Two distinct polypeptides may be translated from a single spliced mRNA of the X genes of human T-cell leukemia and bovine leukemia viruses

Human T-cell leukemia and bovine leukemia viruses have a potential transforming gene, termed X. In addition to the major open reading frame known to encode a functional protein, the X gene harbors another short open reading frame which overlaps this major one. Both of these open reading frames are found on a single spliced X mRNA in a potentially functional form. Circumstantial evidence strongly suggests that they are both translated from the single X mRNA molecule, showing striking similarity to the translation mechanism of an adenovirus Elb gene mRNA. We note that the short open reading frame has the capability to encode a putative nuclear protein with structural features similar to those of an AIDS virus trans-acting protein.     

Bovine leukemia virus post-envelope gene coded protein: evidence for expression in natural infection

Partial sequence analysis of a 14 kilodalton protein (p14), synthesized by in vitro translation of bovine leukemia virus genomic RNA, showed that it is encoded in the 'X' region of proviral DNA, located between the env gene and the 3' long terminal repeat. The 'X' gene contains a short and a long open reading frame (X-SORF and X-LORF) which overlap. BLV p14x is specified by X-SORF and not X-LORF as seen with the related human T-cell leukemia virus which expresses p38-40x. Antibodies in sera from animals with BLV induced tumors were shown to recognize p14x. Expression of this protein in natural infection might be important for virus replication and/or for BLV induced oncogenesis.     

Gene 26 of bacteriophage T4 basal plate. I. Two expression products of the cloned gene

We have cloned the 1.9 kb EcoRV-BglII DNA fragment with T4 genes 51, 26, and 25 into the expression plasmid pT7-5 carrying a T7 promoter. The resulting recombinant plasmid, pRR5-3, contained T4 genes 26 and 25 in the correct orientation for expression. We expressed these genes using the T7 RNA polymerase/promoter system and the synthesis of three polypeptides with the molecular masses of approximately 24, 15, and 8-9 kDa was observed. Expression of genes from the subcloned DNA fragments and from the fragments carrying deletions was studied as well and the 15 kDa protein appeared to be the product of gene 25, while 24 kDa and 8-9 kDa proteins were identified as products of gene 26. The 8-9 kDa protein was shown to be expressed from the end region of gene 26. Having analysed the proteins expressed from the fragments carrying fusion of genes 26 and 25 we supposed two products of gene 26 to be encoded by the same open reading frame.     

Structural characterization of SIL, a gene frequently disrupted in T-cell acute lymphoblastic leukemia.

The SIL (SCL interrupting locus) gene was initially discovered at the site of a genomic rearrangement in a T-cell acute lymphoblastic leukemia cell line. This rearrangement, which occurs in a remarkably site-specific fashion, is present in the leukemic cells of 16 to 26% of patients with T-cell acute lymphoblastic leukemia. We have now cloned a normal SIL cDNA from a cell line which does not carry the rearrangement. The SIL cDNA has a long open reading frame of 1,287 amino acids, with a predicted molecular size of 143 kDa. The predicted protein is not homologous with any previously described protein; however, a potential eukaryotic topoisomerase I active site was identified. Cross-species hybridization using a SIL cDNA probe indicated that the SIL gene was conserved in mammals. A survey of human and murine cell lines and tissues demonstrated SIL mRNA to be ubiquitously expressed, at low levels, in hematopoietic cell lines and tissues. With the exception of 11.5-day-old mouse embryos, SIL mRNA was not detected in nonhematopoietic tissues. The genomic structure of SIL was also analyzed. The gene consists of 18 exons distributed over 70 kb, with the 5' portion of the gene demonstrating alternate exon utilization.     

Identification of a protease gene of human T-cell leukemia virus type I (HTLV-I) and its structural comparison

We determined the nucleotide sequence of a region between the gag and pol genes of a replication-competent proviral clone of a human T-cell leukemia virus type I (HTLV-I) from MT-2 cells. This region overlapping the gag and pol genes contains an open reading frame with a different phase from others. The deduced amino acid sequences show significant homology with the known protease gene of other retroviruses, and harbors highly conserved amino acid sequences that are well conserved in other retroviral protease domains. These results indicate that this open reading frame encodes a HTLV-I protease.     

Role of the 3'' long open reading frame region of bovine leukemia virus in the maintenance of cell transformation.

Viral RNA expression was studied by dot blot hybridization with polyadenylated RNAs extracted from a bovine (YR-1) and an ovine (YR-2) tumor cell clone. Both clones were derived from in vivo bovine leukemia virus-induced tumors. The probes used were either the bovine leukemia virus information or only the long open reading frame sequences. No viral RNA corresponding to the bovine leukemia virus long open reading frame region was detected in YR-2, and a very limited amount of bovine leukemia virus messages was unraveled in YR-1. These results strongly suggest that viral expression, even in the long open reading frame region, is not required to maintain transformation of at least some tumor cells.     

Crp1p,a new cruciform DNA-binding protein in the yeast Saccharomyces cerevisiae

A synthetic cruciform DNA (X-DNA) was used for screening cellular extracts of Saccharomyces cerevisiae for X-DNA-binding activity. Three X-DNA-binding proteins with apparent molecular mass of 28kDa, 26kDa and 24kDa, estimated by SDS-PAGE, were partially purified. They were identified as N-terminal fragments originating from the same putative protein, encoded by the open reading frame YHR146W, which we named CRP1 (cruciform DNA-recognising protein 1). Expression of CRP1 in Escherichia coli showed that Crp1p is subject to efficient proteolysis at one specific site. Cleavage leads to an N-terminal subpeptide of approximately 160 amino acid residues that is capable of binding specifically X-DNA with an estimated dissociation constant (K(d)) of 800nM, and a C-terminal subpeptide of approximately 305 residues without intrinsic X-DNA-binding activity. The N-terminal subpeptide is of a size similarly to that of the fragments identified in yeast, suggesting that the same cleavage process occurs in the yeast and the E.coli background. This makes the action of a site-specific protease unlikely and favours the possibility of an autoproteolytic activity of Crp1p. The DNA-binding domain of Crp1p was mapped to positions 120-141. This domain can act autonomously as an X-DNA-binding peptide and provides a new, lysine-rich DNA-binding domain different from those of known cruciform DNA-binding proteins (CBPs). As reported earlier for several other CBPs, Crp1p exerts an enhancing effect on the cleavage of X-DNA by endonuclease VII from bacteriophage T4.     

The open reading frame I (ORF I)/ORF II part of the human T-cell leukemia virus type I X region is dispensable for p40tax, p27rex, or envelope expression.

The X region of the human T-cell leukemia virus type I contains the second coding exon of the tax and rex regulatory proteins (open reading frame IV [ORF IV] and ORF III, respectively), as well as coding regions for more recently described proteins, p30II (or the tof protein) and p13II in ORF II and the putative rof protein and p12I in ORF I. Deletions and transcomplementation experiments showed that expression of the envelope, as well as that of the tax and rex proteins, was independent of the proteins encoded in the ORF I/ORF II region. Furthermore, p30II and p12I proteins could not replace the rex protein in a rex-dependent envelope or Gag protein expression system.     

In vivo genomic variability of human T-cell leukemia virus type I depends more upon geography than upon pathologies.

To investigate the geography- and disease-associated genomic variation of human T-cell leukemia virus type I (HTLV-I), we studied ex vivo DNA from peripheral blood lymphocytes from nine patients by polymerase chain reaction and direct DNA sequencing. For each viral strain, 1,917 bp was sequenced, including parts of the long terminal repeat, the env gene, and the px II, px III, and px IV coding frames of the px region. The number of genomic variations observed in the U3 region of the long terminal repeat was higher than that seen in the env and px genes. Very few mutations were present in the px II and px III genes. In contrast, the px IV open reading frame exhibited numerous single point mutations. While no specific mutation could be linked to any pathology (adult T-cell leukemia/lymphoma or tropical spastic paraparesis/HTLV-I-associated myelopathy), variations among HTLV-I isolates from different geographic areas (Ivory Coast, Caribbean, and Japan) existed. The Ivory Coast HTLV-I appeared to represent a group by itself.     

Identification of a potential protease-coding gene in the genomes of bovine leukemia and human T-cell leukemia viruses

The genomes of bovine leukemia and human T-cell leukemia viruses both contain an unidentified region between the gag and pol genes. These regions harbor an open reading frame that is in a different phase from the reading frames of the gag and pol genes. Based on the deduced amino acid sequences, we show here that they potentially encode a gag precursor-cleaving protease, which is known to be fused to the gag and pol products of avian and murine retroviruses, respectively. This finding raises the interesting question of the expression and evolution of retroviral genes.     

Expression of the bovine leukemia virus X region in virus-infected cells.

Bovine leukemia virus, like its closest relatives the human T-cell leukemia virus types I and II, contains a 1.8-kilobase X region between the env gene and the 3' long terminal repeat. In this communication, we report the detection and characterization of a subgenomic mRNA from which this X region is presumably translated. This mRNA was produced by a complex splicing mechanism which resulted in juxtaposition of the 5' end of the env gene and the two overlapping X-region open reading frames. Translation of this mRNA could yield at least two distinct proteins depending on which initiation codon is used. Detection of the protein encoded by the BLV X-region long open reading frame has been reported (N. Sagata, J. Tsuzuku-Kawamura, M. Nagayoshi-Aida, F. Shimizu, K.-I. Imagawa, and Y. Ikawa, Proc. Natl. Acad. Sci. USA 82:7879-7883, 1985). Using synthetic peptide antisera, we detected a protein encoded by the short open reading frame in virus-infected cells. The protein migrated in sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of 19,000. It is a nuclear phosphoprotein.     

Comparison of the entire genomes of bovine leukemia virus and human T-cell leukemia virus and characterization of their unidentified open reading frames.

We have compared the sequence of the entire genomes of bovine leukemia virus (BLV) and human T-cell leukemia virus type I (HTLV-I). Both the gag and pol genes show overall strong homologies indicating the close evolutionary relationship of the two retroviruses. However, a surface glycoprotein portion of the env gene shows no appreciable homology, which probably reflects a difference in their host ranges. The 3' end portion of the BLV genome (designated as pXBL) contains an unidentified long open reading frame that has a typical protein-coding property. The potential product of this open reading frame may be a glycoprotein of approximately 40 000 daltons. We note that its amino acid sequence shows low but appreciable homology, especially in its N-terminal quarter, to that of the HTLV-I counterpart (pX product), and we thus suggest that BLV pXBL and HTLV-I pX have diverged from a common ancestral gene. It is tentatively concluded that both the putative pXBL and pX products are respectively produced from a spliced mRNA.