA but rather interacts with cellular CRE-binding proteins to enhance their affinity for the HTLV-I 21-bp repeats. Using the HTLV-I 21-bp repeat and Jurkat T-lymphocyte nuclear extract in a gel electrophoretic mobility-shift assay, we previously detected three protein-DNA complexes that are specific for the CRE in the 21-bp repeat (complexes I, II, and IV). Complexes I and II but not IV interacted with Tax. We now show that complexes I, II, and IV are composed of CREB (CRE binding protein) homodimer, CREB/ATF-1 (activating transcription factor 1) heterodimer, and ATF-1 homodimer, respectively. Tax stabilizes complexes I and II via a direct interaction with the CREB moiety. In the absence of DNA, CREB and Tax continue to form a complex that can be immunoprecipitated by a Tax-specific antibody. These results suggest that one mechanism by which Tax activates transcription may be mediated through the direct interaction with CREB homodimer and/or CREB/ATF-1 heterodimer to stabilize their assembly on the Tax-responsive CRE motifs in the HTLV-I enhancer.

Interaction of the human T-cell lymphotrophic virus type I (HTLV-I) transcriptional activator Tax with cellular factors that bind specifically to the 21-base-pair repeats in the HTLV-I enhancer.

The human T-cell lymphotrophic virus type I (HTLV-I) Tax protein activates transcription from three 21-base-pair (bp) repeat sequences in the viral enhancer. Using gel electrophoretic mobility-shift assays, we now show that Tax interacts directly with the nuclear proteins, Tax activation factors (TAFs), that bind the 21-bp repeats. This interaction is demonstrated by decreased electrophoretic mobilities of the TAFs-21-bp-repeats complexes upon supply of Tax exogenously. Formation of the TAFs-21-bp-repeats and Tax-TAFs-21-bp-repeats complexes correlates with in vivo transactivation by Tax. Furthermore, interaction of Tax with TAFs enhances their binding to the 21-bp repeats. These data indicate that trans-activation by Tax is most likely mediated by interaction of Tax with TAFs.

Distinct regions in human T-cell lymphotropic virus type I tax mediate interactions with activator protein CREB and basal transcription factors.

Human T-cell lymphotropic virus type I (HTLV-I) transactivator Tax augments transcription from three (cyclic AMP response element (CRE)-containing 21-bp repeats in the viral long terminal repeat and several other cis regulatory elements, including the NF-kappa B binding sites and the serum response element. Tax does not bind DNA directly; rather, it acts via cellular sequence-specific DNA binding proteins to stimulate transcription. We have shown recently that Tax forms multiprotein complexes with the heterodimeric and homodimeric forms of a ubiquitous cellular transcription factor, CREB (CRE binding protein). In vitro selection for preferred Tax-CREB binding sites indicates that the Tax-CREB complex exhibits greatly increased DNA recognition specificity and assembles preferentially on CRE motifs, TGACGT/C, flanked by long runs of G (5') and/or C (3') residues, as found in the HTLV-I 21-bp repeats. The indirect tethering of Tax to the 21-bp repeats via CREB is crucial for Tax transactivation. We now report the domain organization of Tax by characterizing its mutants. Tax mutants with alterations in the NH2 terminus, including three deletion mutants, Tax(6-353), Tax(21-353), and Tax(89-353), and two amino acid substitution mutants, M1 (H3S) and M7 (C29A, P30S), all failed to interact with CREB in vitro. In contrast, a short COOH-terminal deletion, Tax(1-319), and a Tax mutant with amino acid substitutions near the COOH end, M47 (L319R, L320S), were able to interact with CREB and the 21-bp repeats to assemble ternary Tax-CREB-DNA complexes. As demonstrated earlier, M1, M7, and M47 all failed to transactivate the HTLV-I long terminal repeat. Our data indicate that the defects in M1 and M7 result from an inability to interact with CREB. In contrast, the COOH-terminal mutations in M47 most likely inactivated the transactivation domain of Tax. As anticipated, a Tax mutant, M22 (G137A, L138S) which activated transcription from the 21-bp repeats with reduced capacity and was defective in trans activating the NF-kappa B binding sites, continued to interact with CREB in vitro, albeit with a lower level of efficiency. Finally, a glutathione S-transferase (GST)-Tax fusion protein with the GST moiety fused to the NH2 terminus of Tax failed to interact with CREB. Removal of the GST domain from GST-Tax by thrombin restores Tax's ability to assemble a ternary Tax-CREB-21-bp-repeat complex.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro association of empty adenovirus capsids with double-stranded DNA.

Several lines of evidence suggest that empty adenovirus capsids are preassembled intermediates in the pathway of virion assembly. We have observed that purified empty capsids of subgroup B adenoviruses have a remarkable affinity for DNA in vitro. The products of capsid-DNA association are sufficiently stable, once formed in low-salt solution, to permit purification and characterization in CsCl density gradients. Neither virions nor the DNA-containing incomplete particles of subgroup B adenoviruses can give rise to such in vitro reaction products. The average molecular weight of the empty adenovirus capsids is about 123 X 10(6), consistent with the absence of viral core peptides and a small deficiency of exterior shell polypeptides. Electron microscopy of negatively stained capsids and the capsids bound to DNA reveals a typical adenovirus size and architecture. The particles appear with a surface discontinuity that is presumed to expose the DNA binding site(s). The DNA molecules associated with the empty capsids are susceptible to the actions of DNase and restriction endonucleases. The dependence of rate of capsid-DNA association on DNA length suggests randomly distributed binding sites on the DNA molecules. Although the DNA molecules can successively acquire additional empty capsids, the empty particles themselves are restricted to interactionwith only one DNA molecule. Electron microscopy of the capsid-DNA complexes spread in cytochrome c films shows that the particles are bo-nd along the contour of extended duplex DNA. The amount of DNA within each bound particle appears to be less than 300 base pairs, as estimated by the length of the DNA molecules visible outside of the bound particle. The empty capsid-DNA association product described in this report provides an interesting substrate for further investigation of the DNA packaging process in a defined in vitro system, with extracts or purified components from infected cells.

Characterization of a novel lipoprotein mutant in Escherichia coli.

Mutants altered in the structural gene for murein lipoprotein in Escherichia coli can be isolated by globomycin selection. We have isolated a unique globomycin-resistant mutant, strain 6-23, which synthesizes a structurally altered, albeit modified and processed, lipoprotein. DNA sequence analysis of the mutant lpp allele and determination of the amino acid composition of the mutant lipoprotein revealed a single amino acid substitution of cysteine for arginine at the 68th amino acid residue of prolipoprotein. Pulse-chase experiments revealed that the kinetics of lipoprotein maturation was affected by this alteration in the structure of lipoprotein.

Expression of the complete human T-cell leukemia virus type I pX coding sequence as a functional protein in Escherichia coli.

Human T-cell leukemia virus type I (HTLV-I), a virus associated with adult T-cell leukemia, contains a long open reading frame (LOR) in the 3' end of its genome between the env region and the 3' long terminal repeat (LTR). This open reading frame encodes a 40-kDa protein (designated p40x) that has been implicated as a positive control element for transcription from the HTLV-I LTR in a phenomenon known as trans-activation. We now report the expression of the complete p40x coding sequence as a 40-kDa protein in Escherichia coli. The p40x protein produced in bacteria is shown, using the protoplast fusion technique, to possess biological activity by its ability to trans-activate a HTLV-I LTR-chloramphenicol acetyltransferase plasmid that is stably integrated into the genome of mouse L cells. This stimulatory activity could be detected within 2 hr after fusion, suggesting the possibility of a direct role for p40x in trans-activation of the HTLV-I LTR. The production of p40x in large quantities in E. coli, together with the rapid protoplast fusion assay for its biological activity, should facilitate the analysis of p40x mutants and the elucidation of the molecular mechanism of trans-activation.

Interaction of bovine leukemia virus transactivator Tax with bZip proteins.

The bovine leukemia virus transactivator, BLV Tax, augments transcription from three imperfect 21-bp repeats in the viral transcriptional regulatory region. Each BLV 21-bp repeat contains a cAMP response element (CRE) in the center and unique 5' and 3' neighboring sequences which are crucial for the transcriptional activation by BLV Tax. Here we describe the interactions of recombinant BLV Tax with cellular bZip proteins. The recombinant BLV Tax, tagged at the carboxy terminus with a hexahistidine extension, was prepared by solubilization in 6 M guanidine hydrochloride and renaturation on the Ni(2+)-chelating Sepharose gel matrix. The renatured BLV TaxH6 activates the BLV LTR when introduced into HeLa cells by scrape loading. Furthermore, the purified BLV TaxH6 enhances binding of members of the CREB/ATF family of bZip proteins to CRE motifs by interacting with their bZip domains in vitro. Chemical cross-linking indicates that dimerization of bZip proteins such as CREB becomes greatly facilitated in the presence of BLV Tax. These results suggest that BLV Tax interacts directly with CREB/ATF-like factors to activate viral mRNA transcription.

Alpha-ketoamide Phe-Pro isostere as a new core structure for the inhibition of HIV protease.

Studies on the inhibition of HIV-1 protease utilizing a core isostere with replacement of the scissle bond for an alpha-amino-ketone have resulted in the development of an alpha-keto-amide isosteric replacement of the Phe-Pro scissle amide bond. The simple dipeptide isostere was shown to be a promising new core structure for the development of the enzyme inhibitors. The Ki of this core structure was determined to be 6 microM, compared to 230 microM and > 50 microM for the corresponding phosphinic acid and hydroxyethylamine isosteres.

Cytoplasmic forms of human T-cell leukemia virus type 1 Tax induce NF-kappaB activation.

Human T-cell leukemia virus type 1 (HTLV-1) Tax targets I-kappaB alpha and I-kappaB beta for phosphorylation, ubiquitination, and proteasome-mediated degradation, causing the nuclear translocation of NF-kappaB/Rel proteins and transcription induction of many cellular genes. The mechanism by which a nuclear protein such as Tax stimulates I-kappaB phosphorylation and degradation remains unclear. Here, we describe two cytoplasmic mutants of Tax, designated TaxDeltaN81 and TaxDeltaN109, from which the domains important for cyclic AMP response element binding factor (CREB) and serum response factor (SRF) binding and nuclear transport have been removed. These mutants were unable to trans activate from the HTLV-1 21-bp repeats or the serum response element in the c-fos promoter. In contrast, they activated NF-kappaB reporters, suggesting that activation of NF-kappaB by Tax occurs in the cytoplasm. Incorporation of the nuclear localization signal (NLS) of the simian virus 40 large T antigen into TaxDeltaN81 and TaxDeltaN109 redirected both proteins predominantly to the nucleus yet did not restore trans activation via CREB or SRF. The NLS fusion had little effect on TaxDeltaN81 but reduced NF-kappaB trans activation by TaxDeltaN109, possibly because of its proximity to the NF-kappaB-activating domain of Tax. In contrast to wild-type Tax, the cytoplasmic TaxDeltaN mutants are not cytotoxic. Stable expression of TaxDeltaN109 in HeLa cells resulted in a significant reduction in the intracellular level of I-kappaB alpha, with the constitutive presence of NF-kappaB in the nucleus and concomitant activation of the NF-kappaB enhancer. These results are suggestive of a potential application of the TaxDeltaN109-like mutants in targeting I-kappaB degradation and NF-kappaB activation. Interestingly, a Tax species with a molecular mass similar to that of TaxDeltaN109 was identified in many HTLV-1-transformed T cells, suggesting that TaxDeltaN109-like species might play a role in HTLV-1-induced leukemogenesis.