Isolation and characterization of a bovine plasma protein which inhibits the replication of bovine leukemia virus

A protein of 155,000 Daltons was isolated from the plasma of a Holstein cow naturally infected with the bovine leukemia virus (BLV) using ammonium sulphate precipitation, anion exchange and gel filtration chromatography. Addition of 5 μg of the protein to 5 × 106 BLV-infected peripheral blood mononuclear cells (PBMC) resulted in marked inhibition of the p24 protein product of the BLV genome as measured by an Enzyme Linked Immunosorbent Assay (ELISA). Cell viability did not vary significantly as compared to control cultures. Amino acid analysis of eight trypsin-digested fragments of the plasma protein revealed 100% homology with bovine fibronectin (FN). Commercially prepared bovine FN was also able to inhibit viral expression in a dose-dependent manner.; Expression of cell surface IgG and IgM on PBMC was found to be significantly reduced after culture with purified FN in comparison to cells cultured in media alone or to freshly isolated cells. To test whether the reduction in viral protein was due to a general block in protein synthesis, de novo protein production was measured in PBMC using 3H leucine uptake. Increased cellular protein synthesis was observed in PBMC cultured with purified plasma protein. A cDNA microarray, done to identify cellular genes up-regulated by a factor of 2 or more, identified a total of 562 genes, including the pro-inflammatory cytokine IL-1β.; Inhibition of viral protein expression occurred at the level of transcription as determined by experiments in which cell lines were transfected with plasmids containing the long terminal repeat (LTR) of the BLV genome fused to the gene for chloramphenicol acetyl transferase (CAT) and cultured with or without the addition of plasma protein. Expression of the CAT gene was significantly diminished in cultures with the added plasma protein as compared to controls.; Cell lines transfected with plasmids containing the LTR of either Human Immunodeficiency Virus I (HIV-I), Human T-cell Leukemia Virus I (HTLV-I) or the NFκB element fused to the CAT gene also produced significantly less CAT gene product when cultured in the presence of this protein. In contrast, the factor did not significantly down-regulate expression of CAT in cells transfected with constructs containing the LTR of Bovine Immunodeficiency Virus (BIV), Feline Immunodeficiency Virus (FIV) or Feline Leukemia Virus (FeLV) associated with the CAT gene. Electrophoretic mobility shift assays confirmed that the isolated factor acted, at least in part, via a cell signalling mechanism which utilized the NFκB response element shared by HIV-I, HTLV-I and BLV.; These results suggest that bovine FN has potential as a novel treatment for retroviral infections.