Gene Expression in Bovine Leukemia Virus-Infected B Lymphocytes

131 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1993.Bovine leukemia virus (BLV) is a type C, chronic transforming retrovirus that infects B-lymphocytes and is associated with a long latency period. We studied the transcriptional activation of BLV in primary cells and also examined whether a measurable increase in BLV expression would effect the transcription of host genes. De novo protein synthesis was inhibited by cycloheximide (CHX) for 6 h after initiating peripheral blood leukocyte (PBL) cultures to eliminate Tax-mediated transcription. Under these conditions, transcription of the doubly spliced tax/rex message was predominant and was induced by the addition of phorbol 12-myristate 13-acetate (PMA), 8-bromo-cyclic AMP (Br-cAMP) and fetal bovine serum. Serum-induced BLV expression was inhibited in PBL in a dose dependent fashion by the protein kinase A (PKA) inhibitor H-89. Host gene expression was classified as labile protein-dependent when transcription was modulated by CHX and protein kinase-mediated when transcription was modulated by PMA, Br-cAMP or serum. Regulation of immunoglobulin-$\mu$ (Ig-$\mu$) was protein kinase-mediated, whereas major histocompatibility complex (MHC) class I and class II genes, heat shock protein 70 and the PKA regulatory subunits were all protein kinase-mediated as well as labile protein-dependent. Compared with animals that were seronegative to BLV antigens and seropositive without persistent lymphocytosis (PL), freshly isolated mIgM$\sp+$ cells obtained from cows with PL expressed an increase in Ig-$\mu$ mRNA and a decrease in the mRNA for Ig-$\lambda$. By contrast, these same cells showed no correlation between BLV-infection status and mRNA expression of MHC class I, class II or PKA genes. After 12 h of cell culture, mIgM$\sp+$ cells from animals with PL expressed high levels of BLV mRNA relative to animals that were seronegative and seropositive without PL. However, there was no apparent effect on host gene expression that correlated with BLV expression since, the MHC class I, class II, Ig-$\mu$, Ig-$\lambda$ and PKA RI$\alpha$ mRNA levels were either unchanged or decreased relative to freshly isolated cells. In previous studies, about one-third of BLV-infected cows with PL were unreactive with the MHC class II DR-specific monoclonal antibody H4 when using the lymphocyte microcytotoxicity test (referred to as "H4$\sp-$" phenotype). By Northern blot analysis we compared mIgM$\sp+$ cells form H4$\sp+$ and H4$\sp-$ animals for steady state levels of DRA, DRB, DQA and DQB mRNA and determined that the H4$\sp-$ phenotype was not transcriptionally regulated. We conclude that BLV expression has no apparent effect on the mRNA levels of Ig, MHC or PKA genes, but that freshly isolated mIgM$\sp+$ cells from BLV-infected PL animals express increased constitutive Ig-$\mu$ mRNA levels. We propose that in vivo, activation of endogenous protein kinase pathways are sufficient to induce a break from retroviral latency by activating BLV transcription and the early stage of the life cycle.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD