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.

Retrovirus-induced target cell activation in the early phases of infection: the mouse mammary tumor virus model.

Mouse mammary tumor virus (MMTV) infects B lymphocytes and expresses a superantigen on the cell surface after integration of its reverse-transcribed genome. Superantigen-dependent B- and T-cell activation becomes detectable 2 to 3 days after infection. We show here that before this event, B cells undergo a polyclonal activation which does not involve massive proliferation. This first phase of B-cell activation is T cell independent. Moreover, during the first phase of activation, when only a small fraction of B cells is infected by MMTV(SW), viral DNA is detected only in activated B cells. Such a B-cell activation is also seen after injection of murine leukemia virus but not after injection of vaccinia virus, despite the very similar kinetics and intensity of the immune response. Since retroviruses require activated target cells to induce efficient infection, these data suggest that the early polyclonal retrovirus-induced target cell activation might play an important role in the establishment of retroviral infections.

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.

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.

The complementary strand of the human T-cell leukemia virus type 1 RNA genome encodes a bZIP transcription factor that down-regulates viral transcription.

The RNA genome of the human T-cell leukemia virus type 1 (HTLV-1) codes for proteins involved in infectivity, replication, and transformation. We report in this study the characterization of a novel viral protein encoded by the complementary strand of the HTLV-1 RNA genome. This protein, designated HBZ (for HTLV-1 bZIP factor), contains a N-terminal transcriptional activation domain and a leucine zipper motif in its C terminus. We show here that HBZ is able to interact with the bZIP transcription factor CREB-2 (also called ATF-4), known to activate the HTLV-1 transcription by recruiting the viral trans-activator Tax on the Tax-responsive elements (TxREs). However, we demonstrate that the HBZ/CREB-2 heterodimers are no more able to bind to the TxRE and cyclic AMP response element sites. Taking these findings together, the functional inactivation of CREB-2 by HBZ is suggested to contribute to regulation of the HTLV-1 transcription. Moreover, the characterization of a minus-strand gene protein encoded by HTLV-1 has never been reported until now.

Collateral methotrexate resistance in cisplatin-selected murine leukemia cells

Resistance to anticancer drugs is a major cause of failure of many therapeutic protocols. A variety of mechanisms have been proposed to explain this phenomenon. The exact mechanism depends upon the drug of interest as well as the tumor type treated. While studying a cell line selected for its resistance to cisplatin we noted that the cells expressed a >25,000-fold collateral resistance to methotrexate. Given the magnitude of this resistance we elected to investigate this intriguing collateral resistance. From a series of investigations we have identified an alteration in a membrane protein of the resistant cell as compared to the sensitive cells that could be the primary mechanism of resistance. Our studies reviewed here indicate decreased tyrosine phosphorylation of a protein (molecular mass = 66) in the resistant cells, which results in little or no transfer of methotrexate from the medium into the cell. Since this is a relatively novel function for tyrosine phosphorylation, this information may provide insight into possible pharmacological approaches to modify therapeutic regimens by analyzing the status of this protein in tumor samples for a better survival of the cancer patients.

A block in virus-like particle maturation following assembly of murine leukaemia virus in insect cells

Expression of the murine leukaemia virus (MLV) major Gag antigen p65(Gag) using the baculovirus expression system leads to efficient assembly and release of virus-like particles (VLP) representative of immature MLV. Expression of P180(Gag-Pol), facilitated normally in mammalian cells by readthrough of the p65(Gag) termination codon, also occurs efficiently in insect cells to provide a source of the MLV protease and a pattern of p65(Gag) processing similar to that observed in mammalian cells. VLP release from P180(Gag-Pol) expressing cells however remains essentially immature with disproportionate levels of the uncleaved p65(Gag) precursor when compared to the intracellular Gag profile. Changing the p65(Gag) termination codon altered the level of p65(Gag) and p180(Gag-Pol) within expressing cells but did not alter the pattern of released VLP, which remained immature. Coexpression of p65(Gag) with a fixed readthrough p180(Gag-Pol) also led to only immature VLP release despite high intracellular protease levels. Our data suggest a mechanism that preferentially selects uncleaved p65(Gag) for the assembly of MLV in this heterologous expression system and implies that, in addition to their relative levels, active sorting of the correct p65(Gag) and p180(Gag-Pol) ratios may occur in producer cells. (C) 2003 Elsevier Inc. All rights reserved.

Coinfection of Leishmania chagasi with Toxoplasma gondii, Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) in cats from an endemic area of zoonotic visceral leishmaniasis

The aim of the present study was to determine the coinfection of Leishmania sp. with Toxoplasma gondii, Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) in a population of cats from an endemic area for zoonotic visceral leishmaniasis. An overall 66/302 (21.85%) cats were found positive for Leishmania sp., with infection determined by direct parasitological examination in 30/302(9.93%), by serology in 46/302(15.23%) and by both in 10/302 (3.31%) cats. Real time PCR followed by amplicon sequencing successfully confirmed Leishmania infantum (syn Leishmania chagasi) infection. Out of the Leishmania infected cats, coinfection with FIV was observed in 12/66(18.18%), with T. gondii in 17/66 (25.75%) and with both agents in 5/66(7.58%) cats. FeLV was found only in a single adult cat with no Leishmania infection. A positive association was observed in coinfection of Leishmania and FIV (p < 0.0001), but not with T. gondii (p > 0.05). In conclusion, cats living in endemic areas of visceral leishmaniasis are significantly more likely to be coinfected with Fly, which may present confounding clinical signs and therefore cats in such areas should be always carefully screened for coinfections. (C) 2012 Elsevier B.V. All rights reserved.

Prevalence of human T cell leukemia virus-I (HTLV-I) antibody among populations living in the Amazon region of Brazil (preliminary report)

Prevalence of human T cell leukemia virus-I (HTLV-I) antibody among populations living in the Amazon region of Brazil (preliminary report)NakauchiC. M.LinharesA. C.MaruyamaK.KanzakiL. I.MacedoJ. E.AzevedoV. N.CassebJ. S. R. Fundação Serviços de Saúde Pública, Seção de Vírus, Instituto Evandro Chagas Belém Brasil Chiba Cancer Research Institute, Department of Pathology Chiba Japan Universidade Federal do Pará Belém Brasil Instituto Offir Loyola Belém Brasil Faculdade Estadual de Medicina Belém Brasil 0319908512933Forty-tree (31.4%) out of 137 serum samples obtained from two Indian communities living in the Amazon region were found to be positive for HTLV-I antibody, as tested by enzyme-linked immunosorbent assay (Elisa). Eighty-two sera were collected from Mekranoiti Indians, yielding 39% of positivity, whereas 11 (20.0%) or the 55 Tiriyo serum samples had antibody to HTLV-I. In addition, positive results occurred in 10 (23.2%) out of 43 sera obtained from patients living in the Belem area, who were suffering from cancer affecting different organs. Five (16.7%) out of 30 Elisa positive specimens were also shown to be positive by either Western blot analysis (WB) or indirect immunogold electron microscopy (IIG-EM).

Coinfection of Leishmania chagasi with Toxoplasma gondii, Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) in cats from an endemic area of zoonotic visceral leishmaniasis

The aim of the present study was to determine the coinfection of Leishmania sp. with Toxoplasma gondii, Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) in a population of cats from an endemic area for zoonotic visceral leishmaniasis. An overall 66/302 (21.85%) cats were found positive for Leishmania sp., with infection determined by direct parasitological examination in 30/302(9.93%), by serology in 46/302(15.23%) and by both in 10/302 (3.31%) cats. Real time PCR followed by amplicon sequencing successfully confirmed Leishmania infantum (syn Leishmania chagasi) infection. Out of the Leishmania infected cats, coinfection with FIV was observed in 12/66(18.18%), with T. gondii in 17/66 (25.75%) and with both agents in 5/66(7.58%) cats. FeLV was found only in a single adult cat with no Leishmania infection. A positive association was observed in coinfection of Leishmania and FIV (p < 0.0001), but not with T. gondii (p > 0.05). In conclusion, cats living in endemic areas of visceral leishmaniasis are significantly more likely to be coinfected with Fly, which may present confounding clinical signs and therefore cats in such areas should be always carefully screened for coinfections. (C) 2012 Elsevier B.V. All rights reserved.

Prevalence of feline leukemia virus infection in domestic cats in Rio de Janeiro

Peripheral blood smears of 1094 domestic cats were collected and tested by indirect immunofluorescence antibody assay for p27 antigen in cells to study the prevalence and risk factors for feline leukemia virus (FeLV) in the state of Rio de Janeiro. Sex, age, breed, outdoor access, neutering status, type of habitation (household, shelter, veterinary clinics and other places), number of household cats and clinical signs were registered on a form. Among the tested samples, 11.52% were positive. Risk factors for FeLV infection included outdoor access, age range between 1 and 5 years old, and cohabitation with numerous cats.

Intracellular Reactive Oxygen Species Production by Polymorphonuclear Leukocytes in Bovine Leukemia Virus-Infected Dairy Cows

The present study assesses the oxidative burst activity from polymorphonuclear leukocytes (PMNLs) from bovine leukemia virus (BLV)-infected cows. Fifteen clinically healthy cows were divided into serologically positive cows without any hematological alteration, serologically positive animals with persistent lymphocytosis (PL) and healthy serologically negative cows. The oxidative burst activity from the PMNLs was evaluated by now cytometry using 2',7'-dichlorofluorescein diacetate as a probe. PMNLs from each cow were incubated with heat-killed Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) to stimulate oxidative burst activity. The results of the present work showed no significant difference in the oxidative burst activity without any stimulus and elicited by S. caucus. Conversely, a decrease in the oxidative burst index induced by E. coli in PMNLs was observed in BLV-infected cows.

Quantification of B cells and T lymphocyte subsets in bovine leukemia virus infected dairy cows

The aim of the present trial was to determine the frequencies and absolute number of B and T lymphocytes subpopulations in bovine leukemia virus (BLV)-infected dairy cows with distinct lymphocyte profile known as non-leukemic (AL) and persistent lymphocytosis (PL). Thus, 15 animals were selected and divided uniformly in three groups (negative, AL, PL). The BLV infection was detected by agar gel immunodiffusion and enzyme-linked immunosorbent-assay. The lymphocytes subsets were evaluated using monoclonal antibodies by flow cytometry. The results of the present study pointed out to an increase in B lymphocytes, and also an augment in CD5(+) and CD11b(+) cells in animals showing PL. Consequently, it can be observed a decrease in the percentage of T cells subsets in these animals. Conversely, no significant alterations in the absolute number of the T lymphocytes, T CD4(+) cells and T CD8(+) lymphocytes were found in BLV-infected dairy cows with PL. Therefore, the correlation between the absolute numbers of B- and T cell subsets in the peripheral blood applied to each group showed a significant and positive strong correlation between numbers of B cells and T cells or T CD8(+) cells in the PL animals, although the same cannot be predicted for T CD4(+) lymphocytes. No such correlation was encountered for the AL and negative-control animals.