A detailed molecular analysis of complete bovine leukemia virus genomes isolated from B-cell lymphosarcomas

Abstract

It is widely accepted that the majority of cancers result from multiple cellular events leading to malignancy after a prolonged period of clinical latency, and that the immune system plays a critical role in the control of cancer progression. Bovine leukemia virus (BLV) is an oncogenic member of the Retroviridae family. Complete genomic sequences of BLV strains isolated from peripheral blood mononuclear cells (PBMC) from cattle have been previously reported. However, a detailed characterization of the complete genome of BLV strains directly isolated from bovine tumors is much needed in order to contribute to the understanding of the mechanisms of leukemogenesis induced by BLV in cattle. In this study, we performed a molecular characterization of BLV complete genomes from bovine B-cell lymphosarcoma isolates. A nucleotide substitution was found in the glucocorticoid response element (GRE) site of the 5' long terminal repeat (5'LTR) of the BLV isolates. All amino acid substitutions in Tax previously found to be related to stimulate high transcriptional activity of 5'LTR were not found in these studies. Amino acid substitutions were found in the nucleocapsid, gp51 and G4 proteins. Premature stop-codons in R3 were observed. Few mutations or amino acid substitutions may be needed to allow BLV provirus to achieve silencing. Substitutions that favor suppression of viral expression in malignant B cells might be a strategy to circumvent effective immune attack.

Figure 1

Alignment of 5^′LTR nucleotide sequences of BLV strains. BLV strains isolated from PBMC cells and previously described are shown by accession number on the left side of the figure. BLV isolates from B-lymphosarcoma tumors (LS1 through LS3) are shown by name. Identity with BLV strain LS1 is indicated by a dot. The U3, R, and U5 regions are indicated on top of the alignment. The three TxRE enhancer regions are shown in green, cyclic AMP- responsive element (CRE) sequences are underlined and E-Box sequences are shown in italics. Binding sites for PU.1/Spi-B are shown in bold. The glucocorticoid responsive element (GRE) binding site is shown in bold, italics and underlined. Nuclear factor κB (NF-κB) binding sites are shown double underlined. The CAT box and GATAA box promoters (PROMT) sequences are indicated in yellow and in bold italics, respectively. The polyadenylation site (PAS) is shown in magenta and the CAP site is shown in light blue. The tRNA proline primer binding sites are shown in red. The downstream activator sequence (DAS) and the interferon regulatory factor (IRF) binding sites are shown in dark and light grey, respectively.

Figure 2

Alignment of amino acid sequences of Gag and Protease (Pro) proteins of BLV isolates. In (A), alignment of Gag polyprotein sequences is shown. Matrix (p15-MA), capsid (p24-CA) and nucleocapsid (p12-NC) protein sequences are shown in green, light blue and magenta, respectively. Zinc finger domains are shown in bold and italics. NC residues in boldface indicate basic residues. Conserved residues among BLV, HTLV-1 and HTLV-2 are shown bold underlined. In (B) alignment of protease (Pro) sequences is shown. Residues proven or predicted to be involved in S2 binding subsites are shown in bold. Residues that appear in HIV-1 drug resistance at the equivalent position in HIV-1 protease [32] are shown in bold underlined. The rest is the same as Figure 1.

Figure 3

Alignment of amino acid sequences of Polymerase (Pol) proteins of BLV isolates. The reverse transcriptase (RT) region is shown in green and the endonuclease region is shown in light blue. The rest is the same as Figure 1.

Figure 4

Alignment of amino acid sequences of Envelope (Env) proteins of BLV isolates. Amino acid residues corresponding to gp51 (SU) and gp30 (TM) proteins are shown on the top of the alignment. Leader peptide of SU is shown in green. Structural strong turn motif GYPD is shown in bold, italics and double underlined. Conformational epitope region is indicated on top of the alignment. Linear epitopes are shown in italics and underlined [41,42]. Receptor-binding domain (RBD) [43] residues are delimited by two triangles. Second strong turn, SSSG, is shown in bold and italics. Amino acids involved in neutralization domains are shown in yellow. CD8 + −T epitope is shown in red. SU transmembrane hydrophobic region (TMHR) is shown in light blue. TM fusion peptide is shown in light grey and residues believed to span host cell membranes are indicated double lined. BLV leash and α-helical region (LHR) [35] is shown in bold, italics and underlined. Epitope GD21 is shown in magenta. TM membrane-spanning region is shown in bold and italics. The cytoplasmic domain is indicated on top of the alignment. The rest is the same as in Figure 1.

Figure 5

Alignment of amino acid sequences of Tax and Rex proteins of BLV isolates. In (A) the alignment of Tax sequences is shown. The region between amino acids 240 and 265, in which missense mutations influence the transactivation activity of the Tax protein [46] is shown in light blue. A putative zinc finger domain is shown in grey, a leucine-rich activation domain in yellow and sites of phosphorylation are indicated in red. Position 303, where a previously described substitution E303K gave rise to a replication-deficient virus [47] is shown in green. Positions where substitutions have been previously reported to have an effect on transactivation activity are shown in bold. In (B) alignment of Rex sequences is shown. Nuclear export signal (NES) is shown in yellow and the nuclear localization signal (NLS) is shown in green. The rest is the same as Figure 1.

Figure 6

Alignment of amino acid sequences of accessory proteins R3 and G4 of BLV isolates. In (A) alignment of G4 sequences is shown. The leader peptide is shown in green, and the two putative cellular protease cleavage sites are indicated by inverted triangles on top of the alignment. The myb-like motif (MYB) and the arginine-rich nucleus targeting RNA-binding region (ARR) are shown in red. In (B) alignment of R3 sequences are shown. Termination codons are shown by an asterisk and indicated by an arrow.