Although sheep are not natural hosts for BLV, studying infection and pathogenesis in this model might be informative for understanding pathogenesis pertaining to other deltaretroviruses. To circumvent the problem of genomic RNA instability, infectious proviruses were cloned and injected into sheep or calves [30
]. Hence, infection of sheep with proviral clone 344 leads to tumor or leukemia after a mean latency period of 33 months [170
]. Since the BLV provirus can be re-amplified from the tumor cells, the three conditions required to fulfill Koch's postulate are demonstrated (i.e. the cloning of the virus, the analysis of its pathogenicity, and its re-isolation from the lesions), clearly establishing viral causality in leukemia/lymphoma.
Among other isolates, clone 395 is deficient for infectivity in vivo
, due to the presence of a E-to-K mutation at codon 303 of the Tax protein [83
]. In cell culture, transfection of provirus 395 yields reduced levels of Tax activity (~ 10 % of wild-type) although the amount of major capsid protein p24 expressed inside the cells and in the supernatant remains unaffected. Adequate levels of transactivation are required for infectivity in vivo
supporting the notion that tax
is an essential gene.
The injection of sheep with provirus 344 fulfills all the requirements of a model system linking fields as diverse as molecular biology, virology and pathogenesis. Therefore, clone 344 has been used to construct a series of derivative proviruses harboring mutations or deletions in different parts of the genome. As expected, large deletions within the structural or enzymatic gag
genes destroy infectivity in vivo
]. Interestingly, co-infection of sheep with two defective recombinants can generate a replication-competent and pathogenic virus by homologous recombination in vivo
. As mentioned earlier (see section 2), several residues/regions in the viral genome are essential for infection: E303 of Tax, Y197 of the TM ITAM motifs and the MHR domain of CA [112
]. Considering that genetic information is highly condensed in the proviral genome, it is surprising to identify a large domain within the provirus that is dispensable for infectivity in vivo
. Indeed, the deletion of the region which expands from the end of the env
gene to the splice acceptor site of the tax/rex
mRNA does not impair infectivity ([83
] and unpublished results). Since these sequences correspond respectively to the third and second exons of the R3 and G4 mRNAs, it appears that these genes are not essential for infectivity in vivo
. Similar conclusions were drawn from HTLV mutant proviruses deleted in the ORFs encoding the p12I
orthologs of R3 and G4 [240
]. Importantly, the R3/G4 deletion greatly interferes with the efficiency of BLV propagation and restricts pathogenesis [170
]. Very recently, however, one out of 20 sheep infected with a R3/G4 mutant developed a lymphoma after 7.5 years of latency, demonstrating that the deleted sequences are not strictly required for pathogenesis (Florins et al, in preparation). It does remain that the integrity of the R3/G4 genes significantly contributes to disease frequency and latency (see Table ).
Summary of unexpected conclusions deduced from the BLV/sheep model
The BLV 344/sheep system has been instrumental for unraveling determinants of the viral replication cycle. Binding of the viral envelope complex to the target cell membrane leads to a process of fusion, allowing subsequent viral entry. The fusion mechanism can be reproduced in vitro
by co-cultivation of fibroblasts or lymphocytes expressing Env proteins at their surface and target cells like CC81, leading to polykaryocytosis [156
]. The fusion process is mediated by the oblique insertion of the TM aminoterminal peptide into the lipid bilayer of the cell membrane. Forcing the peptide to adopt a parallel orientation by mutation abrogates fusion in cell culture and infectivity in vivo
]. In contrast, replacement of the peptide by the corresponding residues derived from SIV (simian immunodeficiency virus) yields a fully fusion-competent envelope. However, a virus carrying this mutation lacks infectivity, suggesting that additional constraints are operative in vivo
. What is even more surprising is that TM mutants (i.e. A60V and A64S) that are deficient for cell fusion in vitro
nevertheless support viral infectivity in vivo
]. And, very unexpectedly, these mutant viruses can propagate at wild-type levels and are pathogenic in sheep (see Table ). Since the A60V and A64S mutants are also impaired for SU/TM interaction, it seems that integrity of the envelope complex is not strictly required in vivo
. If the cell fusion processes in vitro
and in vivo
are indeed impaired equally by these mutations, then the findings offer the unexpected suggestion that BLV replicates by mitotic division of the infected cell rather than by de novo envelope-cell receptor mediated infectious cycle.
gene is assumed to be a major factor required for viral replication and pathogenesis. Tax activates LTR-directed transcription and immortalizes primary cells in culture [190
]. The two activities of Tax can be dissociated; for example, mutations in the zinc finger abrogate transactivation without altering immortalization [194
]. Conversely, substitution of the two major phosphorylation sites in Tax does not alter its transcriptional activity but destroys its oncogenicity in REF cells [200
]. As illustrated by the defect seen with provirus 395, Tax's transactivation activity is required for viral infectivity in vivo
. In contrast, a provirus (Tax106+293) harboring mutated phosphorylation sites remains infectious and propagates at wild-type levels in sheep. In addition, the Tax106+293 mutant is pathogenic despite a loss in its ability to transform primary cells in vitro
] (see Table ). These findings suggest that a deficiency in Tax oncogenic potential as revealed by the REF immortalization does not correlate with leukemogenesis in vivo
As previously mentioned, the BLV transcriptional promoter located in the 5' LTR contains suboptimal binding sequences for the CREB transcription factor. Remarkably, the cyclic-AMP responsive site (CRE) consensus "TGACGTCA" is never strictly conserved in any viral 21 bp element which invariably contains an imperfect substitution (for example, A
TCA). Restoring a perfect CRE sequence into the promoter increases LTR (long terminal repeat) promoter activity, as expected [88
]. However, the proviral loads are drastically reduced in sheep infected with a virus harboring this type of change (see Table ). It is tempting to speculate that BLV may have evolved a self-attenuating process (perhaps for purposes of escaping immunosurveillence) which encourages the virus to maintain a less active promoter through suboptimal use of the CRE-dependent pathway. If this speculation is correct, then one thought is that transcriptional repression of viral expression may be a key factor which regulates viral persistence and spread. As mentioned in section 2, the activity of the viral LTR is also thought to be regulated by E-box motifs which overlap the CRE sites [88
]. However, an E-box mutant virus is infectious, replicates to wild-type levels and is pathogenic in sheep. These observations question the clear significance of the E-box motif in vivo
] (and unpublished results).
Collectively the experimental findings from BLV research emphasize the dichotomy between subgenomic in vitro results and counterpart findings achieved using replicating viruses in vivo; they reinforce the critical need to perform pathogenesis studies in vivo.