Identification and some biochemical properties of the major XBL gene product of bovine leukemia virus.

Abstract

Using a rabbit antiserum directed against a synthetic oligopeptide whose sequence was deduced from the nucleotide sequence of the XBL gene of bovine leukemia virus, we detected a 38-kDa protein in virus-producing cell lines. In vitro translation of hybrid-selected RNA unequivocally demonstrates that this protein, designated p38(XBL), is indeed encoded by the XBL gene. Unlike the other virus-encoded proteins, however, p38(XBL) resides within the cells without being incorporated into virions. It undergoes no gross posttranslational modifications and has a relatively short half-life (5-6 hr) in vivo. Furthermore, cell fractionation combined with pulse-chase experiment reveals that a significant fraction (more than half) of the p38(XBL) localizes to the nucleus of the infected cell after synthesis. We conclude that the XBL gene of bovine leukemia virus is a functional gene encoding a nonvirion protein p38(XBL), which possibly functions within the nucleus of the infected cell to regulate viral or cellular gene expression. P38(XBL) is presumably translated from a doubly spliced, bicistronic mRNA that has the capability to encode another small polypeptide in a different reading frame.Bovine leukemia virus (BLV), an exogenous replicationcompetent retrovirus in cattle, is a causative agent of enzootic bovine leukosis (1) and is evolutionarily related to human T-cell leukemia viruses types I and II (HTLV-I and HTLV-II) (2-5). BLV has no preferred chromosomal site for integration (6,7) and often lacks the 5' half (but not the 3' half) ofits genome in the leukemic cells (8). Furthermore, although BLV carries no typical oncogene of cellular origin (5, 9), it is capable of transforming cells in vitro (10, 11). It is thus possible that the BLV genome itself, possibly its 3' half, harbors a gene(s) that is responsible for cellular transformation, viral replication, or both (4).We and others have recently determined the nucleotide sequence of the integrated BLV provirus (3,5,12), finding that the BLV genome contains an unidentified region (designated XBL gene) between the env gene and the 3' long terminal repeat (LTR) (5). The major long open reading frame (XBL-I) found in the XBL gene has a typical protein-encoding capability (4), and we predicted that its putative product would be produced by a spliced mRNA (4). X or LOR genes of HTLVs are genes equivalent to the XBL gene of BLV (4) and have recently been shown to encode proteins of 38-42 kDa (13-16).Here, we identify the XBL-I product in BLV-infected cell lines, using a rabbit antiserum raised against a synthetic oligopeptide derived from the XBL-I open reading frame. Furthermore, we describe several biochemical properties of the XBL-I product, including its metabolic stability and subcellular localization, to gain insight into its biological function. MATERIALS AND METHODSCells and Metabolic Labeling. BLV-infected fetal lamb kidney cells (FLK) (17) and bat lung cells (Bat2clI) (18) were used as sources of BLV-producing cells. Cells spread at 60-70% confluency were me...

Cooperative Regulation of Bovine Leukaemia Virus Gene Expression by Two Overlapping Open Reading Frames in the XBL Region

“…The expression of the env gene from pLTRenv is markedly enhanced by pSVX-I, which is expected to express only intact XBL-I products (38K protein; Sagata et al, 1985a), but not by pSVfsX-I, which has a + 4 frameshift mutation in XBL-I. The results demonstrate that XBL-I (x-lot) is a trans-acting gene which is essential for the transient LTR-directed expression of the viral env gene and agree with results obtained for the CAT gene expression assay system in BLV (Katoh et al, 1987) and HTLV (Felber et al, 1985;Seiki et al, 1986).…”

Alterations in Humoral Immune Response to Bovine Leukaemia Virus Antigens in Cattle with Lymphoma

“…Recently the Px (also called lor or tat) gene has been identified and partially characterized for BLV (Sagata et al, 1985); the products of this gene mediate trans-acting transcriptional activation (Felber et aL, 1985). The products (p38, p 17 and p 14) are found most frequently in the nucleus of an infected cell where they have a short half-life (Sagata et al,I985).…”

Ribozyme cleaves rex/tax mRNA and inhibits bovine leukemia virus expression.

“…Cells (2.5 x 106) were lysed and sonicated (19), subjected to electrophoresis in 7.5-17.5% polyacrylamide gels, transferred to nitrocellulose, and reacted with Tax monoclonal antibody 5A5 (20) directed at the middle of the tax RNA, failed to cleave its 256-base substrate RNA into the predicted 164-and 92-base products (Fig. 2C), even at a variety of Mg2+ conditions (Fig.…”