INCREASED LYMPHOMAGENICITY AND RESTORED DISEASE SPECIFICITY OF AML1 SITE (CORE) MUTANT SL3-3 MURINE LEUKEMIA-VIRUS BY A 2ND-SITE ENHANCER VARIANT EVOLVED IN-VIVO

SL3-3 is a highly T-lymphomagenic murine retrovirus. The major genetic determinant of disease is the transcriptional enhancer, which consist s of a repeated region with densely packed binding sites for several t ranscription factors, including AML1 (also known as core binding facto r and polyoma enhancer-binding protein 2) and nuclear factor 1 (NF1), Previously, we examined the enhancer structure of proviruses from muri ne tumors induced by SL3-3 with mutated AML1 (core) sites and found a few cases of second-site alterations. These consisted of deletions inv olving the NF1 sites and alterations in overall number of repeat eleme nts, and they conferred increased enhancer strength in transient trans cription assays. We have now tested the pathogenicity of a virus harbo ring one such second-site variant enhancer in inbred NMR1 mice, It ind uced lymphomas with a 100% incidence and a significantly shorter laten cy than the AML1 mutant it evolved from, The enhancer structure thus r epresents the selection for a more tumorigenic virus variant during th e pathogenic process, Sequencing of provirus from the induced tumors s howed the new enhancer variant to be genetically stable. Also, Souther n blotting showed that the tumors induced by the variant were T-cell l ymphomas, as were the wild-type-induced lymphomas, In contrast, tumors induced by the original core/AML1 site I-II mutant appeared to be of non-TT-cell origin and several proviral genomes with altered enhancer regions could be found in the tumors. Moreover, reporter constructs wi th the new tumor-derived variant could not be transactivated by AML1 i n cotransfection experiments as could the wild type. These results emp hasize the importance of both core/AML1 site I and site II for the pat hogenic potential of SL3-3 and at the same time show that second-site alterations can form a viral variant with a substantial pathogenic pot ential although both AML1 sites I and II are nonfunctional.