Prostaglandin E(2) increases bovine leukemia virus tax and pol mRNA levels via cyclooxygenase 2: regulation by interleukin-2, interleukin-10, and bovine leukemia virus

Prostaglandin E(2) (PGE(2)), produced by macrophages, has important immune regulatory functions, suppressing a type 1 immune response and stimulating a type 2 immune response. Type 1 cytokines (interleukin-2 [IL-2], IL-12, and gamma interferon) increase in freshly isolated peripheral blood mononuclear cells (PBMCs) of animals with an early disease stage of bovine leukemia virus (BLV) infection, while IL-10 increases in animals with a late disease stage. Although IL-10 has an immunosuppressive role in the host immune system, IL-10 also inhibits BLV tax and pol mRNA levels in vitro. In contrast, IL-2 stimulates BLV tax and pol mRNA and p24 protein expression in cultured PBMCs. The inhibitory effect of IL-10 on BLV expression depends on soluble factors secreted by macrophages. Thus, we hypothesized that PGE(2), a cyclooxygenase 2 (COX-2) product of macrophages, may regulate BLV expression. Here, we show that the level of COX-2 mRNA was decreased in PBMCs treated with IL-10, while IL-2 enhanced the level of COX-2 mRNA. Addition of PGE(2) stimulated BLV tax and pol mRNA levels and reversed the IL-10 inhibition of BLV mRNA. In addition, the specific COX-2 inhibitor, NS-398, inhibited the amount of BLV mRNA detected. Addition of PGE(2) increased BLV tax mRNA regardless of NS-398 addition. PGE(2) inhibited antigen-specific PBMC stimulation, suggesting that stimulation of BLV tax and pol mRNA levels by PGE(2) is independent of cell proliferation. These findings suggest that macrophage-derived COX-2 products, such as PGE(2), regulate virus expression and disease progression in BLV infection.     

Pathogenicity of molecularly cloned bovine leukemia virus.

To delineate the mechanisms of bovine leukemia virus (BLV) pathogenesis, four full-length BLV clones, 1, 8, 9, and 13, derived from the transformed cell line FLK-BLV and a clone construct, pBLV913, were introduced into bovine spleen cells by microinjection. Microinjected cells exhibited cytopathic effects and produced BLV p24 and gp51 antigens and infectious virus. The construct, pBLV913, was selected for infection of two sheep by inoculation of microinjected cells. After 15 months, peripheral blood mononuclear cells from these sheep served as inocula for the transfer of infection to four additional sheep. All six infected sheep seroconverted to BLV and had detectable BLV DNA in peripheral blood mononuclear cells after amplification by polymerase chain reaction. Four of the six sheep developed altered B/T-lymphocyte ratios between 33 and 53 months postinfection. One sheep died of unrelated causes, and one remained hematologically normal. Two of the affected sheep developed B lymphocytosis comparable to that observed in animals inoculated with peripheral blood mononuclear cells from BLV-infected cattle. This expanded B-lymphocyte population was characterized by elevated expression of B-cell surface markers, spontaneous blastogenesis, virus expression in vitro, and increased, polyclonally integrated provirus. One of these two sheep developed lymphocytic leukemia-lymphoma at 57 months postinfection. Leukemic cells had the same phenotype and harbored a single, monoclonally integrated provirus but produced no virus after in vitro cultivation. The range in clinical response to in vivo infection with cloned BLV suggests an important role for host immune response in the progression of virus replication and pathogenesis.     

Increased interleukin-10 mRNA expression in tumor-bearing or persistently lymphocytotic animals infected with bovine leukemia virus.

Interleukin-10 (IL-10), produced by Th2 helper T cells, B cells, and macrophages, can inhibit cytokine production by Th1 cells and enhance B-cell proliferation and differentiation. Here, we show that peripheral blood mononuclear cells (PBMCs) from bovine leukemia virus-infected animals with late-stage disease express considerably more IL-10 mRNA than animals that are not infected or that are in the early stages of disease. In contrast, the quantities of type 1 cytokines, IL-2 and gamma interferon, decrease with disease progression. In addition, we observed that IL-10 is expressed principally by monocytes/macrophages, not B lymphocytes, in persistently lymphocytotic animals. This observation supports a role for monocytes/macrophages in progression of bovine leukemia virus infection and, of importance, indicates that proliferating B cells are not the source of IL-10 expression. These findings suggest that IL-10 produced by monocytes/macrophages may influence the progression of bovine leukosis in animals that develop persistent lymphocytosis of B cells or B-cell lymphosarcoma.     

Mutational analysis of the human T-cell leukemia virus type I trans-acting rex gene product.

Expression of the human T-cell leukemia virus type I (HTLV-I) rex gene is a prerequisite for the expression of the retroviral structural proteins. We have generated internal deletion mutants of this 27-kDa nucleolar trans-acting gene product to define functional domains in the Rex protein. The phenotype of the various mutant proteins was tested on the homologous HTLV-I rex response element sequence and the heterologous human immunodeficiency virus type 1 (HIV-1) rev response element sequence. Our results indicate that a region between amino acid residues 55 and 132 in the 189-amino-acid Rex protein is required for Rex-mediated trans activation on both retroviral response element sequences. In addition, substitution of the Rex nuclear localization signal by a sequence of the HIV-1 rev gene product targets the Rex protein to the correct subcellular compartment required for Rex function.     

cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA.

The genetic material of all retroviruses examined so far consists of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Since the precise location of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analyzed the dimerization process of Moloney murine leukemia virus (MoMuLV) genomic RNA. For this purpose we derived an in vitro model for RNA dimerization. By using this model, murine leukemia virus RNA was shown to form dimeric molecules. Deletion mutagenesis in the 620-nucleotide leader of MoMuLV RNA showed that the dimer promoting sequences are located within the encapsidation element Psi between positions 215 and 420. Furthermore, hybridization assays in which DNA oligomers were used to probe monomer and dimer forms of MoMuLV RNA indicated that the DLS probably maps between positions 280 and 330 from the RNA 5' end. Also, retroviral nucleocapsid protein was shown to catalyze dimerization of MoMuLV RNA and to be tightly bound to genomic dimer RNA in virions. These results suggest that MoMuLV RNA dimerization and encapsidation are probably controlled by the same cis element, Psi, and trans-acting factor, nucleocapsid protein, and thus might be linked during virion formation.     

Involvement of intracellular Ca2+ in the regulation of bovine leukemia virus expression

The calcium ionophore A23187, which was used to increase the intracellular calcium concentration ([Ca2+]i), was analyzed for effects on bovine leukemia virus (BLV) expression in two BLV infected cell lines. To clarify the role of intracellular free calcium in this response, [Ca2+]i was measured during ionophore treatment with the fluorescent calcium indicator Fura-2. Elevation of intracellular calcium under these conditions caused an enhancement of BLV gp51 and p24 synthesis as well as an activation of the BLV long terminal repeat (LTR) in a dose-dependent manner. Furthermore, it was observed that elevated levels of intracellular calcium following A23187 stimulation lead to activation of NF-kappaB. Based on inhibitor studies, we hypothesize that the effect of A23187 on BLV expression appears to be mediated by PKC.     

No involvement of bovine leukemia virus in sporadic bovine lymphosarcoma

Using various portions of a molecularly cloned bovine leukemia virus (BLV) DNA as probes, the possible integration of a BLV genome or a BLV-related sequence into the chromosomal DNA of sporadic bovine leukosis (SBL) tumor cells was investigated by Southern blotting analysis. Under stringent as well as nonstringent conditions of hybridization, neither BLV nor BLV-related sequence specific to SBL DNAs was detected in any SBL tumor examined. These results provide conclusive evidence for lack of the relation of BLV or a BLV-related agent to SBL.     

Repression of bovine papilloma virus replication is mediated by a virally encoded trans-acting factor

Cells transformed with bovine papilloma virus type 1 mutants in the E6 or E6/7 genes are resistant to high-copy-number amplification of wild-type DNA after supertransfection. Transient and stable replication assays demonstrate this effect. If the supertransfected DNA has a mutation in a newly defined gene (M), this cellular immunity to high-copy-number replication is overcome, resulting in transient replication of the input DNA. In contrast, the resident plasmid does not participate in amplification and is maintained at a constant low copy number. Cotransformation of M- mutants and wild-type DNA into these cells leads to shutoff of replication of both genomes. Thus, M- mutants define a trans-acting negative modulator that regulates viral replication. This function is distinct from the positive factors required for replication. We propose a model that explains why the loss of E6 and E6/7 function leads to immunity of the infected cell.     

Construction of a recombinant bovine leukemia virus vector for analysis of virus infectivity.

A recombinant bovine leukemia virus (BLV) was constructed in which the X region was replaced with the bacterial neomycin resistance gene controlled by the simian virus 40 early promoter. This virus, termed BLV-SVNEO, is a self-packaging, activator-dependent retroviral vector. Introduction of the plasmid pBLV-SVNEO into mammalian cells resulted in constitutive expression of the neo gene, whereas the BLV structural genes, gag, pol, and env, were expressed only in the presence of the two regulatory proteins, Tax and Rex. The production and release of recombinant virus by cells transfected with pBLV-SVNEO were proportional to the number of G418-resistant colonies that developed after susceptible cells were exposed to the filtered culture medium. BLV-SVNEO was able to infect cell lines of human, bovine, canine, feline, and murine origin. BLV-producing cell lines were resistant to superinfection with BLV-SVNEO. This cell-virus system should facilitate molecular genetic studies of BLV and will provide a rapid, quantitative measure of BLV infectivity in a variety of cell types. These studies also demonstrate the feasibility of using activator-dependent retroviral vectors such as BLV-SVNEO to deliver foreign genes into cells and eventually animals.     

A circular trans-acting hepatitis delta virus ribozyme.

A circular trans-acting ribozyme designed to adopt the motif of the hepatitis delta virus (HDV) trans-acting ribozyme was produced. The circular form was generated in vitro by splicing a modified group I intron precursor RNA in which the relative order of the 5' and 3' splice sites, flanking the single HDV-like ribozyme sequence-containing exon, is reversed. Trans-cleavage activity of the circular HDV-like ribozyme was comparable to linear permutations of HDV ribozymes containing the same core sequence, and was shown not to be due to linear contaminants in the circular ribozyme preparation. In nuclear and cytoplasmic extracts from HeLa cells, the circular ribozyme had enhanced resistance to nuclease degradation relative to a linear form of the ribozyme, suggesting that circularization may be a viable alternative to chemical modification as a means of stabilizing ribozymes against nuclease degradation.     

In vivo infection of sheep by bovine leukemia virus mutants.

Direct inoculation of a cloned bovine leukemia virus (BLV) provirus into sheep has allowed study of the viral infectivity of genetic mutants in vivo. Three BLV variants cloned from BLV-induced tumors and 12 in vitro-modified proviruses were isolated and analyzed for viral expression in cell culture. The proviruses were then inoculated into sheep in order to assess viral infectivity in vivo. Of three variants cloned from BLV-induced tumors (344, 395, and 1345), one (344) was found infectious in vivo. This particular provirus was used to engineer 12 BLV mutants. A hybrid between the 5' region of the complete but noninfectious provirus 395 and the 3' end of mutant 344 was infectious in vivo, suggesting that the tax/rex sequences were altered in virus 395. As expected, several regions of the BLV genome appeared to be essential for viral infection: the protease, pol, and env genes. Even discrete modifications in the fusion peptide located at the NH2 end of the transmembrane gp30 glycoprotein destroyed the infectious potential. In contrast, mutations and deletions in the X3 region present between the env gene and the 3' tax/rex region did not interfere with viral infection in vivo. This region of unknown function could thus be used to introduce foreign sequences. A BLV recombinant carrying a ribozyme directed against the tax/rex sequences was still infectious in vivo. Cotransfection of two noninfectious mutants carrying deletions led to infection in two of four independent injections, the infectious virus being then a recombinant between the two deletants. The experimental approach described here should help to gain insight into essential mechanisms such as in vivo viral replication, cooperation between deletants for viral infectivity, and viral superinfections. The gene products in the X3 and X4 region which are dispensable for in vivo infection could be involved in leukemogenesis, and thus proviruses deleted in these sequences could constitute the basis for a live attenuated vaccine.     

trans-acting requirements for replication of Epstein-Barr virus ori-Lyt.

Epstein-Barr virus (EBV) utilizes a completely different mode of DNA replication during the lytic cycle than that employed during latency. The latency origin of replication, ori-P, which functions in the replication of the latent episomal form of the EBV genome, requires only a single virally encoded protein, EBNA-1, for its activity. During the lytic cycle, a separate origin, ori-Lyt, is utilized. Relatively little is known about the trans-acting proteins involved in ori-Lyt replication. We established a cotransfection-replication assay to identify EBV genes whose products are required for replication of ori-Lyt. In this assay, a BamHI-H plasmid containing ori-Lyt was replicated in Vero cells cotransfected with the BamHI-H target, the three EBV lytic-cycle transactivators Zta, Rta, and Mta, and the EBV genome provided in the form of a set of six overlapping cosmid clones. By removing individual cosmids from the cotransfection mixture, we found that only three of the six cosmids were necessary for ori-Lyt replication. Subcloning of the essential cosmids led to the identification of six EBV genes that encode replication proteins. These genes and their functions (either known or predicted on the basis of sequence comparison with herpes simplex virus) are BALF5, the DNA polymerase; BALF2, the single-stranded DNA-binding protein homolog; BMRF1, the DNA polymerase processivity factor; BSLF1 and BBLF4, the primase and helicase homologs; and BBLF2/3, a potential homolog of the third component of the helicase-primase complex. In addition, ori-Lyt replication in this cotransfection assay was also dependent on one or more genes provided by the EBV SalI-F fragment and on the three lytic-cycle transactivators Zta, Rta, and Mta.     

Syncytia infectivity of assay of bovine leukemia virus

Bovine embryonic spleen cell cultures were examined to find several factors influencing the specificity, sensitivity and reproducibility of the syncytia infectivity assay of bovine leukemia virus (BLV). The highest sensitivity of the assay were observed when cell sheets of 30 to 50% confluence were inoculated with a stock of BLV, and when cells containing 4 or more nuclei were counted as syncytial cells. Treatment of the cell sheets with a diethylamino-ethyl-dextran solution (25 micrograms/ml) prior to BLV inoculation was found to be essential for the optimal induction of syncytia. Low-passage cultures were found to be more susceptible to the induction of syncytia by BLV than high-passage cultures. Cell-free BLV preparations decreased in syncytia-inducing ability to some extent by the first cycle of freezing (at -70 degrees C) and thawing. No further decrease, however, was caused by repeated cycles of freezing and thawing or by prolonged incuvation at -80 degrees C. The syncytia-inducing activity of BLV was inhibited by all the BLV-precipitating antibody-positive sera originated from both cases of the adult form of bovine leukosis and cases of persistent lymphocytosis. It was not inhibited by the sera of 16 of 17 cattle apparently healthy and negative for BLV-precipitating antibody. These results indicate that the syncytia infectivity assay and syncytia inhibition test are specific for BLV.     

Genetic analysis of bovine papillomavirus type 1 trans-acting replication factors.

The establishment of bovine papillomavirus type 1 in somatic mammalian cells is mediated by extrachromosomal replication and stable maintenance of the viral genome as a multicopy nuclear plasmid. Previous studies indicated the requirement of viral gene expression for bovine papillomavirus type 1 replication and plasmid maintenance (M. Lusky and M. R. Botchan, Cell 36:391-401, 1984; Turek et al., Proc. Natl. Acad. Sci. U.S.A. 79:7914-7918, 1982). To define the viral genes which are necessary for this process, we constructed a series of specific mutations within the viral genome and assayed the resulting mutants for their ability to replicate extrachromosomally in mouse C127 cells. We report here that the bovine papillomavirus type 1 trans-acting replication factors were encoded by at least two distinct viral genes since the mutants fell into two complementation groups, rep and cop. Mutants (rep-) affecting the E1 open reading frame (ORF) failed to replicate bovine papillomavirus type 1 DNA extrachromosomally and would integrate into chromosomal DNA. We suggest that this gene product is one of the factors required to specifically preclude the integration event. Mutants (cop-) affecting the E7 ORF were maintained in the extrachromosomal state; however, the copy number of the mutant genomes was reduced 100-fold compared with that of wild-type DNA. Analysis of single-cell subclones showed that each cell contained the mutant genomes at a copy number of one to two, indicating that the cop- phenotype did not reflect a simple segregation defect. We propose that the gene defined by mutations in the E7 ORF played a crucial role in stably maintaining the copy number of the viral plasmid at high levels. Genomes with mutations in the cop and rep complementation groups, when cotransfected, rescued the wild-type phenotype, extrachromosomal replication with a high, stable copy number for both types of plasmids. Therefore, the gene products acted in trans, and the mutations were recessive to the wild-type functions. One specific rep- mutant showed a 30-fold-increased transformation efficiency when compared with that of the wild-type genome. In addition, morphological transformation mediated by the cop- mutants appeared to be unstable. These results imply that either or both of the replication functions played some role in regulating the expression of the viral transforming functions.     

The bovine leukemia virus p34 is a transactivator protein.

Recombinant Moloney murine retroviruses containing the BLV post-envelope long open reading frame were constructed and transfected into the psi 2 packaging cell line. They were shown to encode and to express a 34-kd protein able to transactivate the BLV long terminal repeat-directed gene expression in the respective transfected cells. These data demonstrate that the BLV X-LOR gene encodes a p34 transactivator product. Furthermore, the different cell lines produced infectious recombinant retroviruses capable of transferring X-LOR genes into recipient cells. The availability of the BLV transactivator protein should allow us to understand the role of the transactivator protein in BLV-induced leukemogenesis.