The bovine leukemia virus X region encodes a trans-activator of its long terminal repeat

We constructed a fusion plasmid, pMX-I, by which the major open reading frame, X-I, of the bovine leukemia virus (BLV) X gene was expressed under control of the mouse metallothionein promoter. pMX-I was cotransfected into CV1 monkey kidney cells together with another construct containing the BLV long terminal repeat (LTR) linked to the chloramphenicol acetyltransferase (CAT) structural gene. The result of assay of CAT synthesis suggests that the X-I product functions as a trans-acting activation factor of the BLV LTR.     

Myelodysplastic syndromes and acute myeloid leukemia in cats infected with feline leukemia virus clone33 containing a unique long terminal repeat

Feline leukemia virus (FeLV) clone33 was obtained from a domestic cat with acute myeloid leukemia (AML). The long terminal repeat (LTR) of this virus, like the LTRs present in FeLV from other cats with AML, differs from the LTRs of other known FeLV in that it has 3 tandem direct 47‐bp repeats in the upstream region of the enhancer (URE). Here, we injected cats with FeLV clone33 and found 41% developed myelodysplastic syndromes (MDS) characterized by peripheral blood cytopenias and dysplastic changes in the bone marrow. Some of the cats with MDS eventually developed AML. The bone marrow of the majority of cats with FeLV clone33 induced MDS produced fewer erythroid and myeloid colonies upon being cultured with erythropoietin or granulocyte‐macrophage colony‐stimulating factor (GM‐SCF) than bone marrow from normal control cats. Furthermore, the bone marrow of some of the cats expressed high‐levels of the apoptosis‐related genes TNF‐α and survivin. Analysis of the proviral sequences obtained from 13 cats with naturally occurring MDS reveal they also bear the characteristic URE repeats seen in the LTR of FeLV clone33 and other proviruses from cats with AML. Deletions and mutations within the enhancer elements are frequently observed in naturally occurring MDS as well as AML. These results suggest that FeLV variants that bear URE repeats in their LTR strongly associate with the induction of both MDS and AML in cats. © 2008 Wiley‐Liss, Inc.     

Identification of annexin 1 as a PU.1 target gene in leukemia cells

To identify PU.1 downstream target genes, we first established PU.1-knockdown K562 (K562PU.1KD) cells expressing reduced levels of PU.1 by stably transfected PU.1 siRNAs. From microarray analysis, we found that several genes including annexin 1 were markedly induced in K562PU.1KD cells. Annexin 1 is a calcium- and phospholipid-binding protein and increased expression leads to the constitutive activation of extracellular signal-regulated kinase (ERK). Consistent with this, we observed constitutive activation of ERK in K562PU.1KD cells. Furthermore, we revealed the mRNA expression of annexin 1 was negatively correlated with PU.1 mRNA expression in 43 primary AML specimens (R = −0.31, p < 0.042).     

Doxorubicin stimulates transcription from the human immunodeficiency virus long terminal repeat sequences.

A recombinant plasmid carrying the long terminal repeat (LTR) of the human immunodeficiency virus 1 (HIV-1) linked to the reporter chloramphenicol acetyl transferase (CAT) gene was stably introduced into rat liver cells. The transfectant cells expressed CAT activity from the HIV LTR. The response to doxorubicin was studied and it was found that at the optimum concentration of 20 micrograms/ml doxorubicin, the expression of CAT from the HIV LTR was stimulated by 65-fold. Our results suggest caution against therapy including doxorubicin in the treatment of AIDS patients.     

Transforming activity of the RL-akt gene, a c-akt gene activated by long terminal repeat insertion in murine leukemia RL(male symbol)1 cells

The unique antigen peptide pRL1 on BALB/c radiation-induced leukemia RL(male symbol)1 cells is derived from the normally untranslated 5' region of the mouse c-akt gene. Insertion of an endogenous long terminal repeat into the first coding exon of the gene resulted in the enhanced production of an altered akt protein, RL-akt, and creation of the tumor rejection antigen peptide pRL1. In this study, we constructed an RL-akt-expressing vector to investigate the transforming ability and anti-apoptotic activity of RK-akt in NIH/3T3 cells. RL-akt-expressing clones formed more colonies than did c-akt-expressing clones in soft agar and exhibited increased saturation density, a lower serum requirement for growth, and tumorigenicity on athymic nude mice. Immunoblot analysis of subcellular protein distribution showed that a considerable proportion of RL-akt was distributed in the membrane fraction. Thus, RL-akt expressed in NIH/3T3 cells appeared to behave like the v-akt oncoprotein. Furthermore, the RL-akt gene conferred resistance to the apoptosis induced by the calcium ionophore A23187 and by ultraviolet irradiation of NIH/3T3 cells. These findings indicate that the RL-akt gene is able to transform cells and exerts an anti-apoptotic effect on recipient cells, thereby implicating the gene in leukemogenesis of RL(male symbol)1 cells.     

Comparison of DNA sequences of the long terminal repeat of human T lymphotropic virus type I in Japanese and Brazilian Amazonian samples

Antibodies to human T lymphotropic virus type I has been detected in subjects of different human ethnicities all around the world. Etiological relationship between the virus and human diseases has been claimed by many investigators. Amplified and sequenced region of the long terminal repeat of human T lymphotropic virus type I obtained from nucleic acid extracted from serum samples of Japanese and Brazilian patients with cancer of uterine cervix and normal Brazilian subjects, all seropositives for the virus, showed minor genetic variations when compared to the Japanese prototype.     

Detection, quantitation, and characterization of the major internal virion antigen of the bovine leukemia virus by radioimmunoassay.

The major internal polypeptide of the bovine leukemia virus (BLV) was purified to homogeneity with the use of gel filtration and affinity chromatography. Like previous results, the protein had a molecular weight of 25,000 daltons as determined by electrophoresis in polyacrylamide gels with sodium dodecyl sulfate. More than 90% of the 125I-labeled protein was precipitated by bovine sera that reacted in immunofluorescence tests with acetone-fixed BLV-infected cells. In contrast, minimal precipitation (less than 5%) was observed with sera from 36 cattle in leukemia-free herds; these sera, negative by immunofluorescence, included six samples that had high titers of antibodies to the foamy-like bovine syncytia virus (BSV). Antisera prepared against several other oncornaviruses or the Mason-Pfizer monkey virus (M-PMV) did not bind the BLV p25 protein. Conversely, the labeled p30 polypeptides of several oncornaviruses tested did not react with bovine sera that had high titers of antibodies to BLV p25. Competitive radioimmunoassay(s) (RIA) also failed to detect cross-reactions between BLV p25 protein and the internal polypeptides of other mammalian and avian oncornaviruses, M-PMV, or foamy-like BSV. The RIA for BLV p25 antigen was also highly sensitive and specific for the detection and quantitation of the antigen in virus preparations and cell homogenates.     

Detection of bovine leukemia virus in B-lymphocytes by the syncytia induction assay.

Bovine peripheral blood lymphocytes (PBL's) from 3 cows and 1 steer infected with bovine leukemia virus (BLV) were separated by fractionation through nylon wool columns into nylon-adherent and nonadherent cell populations. Nylon-adherent cells were enriched in B-lymphocytes, as determined by the presence of surface membrane immunoglobulins (slg), whereas nylon-nonadherent cells or "non-B-lymphocytes" contained few slg-bearing cells. PBL's and separated B- and non-B-lymphocyte populations were assayed for the presence of BLV by the induction of syncytia in bovine embryonic spleen cells. PBL's and B-lymphocyte populations both produced many syncytia, whereas non-B-lymphocytes yielded few or no syncytia. The specificity of syncytia formation by anti-BLV serum. PBL's from 2 control animals were negative for syncytia induction. This study presents further evidence that B-lymphocytes are the target cells for BLV infection.     

Isolation and characterization of an antigen of the bovine C-type virus.

By means of gel filtration and isoelectric focusing, an antigen of the bovine C-type leukemia virus was isolated in a highly purified form from extracts of infected cells. The antigen has a molecular weight of approximately 25,000 daltons and an isoelectric point of 6.4 to 6.6. In immunodiffusion experiments, the antigen forms a line of identity with an antigen extracted from highly purified bovine C-type leukemia virus by treatment with ether or Triton X-100. As determined by immunodiffusion analyses, the bovine C-type leukemia virus antigen does not have antigenic determinants in common with the murine or feline leukemia viruses, the foamy-like bovine syncytia virus, or the Mason Pfizer monkey virus.     

Development of leukemia and lymphosarcoma induced by bovine leukemia virus in sheep: a hematopathological study

The hematological and neoplastic disorders induced in sheep by experimental bovine leukemia virus (BLV) infection are described. Seventeen of 19 BLV-inoculated sheep developed a marked increase in peripheral blood lymphocytes by 36 months after the intraperitoneal injection of peripheral blood lymphocytes from a BLV-infected cow. This increase correlated with an increase in the number of circulating B lymphocytes as demonstrated by the presence of surface immunoglobulins (SIg) and a high cell proliferative response to lipopolysaccharide and was considered to be a persistent B cell lymphocytosis. Lymphosarcoma developed in five BLV-infected sheep between 19 and 38 months postinoculation and was preceded in four out of five of these cases by an elevation in peripheral blood lymphocytes which began 4 to 26 months before death due to lymphosarcoma. The majority of tumor cells in all lymphosarcoma cases were of the centroblastic type, and in two cases in which the presence of SIg was assayed, the majority of tumor cells were SIg-positive. Thus, BLV-induced lymphosarcoma in sheep seems to be a B lymphocyte-derived tumor.