Various modes of basic helix-loop-helix protein-mediated regulation of murine leukemia virus transcription in lymphoid cell lines

Sørensen

Schmidt

et al. 1998

Akv is an endogenous, ecotropic murine leukemia virus (MuLV) of the AKR strain. It has served as a prototype nonpathogenic or weakly pathogenic reference virus for studies of closely related potent lymphomagenic viruses such as the T-lymphomagenic SL3-3. We here report that Akv and an Akv mutant (Akv1-99) with only one copy of the 99-bp transcriptional enhancer induce malignant lymphomas with nearly 100% incidence and mean latency periods of 12 months after injection into newborn NMRI mice. Molecular analysis of tumor DNA showed that the majority of the tumors were of the B-cell type. Sequence analysis of proviral transcriptional enhancers in DNA of B-cell lymphomas revealed conservation of the enhancer sequence, as well as a lack of sequence duplications of the Akv1-99 variant, while the repeat copy number in Akv was subject to fluctuations. In support of a B-cell specificity of the Akv enhancer, a murine plasmacytoma cell line was found to sustain three- to fivefold-higher transient transcriptional activity upon the Akv and Akv1-99 enhancers than upon the enhancer of the T-lymphomagenic SL3-3 MuLV. Thus, the overall picture is that Akv MuLV possesses a B- lymphomagenic potential and that the second copy of the 99-bp sequence seems to be of minor importance for this potential. However, in one animal the lymphomas induced by Akv1-99 were of the T-cell type. Among the 24 tumors analyzed only this one harbored a clonal proviral integration in the c-myc locus. This provirus had undergone a duplication of a 113-bp sequence of the enhancer region, partly overlapping with the 99-bp repeat of Akv, as well as a few single nucleotide alterations within and outside the repeats. Taken together with previous studies, our results suggest that T- versus B-lymphomagenic specificity of the enhancer is governed by more than one nucleotide difference and that alterations in binding sites for transcription factors of the AML1 and nuclear-factor-1 families may contribute to this specificity.

Section: Viral Enhancer Strength In a B-lymphoid Cell Line The Recovmentioning

confidence: 93%

B-Cell Lymphoma Induction by Akv Murine Leukemia Viruses Harboring One or Both Copies of the Tandem Repeat in the U3 Enhancer

Sørensen

Schmidt

et al. 1998

“…The murine T-lymphoid cell line L691 (15) was grown in RPMI 1640 medium containing Glutamax-1 (Gibco BRL, Life Technologies) and supplemented with 10% newborn calf serum and 100 U of penicillin per ml and 100 g of streptomycin per ml. NIH 3T3 cells and the murine plasmacytoma B-cell line MPC11 (42) were grown in Dulbecco's modified Eagle medium containing Glutamax-1 (Gibco) and supplemented with 10% serum and antibiotics as described above.…”

Section: Methodsmentioning

confidence: 99%

“…Characterized binding sites for transcription factors in this repeat region include the helix-loop-helix proteins SEF2-1 (or E2-2) (12) and ALF1 (43,44) the glucocortocoid receptor (GR) (9), nuclear factor 1 (NF1) (45), and the hematopoietic factors Ets1 (40), Myb (71), and AML1 (65,66,72). Of these factors, ALF1, GR, AML1, and c-Myb have been directly shown to activate the transcription of SL3-3 (9,42,71,72).…”

mentioning

confidence: 99%

Second-site proviral enhancer alterations in lymphomas induced by enhancer mutants of SL3-3 murine leukemia virus: negative effect of nuclear factor 1 binding site

Ethelberg

Hållberg

et al. 1997

Self Cite

SL3-3 is a highly T-lymphomagenic murine retrovirus. Previously, mutation of binding sites in the U3 repeat region for the AML1 transcription factor family (also known as core binding factor [CBF], polyomavirus enhancer binding protein 2 [PEBP2], and SL3-3 enhancer factor 1 [SEF1]) were found to strongly reduce the pathogenicity of SL3-3 (B. Hallberg, J. Schmidt, A. Luz, F. S. Pedersen, and T. Grundström, J. Virol. 65:4177-4181, 1991). We have now examined the few cases in which tumors developed harboring proviruses that besides the AML1 (core) site mutations carried second-site alterations in their U3 repeat structures. In three distinct cases we observed the same type of alteration which involved deletions of regions known to contain binding sites for nuclear factor 1 (NF1) and the addition of extra enhancer repeat elements. In transient-expression experiments in T-lymphoid cells, these new U3 regions acted as stronger enhancers than the U3 regions of the original viruses.This suggests that the altered proviruses represent more-pathogenic variants selected for in the process of tumor formation. To analyze the proviral alterations, we generated a series of different enhancer-promoter reporter constructs. These constructs showed that the additional repeat elements are not critical for enhancer strength, whereas the NF1 sites down-regulate the level of transcription in T-lymphoid cells whether or not the AML1 (core) sites are functional. We therefore also tested SL3-3 viruses with mutated NF1 sites. These viruses have unimpaired pathogenic properties and thereby distinguish SL3-3 from Moloney murine leukemia virus.

“…Each site is thus present two or three times. The interacting factors include the AML1 transcription factor family (45,54), nuclear factor 1 (NF1) (9,29), members of the Ets transcription factor family (28,53), basic helix-loop-helix (bHLH) factor transcription factors (26,27), the glucocorticoid receptor (GR) (6), and the c-Myb transcription factor (28,53). Current thinking holds that the transcription factor binding sites in the SL3-3 repeat region, as in similar regions in other MLVs, match the transcription factor profile of the hematopoietic target cells, enabling the virus to have a high replication rate and to act as a powerful insertional mutagen, thus determining the specificity and strength of the virus as a pathogen.…”

mentioning

confidence: 99%

Increased lymphomagenicity and restored disease specificity of AML1 site (core) mutant SL3-3 murine leukemia virus by a second-site enhancer variant evolved in vivo

Ethelberg

Schmidt

et al. 1997

Self Cite

SL3-3 is a highly T-lymphomagenic murine retrovirus. The major genetic determinant of disease is the transcriptional enhancer, which consists of a repeated region with densely packed binding sites for several transcription factors, including AML1 (also known as core binding factor and polyoma enhancer-binding protein 2) and nuclear factor 1 (NF1). Previously, we examined the enhancer structure of proviruses from murine tumors induced by SL3-3 with mutated AML1 (core) sites and found a few cases of second-site alterations. These consisted of deletions involving the NF1 sites and alterations in overall number of repeat elements, and they conferred increased enhancer strength in transient transcription assays. We have now tested the pathogenicity of a virus harboring one such second-site variant enhancer in inbred NMRI mice. It induced lymphomas with a 100% incidence and a significantly shorter latency than the AML1 mutant it evolved from. The enhancer structure thus represents the selection for a more tumorigenic virus variant during the pathogenic process. Sequencing of provirus from the induced tumors showed the new enhancer variant to be genetically stable. Also, Southern blotting showed that the tumors induced by the variant were T-cell lymphomas, 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-T-cell origin and several proviral genomes with altered enhancer regions could be found in the tumors. Moreover, reporter constructs with the new tumor-derived variant could not be transactivated by AML1 in cotransfection experiments as could the wild type. These results emphasize the importance of both core/AML1 site I and site II for the pathogenic potential of SL3-3 and at the same time show that second-site alterations can form a viral variant with a substantial pathogenic potential although both AML1 sites I and II are nonfunctional.