Suppressor Mutations within the Core Binding Factor (CBF/AML1) Binding Site of a T-Cell Lymphomagenic Retrovirus

Abstract

The transcriptional enhancer of the lymphomagenic mouse retrovirus SL3 contains a binding site for the transcription factor core binding factor (CBF; also called AML1, PEBP2, and SEF1). The SL3 CBF binding site is called the core. It differs from the core of the weakly lymphomagenic mouse retrovirus Akv by one nucleotide (the sequences are TGTGGTTAA and TGTGGTCAA, respectively). A mutant virus called SAA that was identical to SL3 except that its core was mutated to the Akv sequence was only moderately attenuated for lymphomagenicity. In most SAA-infected mice, tumor proviruses contained either reversions of the original mutation or one of two novel core sequences. In 20% of the SAA-infected mice, tumor proviruses retained the original SAA/Akv core mutation but acquired one of two additional mutations (underlined), TGCGGTCAA or TGTGGTCTA, that generated core elements called So and T*, respectively. We tested whether the novel base changes in the So and T* cores were suppressor mutations. SL3 mutants that contained So or T* cores in place of the wild-type sequence were generated. These viruses induced T-cell lymphomas in mice more quickly than SAA. Therefore, the mutations in the So and T* cores are indeed second-site suppressor mutations. The suppressor mutations increased CBF binding in vitro and transcriptional activity of the viral long terminal repeats (LTRs) in T lymphocytes to levels comparable to those of SL3. Thus, CBF binding was increased by any of three different nucleotide changes within the sequence of the SAA core. Increased CBF binding resulted in increased LTR transcriptional activity in T cells and in increased viral lymphomagenicity.

Selection of Reversions and Suppressors of a Mutation in the CBF Binding Site of a Lymphomagenic Retrovirus

“…The suppressor mutations also restored transcriptional activity of the viral LTR to levels comparable to that of SL3 in T cells. In addition, the mutations in the So and T* cores also restored CBF binding activity to SL3 levels (17). Thus, viruses with either reversions or suppressor mutations within the core were selected during the course of lymphomagenesis by SAA, presumably as a function of increased transcriptional activity in T cells.…”

“…Viral constructs with cores called So (TGCGGTCAA) or T* (TGT GGTCTA) that contained the second-site mutations were engineered. The mutations in the So and T* cores were found to be second-site suppressor mutations, because viruses containing them were significantly more pathogenic than the SAA virus (17). The suppressor mutations also restored transcriptional activity of the viral LTR to levels comparable to that of SL3 in T cells.…”

“…The core exhibited about a fivefoldhigher binding affinity for CBF than a second CBF binding site in each 72-bp repeat known as core II (25). Mutations in the SL3 core element reduced transcription in T cells about fourfold in most T-cell lines (3,17,19,24,30). Mutations of core II by itself reduced transcription twofold or less in T cells (30).…”

“…Akv is an endogenous ecotropic MuLV from AKR mice that is relatively weakly pathogenic (15). Although the Akv core bound CBF in electrophoretic mobility shift assays about twofold less efficiently than the SL3 core (29), a mutant of SL3 containing the Akv sequence in both enhancer cores had significantly reduced lymphomagenicity and transcriptional activity in T cells (17,19). The mutant virus, referred to as SAA, was identical to SL3 except for the T-to-C change in the enhancer cores of the LTR in both 72-bp repeats.…”

“…The mutant virus, referred to as SAA, was identical to SL3 except for the T-to-C change in the enhancer cores of the LTR in both 72-bp repeats. SAA exhibited an increased latency period prior to the appearance of lymphomas relative to SL3, and/or the incidence of the disease was decreased, depending on the mouse strain utilized (17,19). Thus, the precise sequence of the SL3 core element was crucial for maximum pathogenicity of the virus.…”