Burkholderia pseudomallei-induced expression of a negative regulator, sterile-alpha and Armadillo motif-containing protein, in mouse macrophages: a possible mechanism for suppression of the MyD88-independent pathway.

Burkholderia pseudomallei, a causative agent of melioidosis, is a Gram-negative facultative intracellular bacterium that can survive and multiply in macrophages. Previously, we demonstrated that B. pseudomallei failed to activate gene expression downstream of the MyD88-independent pathway, particularly the expression of beta interferon (IFN-ß) and inducible nitric oxide synthase (iNOS), leading to the inability of macrophages to kill this bacterium. In the present report, we extended our study to show that B. pseudomallei was able to activate sterile-α and Armadillo motif (SARM)-containing protein, a known negative regulator of the MyD88-independent pathway. Both live B. pseudomallei and heat-killed B. pseudomallei were able to upregulate SARM expression in a time-dependent manner in mouse macrophage cell line RAW 264.7. The expression of SARM required bacterial internalization, as it could be inhibited by cytochalasin D. In addition, the intracellular survival of B. pseudomallei was suppressed in SARM-deficient macrophages. Increased expression of IFN-ß and iNOS and degradation of IκBα correlated with enhanced macrophage killing capability. These results demonstrated that B. pseudomallei modulated macrophage defense mechanisms by upregulating SARM, thus leading to the suppression of IFN-ß and iNOS needed for bacterial elimination.

Involvement of the MyD88-independent pathway in controlling the intracellular fate of Burkholderia pseudomallei infection in the mouse macrophage cell line RAW 264.7.

Burkholderia pseudomallei is a facultative intracellular Gram-negative bacterium which is capable of surviving and multiplying inside macrophages. B. pseudomallei strain SRM117, a LPS mutant which lacks the O-antigenic polysaccharide moiety, is more susceptible to macrophage killing during the early phase of infection than is its parental wild type strain (1026b). In this study, it was shown that the wild type is able to induce expression of genes downstream of the MyD88-dependent (ikappabzeta, il-6 and tnf-alpha), but not of the MyD88-independent (inos, ifn-beta and irg-1), pathways in the mouse macrophage cell line RAW 264.7. In contrast, LPS mutant-infected macrophages were able to express genes downstream of both pathways. To elucidate the significance of activation of the MyD88-independent pathway in B. pseudomallei-infected macrophages, the expression of TBK1, an essential protein in the MyD88-independent pathway, was silenced prior to the infection. The results showed that silencing the tbk1 expression interferes with the gene expression profile in LPS mutant-infected macrophages and allows the bacteria to replicate intracellularly, thus suggesting that the MyD88-independent pathway plays an essential role in controlling intracellular survival of the LPS mutant. Moreover, exogenous IFN-gamma upregulated gene expression downstream of the MyD88-independent pathway, and interfered with intracellular survival in both wild type and tbk1-knockdown macrophages infected with either the wild type or the LPS mutant. These results suggest that gene expression downstream of the MyD88-independent pathway is essential in regulating the intracellular fate of B. pseudomallei, and that IFN-gamma regulates gene expression through the TBK1-independent pathway.

Involvement of B. pseudomallei RpoS in apoptotic cell death in mouse macrophages.

Burkholderia pseudomallei, the causative agent of melioidosis, is a facultative intracellular Gram-negative bacillus which can survive and multiply in both phagocytic and nonphagocytic cells. This bacterium could also induce apoptosis in various cell types. In the present study, we extend our finding to demonstrate the role of RpoS of B. pseudomallei in apoptosis induction. Unlike the wild-type strain, the B. pseudomallei rpoS mutant strain failed to induce cytotoxicity in mouse macrophages (RAW264.7). Furthermore, the mutant produced less extensive mitochondrial membrane potential changes and caspase-3 activation in the macrophages than did the wild-type strain. These data suggest that the RpoS of B. pseudomallei plays an essential role in the regulation of cell death in mouse macrophages.

Fate of a Burkholderia pseudomallei lipopolysaccharide mutant in the mouse macrophage cell line RAW 264.7: possible role for the O-antigenic polysaccharide moiety of lipopolysaccharide in internalization and intracellular survival.

Burkholderia pseudomallei is a facultative intracellular gram-negative bacterium that can survive and multiply inside macrophages. One of the mechanisms by which B. pseudomallei escapes macrophage killing is by interfering with the expression of inducible nitric oxide synthase (iNOS). However, the bacterial components that modulate antimicrobial activity of the macrophage have not been fully elucidated. In the present study, we demonstrated that B. pseudomallei strain SRM117, a lipopolysaccharide (LPS) mutant that lacks the O-antigenic polysaccharide moiety, was more susceptible to macrophage killing during the early phase of infection than the parental wild-type strain (1026b). Unlike the wild type, the LPS mutant could readily stimulate Y701-STAT-1 phosphorylation (pY701-STAT-1) and interferon-regulatory factor 1 (IRF-1) expression, both of which are essential transcription factors of iNOS. Neutralizing antibody against beta interferon was able to inhibit the phosphorylation of Y701-STAT-1 and the expression of IRF-1 and iNOS, all of which resulted in an increased rate of intracellular replication. These data suggest that the O-antigenic polysaccharide moiety of B. pseudomallei modulates the host cell response, which in turn controls the intracellular fate of B. pseudomallei inside macrophages.

Expression of suppressor of cytokine signaling 3 (SOCS3) and cytokine-inducible Src homology 2-containing protein (CIS) induced in Burkholderia pseudomallei--infected mouse macrophages requires bacterial internalization.

We recently reported that Burkholderia pseudomallei was able to activate the expression of suppressor of cytokine signaling 3 (SOCS3) and cytokine-inducible Src homology 2-containing protein (CIS). In the present study, we presented evidence showing that the induction of these negative regulators was most probably triggered from within rather than at the cell surface of mouse macrophage cell line (RAW264.7) suggesting that macrophage activation most likely requires the interaction of bacteria with a putative host cell cytoplasmic component(s).

Burkholderia pseudomallei RpoS regulates multinucleated giant cell formation and inducible nitric oxide synthase expression in mouse macrophage cell line (RAW 264.7).

Burkholderia pseudomallei is the causative agent of melioidosis. This bacterium can invade and survive inside the phagocytic and nonphagocytic cells. After internalization, the bacteria can escape from the membrane-bound phagosome into the cytoplasm. Internalised B. pseudomallei can also induce a cell-to-cell fusion, resulting in a multinucleated giant cell (MNGC) formation. In the present study, we demonstrated that B. pseudomallei rpoS null mutant was similar to its wild type parent in its ability to survive and multiply inside the mouse macrophages, but it failed to stimulate MNGC formation. The rpoS mutant was also unable to activate inducible Nitric Oxide Synthase (iNOS) in resting mouse macrophages but in gamma interferon (IFN-gamma)-activated macrophages, the mutant was able to induce significantly higher levels of iNOS and NO when compared with its wild-type counterpart, resulting in a significantly lower number of bacteria inside the infected host cells.

Burkholderia pseudomallei-induced expression of suppressor of cytokine signaling 3 and cytokine-inducible src homology 2-containing protein in mouse macrophages: a possible mechanism for suppression of the response to gamma interferon stimulation.

Burkholderia pseudomallei, the causative agent of melioidosis, is a facultative intracellular gram-negative bacterium that is able to survive and multiply in macrophages. Previously, we reported that B. pseudomallei was able to escape macrophage killing by interfering with the expression of inducible nitric oxide synthase (iNOS). In the present study, we extended this finding and demonstrated that B. pseudomallei was able to activate the expression of suppressor of cytokine signaling 3 (SOCS3) and cytokine-inducible Src homology 2-containing protein (CIS) but not SOCS1 in a mouse macrophage cell line (RAW 264.7). The expression of SOCS3 and CIS in B. pseudomallei-infected macrophages directly correlated with a decreased gamma interferon (IFN-gamma) signaling response, as indicated by a reduction in Y701-STAT-1 phosphorylation (pY701-STAT-1). Moreover, a reduction in the expression of IFN-gamma-induced proteins, such as interferon regulatory factor 1 (IRF-1), was observed in B. pseudomallei-infected macrophages that were treated with IFN-gamma. Since pY701-STAT-1 and IRF-1 are essential transcription factors for regulating iNOS expression, the failure to activate these factors could also result in depression of iNOS expression and a loss of macrophage killing capacity. Taken together, the data indicate that the activation of SOCS3 and CIS expression in B. pseudomallei-infected macrophages interfered with IFN-gamma signaling, thus allowing the bacteria to escape killing by these phagocytic cells.

Burkholderia pseudomallei invasion and activation of epithelial cells requires activation of p38 mitogen-activated protein kinase.

Burkholderia pseudomallei is a causative agent of melioidosis. This gram-negative bacterium is able to survive inside the macrophages and also able to invade non-phagocytic cells including epithelial cells. Interaction of pathogenic bacteria to the host cells is frequently associated with activation of mitogen-activated protein (MAP) kinases signaling activity. In this study, we demonstrated that B. pseudomallei stimulated p38 MAP kinase of human alveolar lung epithelial cell line (A549). Phosphorylation of p38 was observed after 15 min, attained a maximal level at 60 min after the infection. A specific inhibitor of p38 phosphorylation, SB 203580, was able to inhibit invasion of this bacterium into the cells suggesting that invasion of B. pseudomallei required activation of p38. In contrast, wortmannin which is a specific inhibitor of phosphoinositide 3-kinase (PI3-kinase) failed to inhibit the invasion. Moreover, SB 203580 can also interfere with IkappaBalpha degradation and IL-8 mRNA expression, indicating that the phosphorylation of p38 occurred upstream of NF-kappaB activation. Cytochalasin D, an inhibitor of actin polymerization needed for internalisation of bacteria, did not have any effect on the phosphorylation of p38. These results indicate that B. pseudomallei stimulate phosphorylation of p38 making by initial contact with the cell surface components and do not require internalisation and interaction with intracellular cytoplasmic components of the cells.

Burkholderia pseudomallei stimulates low interleukin-8 production in the human lung epithelial cell line A549.

Melioidosis is a life-threatening disease caused by Burkholderia pseudomallei. The lung is the most commonly affected organ, resulting in abscess formation in patients with chronic melioidosis. Previous study has shown that B. pseudomallei was able to invade and multiply in epithelial cells. In the present study, we have demonstrated that B. pseudomallei is able to stimulate interleukin 8 (IL-8) production from the human alveolar lung epithelium cell line A549. However, the level of IL-8 production was significantly lower than when the cells were infected with other Gram-negative bacteria such as Salmonella enterica serovar Typhi (S. typhi) which were used for comparison. The degree of IkappaBalpha degradation in the B. pseudomallei-infected cells was lower than that of the S. typhi-infected cells, suggesting that B. pseudomallei is also a poorer cell activator. Inhibition of B. pseudomallei invasion by cytochalasin D did not interfere with either IL-8 production or IkappaBalpha degradation, indicating that bacterial uptake is not required for the production of this chemokine. Thus, it appears that the signalling initiated by the interaction of B. pseudomallei with the epithelial cell surface is sufficient for epithelial cell activation.

Induction of iNOS expression and antimicrobial activity by interferon (IFN)-beta is distinct from IFN-gamma in Burkholderia pseudomallei-infected mouse macrophages.

Burkholderia pseudomallei is a causative agent of melioidosis. This Gram-negative bacterium is able to survive and multiple inside both phagocytic and nonphagocytic cells. We previously reported that exogenous interferons (both type I and type II) enhanced antimicrobial activity of the macrophages infected with B. pseudomallei by up-regulating inducible nitric oxide synthase (iNOS). This enzyme thus plays an essential role in controlling intracellular growth of bacteria. In the present study we extended our investigation, analysing the mechanism(s) by which the two types of interferons (IFNs) regulate antimicrobial activity in the B. pseudomallei-infected macrophages. Mouse macrophage cell line (RAW 264.7) that was exposed simultaneously to B. pseudomallei and type I IFN (IFN-beta) expressed high levels of iNOS, leading to enhanced intracellular killing of the bacteria. However, neither enhanced iNOS expression nor intracellular bacterial killing was observed when the macrophages were preactivated with IFN-beta prior to being infected with B. pseudomallei. On the contrary, the timing of exposure was not critical for the type II IFN (IFN-gamma) because when the cells were either prestimulated or co-stimulated with IFN-gamma, both iNOS expression and intracellular killing capacity were enhanced. The differences by which these two IFNs regulate antimicrobial activity may be related to the fact that IFN-gamma was able to induce more sustained interferon regulatory factor-1 (IRF-1) expression compared with the cells activated with IFN-beta.

Involvement of beta interferon in enhancing inducible nitric oxide synthase production and antimicrobial activity of Burkholderia pseudomallei-infected macrophages.

Burkholderia pseudomallei is the causative agent of melioidosis, a life-threatening disease that affects both humans and animals. This bacterium is able to survive and multiply inside both phagocytic and nonphagocytic cells. We recently reported that mouse macrophages infected with B. pseudomallei fail to produce a significant level of inducible nitric oxide synthase (iNOS), a crucial enzyme needed for the cells to control the intracellular growth of this bacterium. In the present study, we extended our investigation to demonstrate that, unlike other gram-negative bacteria that have been investigated, B. pseudomallei only minimally activates beta interferon (IFN-beta) production; this minimal activation leads to a low level of interferon regulating factor 1 (IRF-1) in the macrophages, in parallel with poor iNOS expression. Adding exogenous IFN-beta to the system could upregulate IRF-1 production, which in turn could enhance iNOS expression in the B. pseudomallei-infected macrophages and lead to suppression of the intracellular growth of this bacterium. Taken together, these results imply that the failure of macrophages to successfully control the growth and survival of intracellular B. pseudomallei is related, at least in part, to the defective production of IFN-beta, which modulates the ability of macrophages to synthesize iNOS.

CpG ODN enhances uptake of bacteria by mouse macrophages.

Unmethylated CpG motif in synthetic oligodeoxynucleotide (CpG ODN) or bacterial DNA is well recognized for its role in innate immunity, including enhancing production of NO and cytokines by macrophages. In the present study, we demonstrated the effect of CpG ODN on the phagocytic uptake of bacteria by macrophages. Flow cytometric analysis of mouse macrophages (RAW 264.7) incubated with fluorescein isothiocyanate (FITC)-labelled Burkholderia pseudomallei, Salmonella enterica serovar Typhi or Escherichia coli showed that CpG ODN increased the uptake of these bacteria by mouse macrophages. The enhancement of bacterial uptake by CpG ODN was concentration-dependent. The increase of bacterial uptake by CpG ODN-activated macrophages shown above is consistent with the result of bacteria internalization study using a standard antibiotic protection assay. There was also an increase in the rate and degree of multi-nucleated giant cell formation, phenomena which have been shown previously to be unique when the cells were infected with B. pseudomallei. These observations may provide significant insights for future investigation into host cell-pathogen interaction.

CpG ODN activates NO and iNOS production in mouse macrophage cell line (RAW 264.7).

Synthetic CpG containing oligodeoxynucleotide (CpG ODN) is recognized for its ability to activate cells to produce several cytokines, such as IL-12 and TNF-alpha. In the present study we have demonstrated that CpG ODN 1826, known for its immunostimulatory activity in the mouse system could, by itself, induce nitric oxide (NO) and inducible nitric oxide synthase (iNOS) production from mouse macrophage cell line (RAW 264.7). Neutralizing antibody against TNF-alpha was not able to inhibit NO or iNOS production from the CpG ODN 1826-activated macrophages, suggesting that although the TNF-alpha was also produced by CpG ODN-activated macrophages, the production of iNOS was not mediated through TNF-alpha. Although both CpG ODN 1826 and lipopolysaccharide (LPS) were able to stimulate NO and iNOS production, the exposure time required for maximum production of NO and iNOS for the CpG ODN 1826-activated macrophages was significantly longer than those activated with LPS. These results were due probably to a delay of NF-kappaB translocation, as indicated by the delay of IkappaBalpha degradation. Moreover, the fact that chloroquine abolished NO and iNOS production from the cells treated with CpG ODN 1826 but not from those treated with LPS suggested that the induction of NO and iNOS production from the cells stimulated with CpG ODN (1826) also required endosomal maturation/acidification.

A radical form of nitric oxide suppresses RNA synthesis of rabies virus.

Hydrophobia is an incurable disease of the central nervous system. Therefore, every mode of the immune response is important to inhibit and clear infection. Innate immunity such as nitric oxide is significantly upregulated during rabies virus infection in vivo. In this report, the possible role of nitric oxide in inhibition of rabies virus replication was studied. Rabies virus infected neuroblastoma cells were treated with nitric oxide generated from SNP or SNP in the presence of ascorbate. SNP-ascorbate generates mainly NO* in culture medium while NO(+) is the major product of SNP alone. Treatment with SNP-ascorbate resulted in delay and suppression of infectious viral particle production. In contrast, treatment with SNP alone did not interfere with multiplication of this virus. The mechanism of inhibition by NO was at the level of gene expression, which was demonstrated by reduction in the level of N, G and L gene expression. The effect of SNP-ascorbate generated NO on rabies virus protein synthesis was also investigated. Synthesis of N protein in the presence of NO was suppressed which correlated to down regulation of N gene expression. We hypothesize that one of the roles of NO in the central nervous system during rabies virus infection is to limit viral dissemination by down-regulating rabies virus production through transcription inhibition.

Burkholderia pseudomallei interferes with inducible nitric oxide synthase (iNOS) production: a possible mechanism of evading macrophage killing.

Burkholderia pseudomallei is a causative agent of melioidosis, a life threatening disease which affects humans and animals in tropical and subtropical areas. This bacterium is known to survive and multiply inside cells such as macrophages. The mechanism of host defense against this bacterium is still unknown. In this study, we demonstrated that B. pseudomallei exhibited unique macrophage activation activity compared with Escherichia coli and Salmonella typhi. The mouse macrophage cell line (RAW 264.7) infected with B. pseudomallei at MOI of 0.1:1, 1:1 and 10:1 did not express a detectable level of inducible nitric oxide synthase (iNOS). Moreover, the B. pseudomallei infected cells released TNF-alpha only when they were infected with high MOI (10:1). Unlike the cells infected with B. pseudomallei, the cells infected with E. coli, and S. typhi expressed iNOS even at MOI of 0.1:1. These infected cells also released a significantly higher level of TNF-alpha at the low MOI ratio. The cells that were preactivated with IFN-gamma prior to being infected with B. pseudomallei exhibited an enhanced production of iNOS and TNF-alpha release. The increased macrophage activation activity in the presence of IFN-gamma also correlated with the restriction of the intracellular bacteria survival. Moreover, IFN-gamma also prevented cell fusion and multinucleated cell formation induced by B. pseudomallei, a phenomenon recently described by our group. Altogether, these results indicate that internalization of B. pseudomallei failed to trigger substantial macrophage activation, a phenomenon which could prolong their survival inside the phagocytic cells and facilitate a direct cell to cell spreading of B. pseudomallei to neighboring cells.

Kinetic studies of the production of nitric oxide (NO) and tumour necrosis factor-alpha (TNF-alpha) in macrophages stimulated with Burkholderia pseudomallei endotoxin.

The mechanism by which Burkholderia pseudomallei survives in macrophages is not clearly understood. In this study, we demonstrated that the mouse macrophage cell line (RAW 264.7) treated with lipopolysaccharide (LPS) from B. pseudomallei (BP-LPS) produced significantly less NO and TNF-alpha compared with those stimulated with the LPS from Escherichia coli and Salmonella typhi. The time required for the BP-LPS to trigger substantial NO and TNF-alpha release was at least 30 min, compared with < 5 min for the E. coli-LPS. A time course study of inducible nitric oxide synthase (iNOS) protein expression also indicated that the time required for macrophages stimulated with the BP-LPS to up-regulate iNOS was longer. The longer time lag for the BP-LPS to activate macrophages was probably due to the delay in up-regulation of iNOS and TNF-alpha mRNA transcription. These results indirectly suggest that the delay of the mediators' production may be due to a reduced rate of signal transduction initiated by the interaction of BP-LPS with the macrophage cell surface. The use of MoAb to phosphorylated p38 in a Western blot analysis provided data compatible with the notion that the maximum level of phosphorylated p38 from the cells activated with BP-LPS was attained at a slower rate. These results suggest that the unique structure of BP-LPS exhibits a property which may interfere with macrophage cell activation.

TNF-alpha induces caspase 3 (CPP 32) dependent apoptosis in human cholangiocarcinoma cell line.

Cholangiocarcinoma (CCA), a malignant tumor derived from bile duct epithelium, occurs with a higher incidence in tropical countires especially in some areas of Southeast Asian countries such as Thailand. This tumor is relatively resistant to chemotherapy. In this study, molecular mechanism of killing of this tumor by TNF-alpha was investigated. Human cholangiocarcinoma cell line (HuCCA-1) was developed and used as a model for treatment. Activation of HuCCA-1 with TNF-alpha in the present of actinomycin D (1 microg/ml) caused death of the tumor cells. Western blotting analysis of the cells extracted demonstrated the cleavage of poly (ADP-ribose) polymerase (PARP) within 6-8 hours following TNF-alpha treatment indicating apoptotic death. The cleavage of PARP was inhibited when the cell line was pretreated with peptide inhibitor, Ac-DEVD-CHO, suggesting that apoptosis induced by TNF-alpha of this cell line involves activation of caspase II subfamily. The procaspase 3 (proCPP-32), one of the caspase group II subfamily was degraded after the HuCCA- I cell line was treated with TNF-alpha. Furthermore, Gelsolin, an 83 kDa protein which is identified as caspase 3 substrate, was cleaved to 43 kDa fragments after the cells were treated with TNF-alpha. These results indicate that apoptosis of human cholangiocarcinoma cell line as induced by TNF-alpha treatment is mediated through caspase 3.

Binding of tumour necrosis factor-alpha (TNF-alpha) to TNF-RI induces caspase(s)-dependent apoptosis in human cholangiocarcinoma cell lines.

Cholangiocarcinoma (CCA), a tumour of the bile duct epithelium, occurs with a higher incidence in South-east Asian countries than in Europe and North America. The prognosis is poor, due to the unavailability of early diagnosis and the tumours being relatively resistant to chemotherapy. In the present study one of the fatal routes of this tumour was studied. This death was stimulated by TNF-alpha. TNF-alpha at a concentration of 760 pg/ml and 100 pg/ml in the presence of 1 microgram/ml actinomycin D induced 50% cell death of the two established human cholangiocarcinoma cell lines HuCCA-1 and HuCCA-INu, respectively. Preincubation of both cell lines with MoAb to TNF-RI or TNF-RII before TNF-alpha treatment showed that only the MoAb specific to TNF-RI inhibited death. The death of these two cell lines was proved to be apoptosis. Western blot analysis of extracts from both cell lines demonstrated a cleavage of poly (ADP-ribose) polymerase (PARP) within 6-8 h following TNF-alpha treatment. The degradation of PARP was prevented by a MoAb to TNF-RI indicating that the TNF-RI but not TNF-RII was involved in TNF-induced apoptosis in these two human cholangiocarcinoma cell lines. Moreover, peptide inhibitor for caspase II subfamily, Ac-DEVD-CHO, reduced the cytolysis of TNF-alpha-treated cholangiocarcinoma cells. The inhibitor also prevented degradation of PARP. These results indicate that the interaction between TNF-alpha and TNF-RI alone generated a sufficient signal to activate a caspase II subfamily-dependent apoptosis in human cholangiocarcinoma cell lines.

Rabies virus replication induces Bax-related, caspase dependent apoptosis in mouse neuroblastoma cells.

Rabies virus has been shown to induce apoptosis in infected cells, but the intracellular pathway of cell killing is unknown. In this report, we show that rabies virus infected mouse neuroblastoma cells underwent chromatin condensation and DNA fragmentation within 48 h post-infection. An increased level of the apoptotic enhancer, Bax, was detected within 24 h after infection. In contrast to Bax, the production of the apoptotic antagonist, Bcl-2, remained unchanged. Shortly after detection of Bax, caspase 1 (ICE) was upregulated. Reduction of DNA fragmentation in rabies virus infected cultures pretreated with YVAD and DEVD suggested that more than one subfamily of caspase functioned in the death process. Significant degradation of the DNA repair enzyme, poly ADP-ribose polymerase (PARP), was revealed after caspase upregulation. This study showed that replication of rabies viruses in mouse neuroblastoma cells induced the Bax-related death program leading to destruction of the DNA repair system probably by caspase activity.

Transactivation by the human T-cell leukemia virus Tax protein is mediated through enhanced binding of activating transcription factor-2 (ATF-2) ATF-2 response and cAMP element-binding protein (CREB).

The human T-cell leukemia virus type I (HTLV-I)-encoded transcriptional activator protein Tax is strongly implicated in HTLV-I pathogenesis. Tax regulates HTLV-I gene expression through three 21-base pair (bp) repeat enhancer elements located in the transcriptional control region of the virus. Tax does not bind these elements directly, but mediates transactivation through the cellular transcription factors that recognize a cAMP response element (CRE)-like sequence centered within each of the 21-bp repeats. In this report, we identify activating transcription factor-2 (ATF-2) and CRE-binding protein (CREB) as the principal T-cell proteins that bind the three 21-bp repeats in vitro. Purified Tax protein augments the level of RNA synthesis induced by ATF-2 and CREB in a cell-free transcription assay, providing evidence that Tax cooperates with these cellular proteins to activate HTLV-I transcription. Furthermore, Tax dramatically increases the binding of both the T-cell-derived and recombinant forms of ATF-2 and CREB to each of the 21-bp repeats. The target sequences for this enhancement reside within the DNA binding/dimerization domains of these proteins. These data suggest that Tax transactivates HTLV-I gene expression by increasing the number of bound ATF-2 and CREB molecules at the viral promoter.