Mechanisms of leukemogenesis induced by bovine leukemia virus: prospects for novel anti-retroviral therapies in human

Abstract

In 1871, the observation of yellowish nodules in the enlarged spleen of a cow was considered to be the first reported case of bovine leukemia. The etiological agent of this lymphoproliferative disease, bovine leukemia virus (BLV), belongs to the deltaretrovirus genus which also includes the related human T-lymphotropic virus type 1 (HTLV-1). This review summarizes current knowledge of this viral system, which is important as a model for leukemogenesis. Recently, the BLV model has also cast light onto novel prospects for therapies of HTLV induced diseases, for which no satisfactory treatment exists so far.

Figure 1

Schematic representation of the BLV viral particle. Two copies of single stranded genomic RNA are packaged in a viral particle. The CA (p24) proteins form a capsid that contains the viral RNA in interaction with nucleocapsid NC (p12). Two enzymatic proteins (RT and IN) required, respectively, for reverse transcription and integration of the viral genome are also packaged into the capsid. The matrix protein MA (p15) interconnects the capsid and the outer envelope that is formed by a lipid bilayer of cellular origin in which a complex of viral proteins (gp51 SU and gp30 TM) are inserted.

Figure 2

Structure of the BLV provirus: genes, RNA transcripts and viral protein. The provirus is flanked by two identical long terminal repeat sequences (LTRs) and contains the open reading frames (orfs) corresponding to gag, prt (protease), pol and env. Several orfs coding for Tax, Rex, R3 and G4 are present in the X region between env and the 3'LTR. The genomic RNA transcript initiates and terminates in the 5' and 3' LTRs, respectively. This genomic RNA serves as a template for the expression of the gag-prt-pol precursors (pr145, pr66 and pr44) that are processed in structure and enzymatic proteins: matrix (MA) p15, capsid (CA) p24, nucleocapsid (NC) p12, protease (PRT) p14 and, p80 (RT/IN) harboring reverse transcriptase, RNAse H and integrase activities. A large intron corresponding to gag-prt-pol is excised to yield the envelope (env) RNA. After translation, the pr72 precursor is cleaved in two subunits: the extracellular (SU) gp51 and the transmembrane (TM) gp30 glycoproteins. To generate the Tax/Rex messenger RNA, a second intron is cleaved. This double-spliced RNA encodes both the p34 Tax protein using an initiation codon at the end of pol and Rex which shares the same AUG as Env pr72. Two minor RNAs identified by RT/PCR code for p5 (R3) and p11 (G4). The R3 RNA is similar to the double-spliced Tax/Rex message but the second intron is shorter and splicing occurs at the 5' end of the R3 frame. The R3 protein shares its aminoterminal end with Rex and pr72. In the G4 message, a very large intron is excised between a particular splice donor site different from that of the other viral RNAs and an acceptor just 5' to the G4 frame. The G4 protein initiates at a suboptimal CUG codon located in R of the 5'LTR.

Figure 3

Subcellular localisation of the Tax, Rex, R3 and G4 proteins. Hela cells were transfected with expression vectors for Tax, Rex, R3 and G4, cultivated during 24 hours, indirectly marked with FITC-conjugated antibodies and visualized under a fluorescent microscope.

Figure 4

Cells expressing CA are not prone to undergo apoptosis. Dot plot resulting from a flow cytometry analysis of peripheral blood mononuclear cells isolated from a lymphocytic BLV-infected sheep and transiently cultivated during 24 hours. Cells were labeled with a monoclonal antibody specific for the viral major capsid protein p24gag (X axis: viral expression) and by TUNEL (Y axis: apoptosis).

Figure 5

Canulation of mesenteric lymphatic vessels. The lymphatic vessels and the lymph nodes were colored by injection of Evans blue and dwelling catheters were inserted into the efferent lymphatics.

Figure 6

Hypothetical mechanism of BLV replication. Normal B cell activation and proliferation depend on a variety of immune stimuli, involving the BCR (and possibly CD5, section 3), CD40 ligand expressed by T cells, various cytokines or even autoantigens (see section 5). We hypothesize that BLV replication is initiated by these classical regulatory mechanisms because viral expression can be augmented by molecules that mimic B cell activation by immune cells. Tax expression precedes that of structural and enzymatic proteins and promotes entry into the cell cycle, providing a selective proliferative advantage of the infected cells (see section 4, The Tax transactivator). Uneven distribution of the viral proteins upon mitosis would generate two types of infected cells containing or not BLV antigens (see section 4, The envelope gene). Other processes might account for silencing of viral expression in one daugther cell such as a specific inhibition by a viral factor such as HTLV p30 or HBZ (still to be discovered for BLV). We think that BLV expression is ongoing continuously in vivo because viral transcripts are detected in whole blood immediately upon incubation at 37°C in the absence of any exogenous factors (see section 5, Low levels of viral expression are detected in vivo). Virus-positive cells would be destroyed by the immune response (see section 5, Host humoral and cytotoxic immune responses) or would undergo apoptosis via intrinsic or extrinsic pathways (section 6: Is BLV inhibiting apoptosis?). These cells would thus permanently stimulate the host's immune response. Cells in which viral expression has been shut off or lacking viral antigens after mitosis would enter a resting stage in the absence of Tax and/or immune stimulation. These cells surviving destruction by the immune response can be isolated and observed ex vivo.

Figure 7

Valproate, an inhibitor of deacetylases, activates gene transcription and induces apoptosis. Reciprocal reactions, acetylation and deacetylation are catalyzed by histone acetyltransferase (HAT) and histone deacetylase (HDAC). Acetylation of chromatin leads to deconsendation and activates transcription of a subset of genes. Valproate induces hyperacetylation, activates BLV expression and triggers apoptosis in cell culture. Valproate treatment of sheep with leukemia/lymphoma reduces the number of tumor cells.