HBZ-mediated shift of JunD from growth suppressor to tumor promoter in leukemic cells by inhibition of ribosomal protein S25 expression.

Human T-cell leukemia virus type 1 (HTLV-1) basic-leucine zipper (bZIP) factor (HBZ) is a key player in proliferation and transformation of HTLV-1-infected cells, thus contributing to adult T-cell leukemia (ATL) development. HBZ deregulates gene expression within the host cell by interacting with several cellular partners. Through its C-terminal ZIP domain, HBZ is able to contact and activate JunD, a transcription factor of the AP-1 family. JunD mRNA is intronless but can generate two protein isoforms by alternative translation initiation: JunD full-length and Δ JunD, an N-terminal truncated form unresponsive to the tumor suppressor menin. Using various cell lines and primary T-lymphocytes, we show that after serum deprivation HBZ induces the expression of Δ JunD isoform. We demonstrate that, unlike JunD, Δ JunD induces proliferation and transformation of cells. To decipher the mechanisms for Δ JunD production, we looked into the translational machinery and observed that HBZ induces nuclear retention of RPS25 mRNA and loss of RPS25 protein expression, a component of the small ribosomal subunit. Therefore, HBZ bypasses translational control of JunD uORF and favors the expression of Δ JunD. In conclusion, we provide strong evidences that HBZ induces Δ JunD expression through alteration of the cellular translational machinery and that the truncated isoform Δ JunD has a central role in the oncogenic process leading to ATL.

Interaction between C/EBPbeta and Tax down-regulates human T-cell leukemia virus type I transcription.

The human T-cell leukemia virus type I (HTLV-I) Tax protein trans-activates viral transcription through three imperfect tandem repeats of a 21-bp sequence called Tax-responsive element (TxRE). Tax regulates transcription via direct interaction with some members of the activating transcription factor/CRE-binding protein (ATF/CREB) family including CREM, CREB, and CREB-2. By interacting with their ZIP domain, Tax stimulates the binding of these cellular factors to the CRE-like sequence present in the TxREs. Recent observations have shown that CCAAT/enhancer binding protein beta (C/EBPbeta) forms stable complexes on the CRE site in the presence of CREB-2. Given that C/EBPbeta has also been found to interact with Tax, we analyzed the effects of C/EBPbeta on viral Tax-dependent transcription. We show here that C/EBPbeta represses viral transcription and that Tax is no more able to form a stable complex with CREB-2 on the TxRE site in the presence of C/EBPbeta. We also analyzed the physical interactions between Tax and C/EBPbeta and found that the central region of C/EBPbeta, excluding its ZIP domain, is required for direct interaction with Tax. It is the first time that Tax is described to interact with a basic leucine-zipper (bZIP) factor without recognizing its ZIP domain. Although unexpected, this result explains why C/EBPbeta would be unable to form a stable complex with Tax on the TxRE site and could then down-regulate viral transcription. Lastly, we found that C/EBPbeta was able to inhibit Tax expression in vivo from an infectious HTLV-I molecular clone. In conclusion, we propose that during cell activation events, which stimulate the Tax synthesis, C/EBPbeta may down-regulate the level of HTLV-I expression to escape the cytotoxic-T-lymphocyte response.

Activation of HTLV-I transcription in the presence of Tax is independent of the acetylation of CREB-2 (ATF-4).

We have previously demonstrated that the bZIP transcription factor CREB-2, also called ATF-4, trans-activates, in association with the viral protein Tax, the human T-cell leukemia virus type I (HTLV-I) promoter. In this study, we have examined whether CREB-2 acetylation affects transcriptional activation mediated by Tax. We present evidence that CREB-2 is acetylated in vitro and in vivo. CREB-2 is acetylated in two regions: the basic domain of the bZIP (from amino acid residue 270 to 300) and the short basic domain (from 342 to 351) located downstream from the bZIP. We also demonstrate that CREB-2 is acetylated by p300/CBP but not by p/CAF. Moreover, replacement of lysine by arginine in the basic domains decreases the trans-activating capacity of CREB-2. However, in the presence of Tax, the HTLV-I transcription remains fully activated by these CREB-2 mutants. Although we cannot totally exclude that the mutations could also affect CREB-2 structure and activity independent of acetylation, our results suggest that activation of the viral promoter in the presence of Tax is independent of the CREB-2 acetylation.

How the sequestration of a protein interferes with its mechanism of action: example of a new family of proteins characterized by a particular cysteine-rich carboxy-terminal domain involved in gene expression regulation.

We describe here a new family of proteins characterized by a particular cysteine-rich carboxy-terminal domain and involved in gene expression regulation. This family presently includes three members: I-mfa (inhibitor of MyoD family), HIC p40 and HIC p32 (human I-mfa domain-containing protein). I-mfa, by interacting with MyoD family members, represses both transcriptional activation and myogenesis mediated by these factors. HIC two isoforms, HIC p40 and HIC p32, are involved in the positive regulation of Tax-mediated HTLV-I (human T-cell leukemia virus type 1) promoter activation and in the negative regulation of Tat-mediated HIV-1 (human immunodeficiency virus type 1) promoter transcription. The common carboxy-terminal region of HIC p40 and HIC p32, which is clearly involved in these regulations, shares 77% homology with the carboxy-terminal domain of I-mfa. This suggests that I-mfa, HIC p40 and HIC p32 are part of a new family of proteins involved in gene expression regulation and characterized by a specific cysteine-rich carboxy-terminal domain. Moreover, the three proteins present different subcellular localizations: I-mfa and HIC p32 are mainly cytoplasmic while HIC p40 is mainly nucleolar. The specific localization of each member of this new family will be discussed, possibly explaining how they work. Effectively, a mechanism of protein sequestration in a particular compartment, cytoplasm or nucleolus, could be involved in their function, as it is the case for many other proteins. This relationship between sequestration and function regulation will be exemplified for several cellular factors.

Molecular interactions involved in the transactivation of the human T-cell leukemia virus type 1 promoter mediated by Tax and CREB-2 (ATF-4).

The human T-cell leukemia virus type 1 (HTLV-1) Tax protein activates viral transcription through three 21-bp repeats located in the U3 region of the HTLV-1 long terminal repeat and called Tax-responsive elements (TxREs). Each TxRE contains nucleotide sequences corresponding to imperfect cyclic AMP response elements (CRE). In this study, we demonstrate that the bZIP transcriptional factor CREB-2 is able to bind in vitro to the TxREs and that CREB-2 binding to each of the 21-bp motifs is enhanced by Tax. We also demonstrate that Tax can weakly interact with CREB-2 bound to a cellular palindromic CRE motif such as that found in the somatostatin promoter. Mutagenesis of Tax and CREB-2 demonstrates that both N- and C-terminal domains of Tax and the C-terminal region of CREB-2 are required for direct interaction between the two proteins. In addition, the Tax mutant M47, defective for HTLV-1 activation, is unable to form in vitro a ternary complex with CREB-2 and TxRE. In agreement with recent results suggesting that Tax can recruit the coactivator CREB-binding protein (CBP) on the HTLV-1 promoter, we provide evidence that Tax, CREB-2, and CBP are capable of cooperating to stimulate viral transcription. Taken together, our data highlight the major role played by CREB-2 in Tax-mediated transactivation.

Molecular cloning of a novel human I-mfa domain-containing protein that differently regulates human T-cell leukemia virus type I and HIV-1 expression.

Regulation of viral genome expression is the result of complex cooperation between viral proteins and host cell factors. We report here the characterization of a novel cellular factor sharing homology with the specific cysteine-rich C-terminal domain of the basic helix-loop-helix repressor protein I-mfa. The synthesis of this new factor, called HIC for Human I-mfa domain-Containing protein, is controlled at the translational level by two different codons, an ATG and an upstream non-ATG translational initiator, allowing the production of two protein isoforms, p32 and p40, respectively. We show that the HIC protein isoforms present different subcellular localizations, p32 being mainly distributed throughout the cytoplasm, whereas p40 is targeted to the nucleolus. Moreover, in trying to understand the function of HIC, we have found that both isoforms stimulate in T-cells the expression of a luciferase reporter gene driven by the human T-cell leukemia virus type I-long terminal repeat in the presence of the viral transactivator Tax. We demonstrate by mutagenesis that the I-mfa-like domain of HIC is involved in this regulation. Finally, we also show that HIC is able to down-regulate the luciferase expression from the human immunodeficiency virus type 1-long terminal repeat induced by the viral transactivator Tat. From these results, we propose that HIC and I-mfa represent two members of a new family of proteins regulating gene expression and characterized by a particular cysteine-rich C-terminal domain.

Sequence requirement for the nucleolar localization of human I-mfa domain-containing protein (HIC p40).

The human I-mfa domain-containing protein (HIC) mRNA produces two protein isoforms, HIC p32 and p40, synthesized from alternative translational initiations. p32 translation is initiated from a standard AUG codon and p40 is an N-terminal extension of p32 generated from an upstream GUG codon. The two isoforms show different subcellular localization: p32 is distributed throughout the cytoplasm whereas p40 can be found both in the cytoplasm and the nucleolus. To investigate the possibility that p40 contains a nucleolus targeting sequence in its N-terminal region, COS cells were transfected with an eukaryotic expression vector coding for green fluorescent protein (GFP) fused to the p40 N terminus. The localization of this fusion protein in the nucleolus indicated that the N-terminal amino acids of p40 probably contain a nucleolar localization signal (NoLS). To find the structural motifs required for nucleolar localization of p40, deletion mutants were expressed in COS cells as fusion polypeptides with GFP. We defined a domain of 19 amino acids near the N terminus that contains an arginine-rich subdomain that conforms to other known NoLS. To demonstrate that this sequence is an authentic NoLS, the sequence was fused to GFP. This fusion protein was observed to migrate into the nucleolus. Taken together, our studies demonstrate that p40 contains a NoLS.

CREB-2, a cellular CRE-dependent transcription repressor, functions in association with Tax as an activator of the human T-cell leukemia virus type 1 promoter.

The Tax protein of the human T-cell leukemia virus type 1 (HTLV-1) has been implicated in human T-cell immortalization. The primary function of Tax is to transcriptionally activate the HTLV-1 promoter, but Tax is also known to stimulate expression of cellular genes. It has been reported to associate with several transcription factors, as well as proteins not involved in transcription. To better characterize potential cellular targets of Tax present in infected cells, a Saccharomyces cerevisiae two-hybrid screening was performed with a cDNA library constructed from the HTLV-1-infected MT2 cell line. From this study, we found 158 positive clones representing seven different cDNAs. We focused our attention on the cDNA encoding the transcription factor CREB-2. CREB-2 is an unconventional member of the ATF/CREB family in that it lacks a protein kinase A (PKA) phosphorylation site and has been reported to negatively regulate transcription from the cyclic AMP response element of the human enkephalin promoter. In this study, we demonstrate that CREB-2 cooperates with Tax to enhance viral transcription and that its basic-leucine zipper C-terminal domain is required for both in vitro and in vivo interactions with Tax. Our results confirm that the activation of the HTLV-1 promoter through Tax and factors of the ATF/CREB family is PKA independent.

Activation of E2F-mediated transcription by human T-cell leukemia virus type I Tax protein in a p16(INK4A)-negative T-cell line.

The human T-cell leukemia virus type I (HTLV-I) is a causative agent of adult T-cell leukemia. Although the exact mechanism by which HTLV-I contributes to leukemogenesis is still unclear, the Tax protein is thought to play a major role in this process. This 40-kDa polypeptide is able to interact with the tumor suppressor p16(INK4A). Consequently, Tax can activate the signaling pathway that lead to the release of E2F that in turn induces expression of factors required for cell cycle progression. In this paper, we demonstrate that Tax can also activate E2F-mediated transcription independently of p16(INK4A). Indeed, when Tax is coexpressed with the E2F-1 transcription factor in CEM T-cells, which lack expression of p16(INK4A), it strongly potentiates the E2F-dependent activation of a reporter construct driven by a promoter containing E2F binding sites. This stimulation is abrogated by mutations affecting the E2F-binding sites. In addition, Tax also stimulates the transcription of the E2F-1 gene itself. Using Tax mutants that fail to activate either ATF- or NF-kappaB-dependent promoters and different 5' truncation mutants of the E2F-1 promoter, we show that the Tax-dependent transcriptional control of the E2F1 gene involves, at least in part, the ATF binding site located in the E2F-1 promoter.

The Ick protein tyrosine kinase is not involved in antibody-mediated CD4 (CDR3-loop) signal transduction that inhibits HIV-1 transcription.

Monoclonal antibodies (mAb) that bind to the immunoglobulin CDR3-like region in the D1 domain of the CD4 molecule can inhibit the HIV-1 life cycle in CD4-positive T cells and lymphoblastoid cell lines at the stage of transcription. This antiviral effect requires the integrity of the cytoplasmic tail of CD4 which is known to act as a signal transduction region through its association with the protein tyrosine kinase (PTK) p56lck. In this study, we investigated the putative role of this PTK in transducing inhibitory signals that act on HIV-1 replication after triggering by anti-CDR3-like region antibody treatment of infected T cell lines. CEM (CD4+/p56lck + inducible), MT2 (CD4+/p56lck - repressed), HSB-2 (CD4-/p56lck + constitutively), HSB-2 WTCD4 (CD4+/p56lck + constitutively), HSB-2 CD4.402 (CD4+ truncated form which lacks the cytoplasmic domain/p56lck + constitutively), and HSB-2 CD4mut (CD4+ unable to bind lck/p56lck + constitutively) were exposed to HIV-1 and cultured in medium supplemented with an anti-CDR3-like region-specific antibody or a control anti-CD4 mAb which does not inhibit HIV-1 transcription. We found that CDR3-loop-mediated inhibitory signals are efficiently transduced in CD4-positive cells which demonstrate a constitutive activation of p56lck or in CD4-positive cells lacking p56lck expression. Moreover, inhibitory signals were transduced in HSB-2 CD4mut cells expressing a cell surface CD4 with a double cysteine mutation in its cytoplasmic tail that renders the molecule unable to bind p56lck, but not HSB-2 CD4.402 cells expressing a truncated form of CD4 which lacks the cytoplasmic domain. These results indicate that the p56lck plays no direct role in this process and suggests the existence of another signaling partner for CD4.

Comparison of packaging strategy in retroviruses and pararetroviruses.

Reverse transcription is not solely a retroviral mechanism. Animal hepadnaviruses, plant caulimoviruses, and badnaviruses have a RNA intermediate which is reverse transcribed into double-stranded DNA genome. Based on this fact, these three groups of viruses have been regrouped under the name of pararetroviruses. Although each one has developed its own strategy to assure an efficient packaging of their genome, it is clear that they have adopted a strategy where encapsidation prepares for initiation of reverse transcription. This is discussed in this review.

The cowpea mosaic virus RNA 1-encoded 112 kDa protein may function as a VPg precursor in vivo.

Processing of the 112 kDa ('112K') protein encoded by cowpea mosaic virus RNA 1 was examined in cowpea mesophyll protoplasts using a transient expression system. Cleavage of the 112K protein occurred via two alternative pathways either into VPg and 110K (24K + 87K) or into 26K (VPg + 24K) and 87K proteins. The 26K protein can be further cleaved into VPg and 24K proteins. The results support a model in which the 112K protein functions as the precursor of VPg during initiation of replication.

Sequence of a cauliflower mosaic virus strain infecting solanaceous plants.

The complete nucleotide sequence (8031 bp) of the DNA of cauliflower mosaic virus (CaMV) strain B29 is reported. This strain is unusual, since it infects both cruciferous and solanaceous plants. So far, from data of sequence comparisons between B29 and other CaMV strains there is no evidence for any obvious correlation between host range and distinct sequence features.

The full-length product of cauliflower mosaic virus open reading frame III is associated with the viral particle.

The gene III product (P15) of cauliflower mosaic virus (CaMV) is a DNA binding protein in which the DNA binding activity is located on its C-terminal part. In previous work, a C-terminal processed form of P15 (P11) was detected in purified viral particles as a minor component. The full-length P15 was shown to be present and to be matured, possibly by a cysteine proteinase, in CaMV replication complexes isolated from infected turnip leaves. In this paper, we have shown that a virion-enriched fraction obtained from such replication complexes by size exclusion chromatography contained most of the P15 in its uncleaved form and was enriched in the activity responsible for its proteolysis. This enabled us to characterize better the proteinase activity (temperature and pH optimum; effect of specific inhibitors) responsible for P15 cleavage and to confirm that it corresponds to a cysteine proteinase. Based upon these observations, a purification procedure for CaMV particles was devised which impaired the cleavage of P15 into P11 and allowed the isolation of virions containing almost exclusively the noncleaved form. This finding supports our hypothesis that the CaMV gene III product could be involved in the folding of the viral genome during encapsidation.

Identification of C-terminal amino acid residues of cauliflower mosaic virus open reading frame III protein responsible for its DNA binding activity.

We cloned in Escherichia coli truncated versions of the protein p15 encoded by open reading frame III of cauliflower mosaic virus. We then compared the ability of the wild-type p15 (129 amino acids) and the deleted p15 to bind viral double-stranded DNA genome. Deletions of > 11 amino acids in the C-terminal proline-rich region resulted in loss of DNA binding activity of wild-type p15. Moreover, a point mutation of the proline at position 118 sharply reduced the interaction between the viral protein and DNA. These results suggest that cauliflower mosaic virus p15 belongs to the family of DNA binding proteins having a proline-rich motif involved in interaction with double-stranded DNA.

Characterization of different electrophoretic forms of cauliflower mosaic virus virions (strain Cabb-S).

The electrophoretic forms of purified cauliflower mosaic virus (CaMV), strain Cabb-S, were examined by electrophoresis on agarose gels. Three populations of viral particles were identified: a faster migrating component (the form F) and two slower migrating components (the forms S and S'). When the different forms of virions, after excision from gels, were subjected to analysis in SDS-polyacrylamide gel, the fast component consisted of the 37 and 42 kDa coat proteins whereas the slow components contained mainly the 39 kDa coat protein. However, there was no difference among the nucleic acids associated within the three forms. The biological significance of the different components is discussed.

Processing of the minor capsid protein of the cauliflower mosaic virus requires a cysteine proteinase.

The major capsid protein of the cauliflower mosaic virus (CaMV) is processed in vivo. The viral aspartic proteinase that catalyses this maturation has been characterized previously and is coded by the CaMV gene V. This virus has a second capsid protein, a minor component, encoded by gene III. This protein, P3, is also processed at its C-terminus in vivo. To determine whether P3 is matured by the CaMV proteinase P5, we expressed, in Saccharomyces cerevisiae, P3, P5 and a fusion protein P7-P4, containing potential sites of cleavage. P5 was found to be involved in maturation of P7-P4 but did not cleave P3. The latter result was confirmed by experiments carried out with an in vitro translation system (the reticulocyte lysate) and with preparations of replication complexes purified from infected plants. Moreover, [N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leu cyl]-amido(4-guanido)butane, a specific inhibitor of cysteine proteinases, inhibited the maturation of P3, suggesting that the two CaMV capsid proteins are not processed by the same proteolytic event.

How do viral reverse transcriptases recognize their RNA genome?

Reverse transcription is not solely a retroviral mechanism. Hepadnaviruses and caulimoviruses have RNA intermediates that are reverse transcribed into DNA. Moreover non-viral retroelements, retrotransposons, use reverse transcription in their transposition. All these retroelements encode reverse transcriptase but each group developed their own expression modes capable of assuring a specific and efficient replication of their genomes.

Expression of cauliflower mosaic virus gene I in Saccharomyces cerevisiae.

Cauliflower mosaic virus (CaMV) gene I encodes a 40-kDa protein, P1, which is thought to be involved in the cell-to-cell movement of the virus. In order to investigate its functioning, P1 was expressed in Saccharomyces cerevisiae transformed by an expression vector containing CaMV gene I. When produced in yeast, PI was 40 kDa in size and not N-glycosylated.