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

Abstract

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.

Stability of AML1 (core) site enhancer mutations in T lymphomas induced by attenuated SL3-3 murine leukemia virus mutants

“…Our results have confirmed previous reports on fluctuations in the number of U3 tandem repeats (8,19). Moreover, no differences in repeat numbers between 5Ј and 3Ј LTRs of the same provirus were found, supporting the hypothesis that fluctuations occur at a preintegration step, most likely reverse transcription.…”

“…Agarose gel electrophoretic analysis of the PCR products identified one or more provirus-specific bands (data not shown). By this kind of analysis, the appearance of more than one band has been found to reflect the presence of more than one type of U3 structure in a given tumor (8,19). Our data are compatible with a fluctuation in the number of U3 tandem repeats in the proviruses of a tumor, an interpretation which is supported by analysis of c-myc-integrated proviruses (see below).…”

“…These were from a GTT-mutant virus-induced tumor not included in the present study and from two tumors induced by another 3-bp transversion mutation at the same site (the ATC mutation) and by a mutant harboring the GTT mutation as well as a 3-bp transversion of the weaker AML1 site II. In all three cases the second-site deletions were found to confer increased T-lymphoid strength and specificity of the transcriptional enhancer (8).…”

“…Since we found no evidence of selection for mutational alterations within the sequence of the impaired AML1 site, the possibility of selection for alterations outside this sequence was examined. In fact, we have previously reported on three cases of tandem repeat sequence deletions in tumors induced by SL3-3 viruses with mutations at the AML1 site (8). These were from a GTT-mutant virus-induced tumor not included in the present study and from two tumors induced by another 3-bp transversion mutation at the same site (the ATC mutation) and by a mutant harboring the GTT mutation as well as a 3-bp transversion of the weaker AML1 site II.…”

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

“…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.…”