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F S Pedersen

Publications and source records attributed to F S Pedersen.

At least 37 records · Page 2Linked to original sources
PubMed1999

The nucleotide sequence of the high-leukemogenic murine retrovirus SL3-3 reveals a patch of mink cell focus forming-like sequences upstream of the ecotropic envelope gene. Brief report.

We report the complete nucleotide sequence of the potent T-lymphomagenic murine retrovirus SL3-3. The non-LTR regions of the virus show 98% sequence identity to the endogenous ecotropic Akv murine leukemia virus. While the region encoding the surface envelope protein is completely identical to that of Akv, a approximately 200 nucleotide stretch in the integrase encoding region upstream of env is similar to the sequence of mink cell focus forming (MCF) viruses and shows a complete match with the mouse retrovirus 10A1. The history of SL3-3 may therefore include recombination involving an Akv-like virus and a virus with MCF-like sequences.

Animals↗
PubMed1999

The kissing-loop motif is a preferred site of 5' leader recombination during replication of SL3-3 murine leukemia viruses in mice.

A panel of mouse T-cell lymphomas induced by SL3-3 murine leukemia virus (MLV) and three primer binding site mutants thereof (A. H. Lund, J. Schmidt, A. Luz, A. B. Sorensen, M. Duch, and F. S. Pedersen, J. Virol. 73:6117-6122, 1999) were analyzed for the occurrence of recombination between the exogenous input virus and endogenous MLV-like sequences within the 5' leader region. Evidence of recombination within the region studied was found in 14 of 52 tumors analyzed. Sequence analysis of a approximately 330-bp fragment of 44 chimeric proviruses, encompassing the U5, the primer binding site, and the upstream part of the 5' untranslated region, enabled us to map recombination sites, guided by distinct scattered nucleotide differences. In 30 of 44 analyzed sequences, recombination was mapped to a 33-nucleotide similarity window coinciding with the kissing-loop stem-loop motif implicated in dimerization of the diploid genome. Interestingly, the recombination pattern preference found in replication-competent viruses from T-cell tumors is very similar to the pattern previously reported for retroviral vectors in cell culture experiments. The data therefore sustain the hypothesis that the kissing loop, presumably via a role in RNA dimer formation, constitutes a hot spot for reverse transcriptase-mediated recombination in MLV.

5' Untranslated Regions↗
PubMed1999

Increased induction of osteopetrosis, but unaltered lymphomagenicity, by murine leukemia virus SL3-3 after mutation of a nuclear factor 1 site in the enhancer.

SL3-3 is a murine leukemia virus which is only weakly bone pathogenic but highly T-cell lymphomagenic. A major pathogenic determinant is the transcriptional enhancer comprising several transcription factor binding sites, among which are three identical sites for nuclear factor 1 (NF1). We have investigated the pathogenic properties of NF1 site enhancer mutants of SL3-3. Two different mutants carrying a 3-bp mutation either in all three NF1 sites or in the central site alone were constructed and assayed in inbred NMRI mice. The wild type and both mutants induced lymphomas in all mice, with a mean latency period of 9 weeks. However, there was a considerable difference in osteopetrosis induction. Wild-type SL3-3 induced osteopetrosis in 11% of the mice (2 of 19), and the triple NF1 site mutant induced osteopetrosis in none of the mice (0 of 19), whereas the single NF1 site mutant induced osteopetrosis in 56% (10 of 18) of the mice, as determined by X-ray analysis. A detailed histological examination of the femurs of the mice was carried out and found to support this diagnosis. Thus, the NF1 sites of SL3-3 are major determinants of osteopetrosis induction, without determining lymphomagenesis. This conclusion was further supported by evaluation of the bone pathogenicity of other SL3-3 enhancer variants, the lymphomagenicity of which had been examined previously. This evaluation furthermore strongly indicated that the core sites, a second group of transcription factor binding sites in the viral enhancer, are necessary for the osteopetrosis induction potential of SL3-3.

Animals↗
PubMed1999

Amphotropic murine leukemia virus entry is determined by specific combinations of residues from receptor loops 2 and 4.

Pit2 is the human receptor for amphotropic murine leukemia virus (A-MuLV); the related human protein Pit1 does not support A-MuLV entry. Interestingly, chimeric proteins in which either the N-terminal or the C-terminal part of Pit2 was replaced by the Pit1 sequence all retained A-MuLV receptor function. A possible interpretation of these observations is that Pit1 harbors sequences which can specify A-MuLV receptor function when presented in a protein context other than Pit1, e.g., in Pit1-Pit2 hybrids. We reasoned that such Pit1 sequences might be identified if presented in the Neurospora crassa protein Pho-4. This protein is distantly related to Pit1 and Pit2, predicted to have a similar membrane topology with five extracellular loops, and does not support A-MuLV entry. We show here that introduction of the Pit1-specific loop 2 sequence conferred A-MuLV receptor function upon Pho-4. Therefore, we conclude that (i) a functional A-MuLV receptor can be constructed by combining sequences from two proteins each lacking A-MuLV receptor function and that (ii) a Pit1 sequence can specify A-MuLV receptor function when presented in another protein context than that provided by Pit1 itself. Previous results indicated a role of loop 4 residues in A-MuLV entry, and the presence of a Pit2-specific loop 4 sequence was found here to confer A-MuLV receptor function upon Pho-4. Moreover, the introduction of a Pit1-specific loop 4 sequence, but not of a Pit2-specific loop 4 sequence, abolished the A-MuLV receptor function of a Pho-4 chimera harboring the Pit1-specific loop 2 sequence. Together, these data suggest that residues in both loop 2 and loop 4 play a role in A-MuLV receptor function. A-MuLV is, however, not dependent on the specific Pit2 loop 2 and Pit2 loop 4 sequences for entry; rather, the role played by loops 2 and 4 in A-MuLV entry can be fulfilled by several different combinations of loop 2 and loop 4 sequences. We predict that the residues in loops 2 and 4, identified in this study as specifying A-MuLV receptor function, are to be found among those not conserved among Pho-4, Pit1, and Pit2.

Amino Acid Sequence↗
PubMed1999

Replication and pathogenicity of primer binding site mutants of SL3-3 murine leukemia viruses.

Retroviral reverse transcription is primed by a cellular tRNA molecule annealed to an 18-bp primer binding site sequence. The sequence of the primer binding site coincides with that of a negatively acting cis element that mediates transcriptional silencing of murine leukemia virus (MLV) in undifferentiated embryonic cells. In this study we test whether SL3-3 MLV can replicate stably using tRNA primers other than the cognate tRNAPro and analyze the effect of altering the primer binding site sequence to match the 3' end of tRNA1Gln, tRNA3Lys, or tRNA1,2Arg in a mouse pathogenicity model. Contrary to findings from cell culture studies of primer binding site-modified human immunodeficiency virus type 1 and avian retroviruses, our findings were that SL3-3 MLV may stably and efficiently replicate with tRNA primers other than tRNAPro. Although lymphoma induction of the SL3-3 Lys3 mutant was significantly delayed relative to that of the wild-type virus, molecular tumor analysis indicated that all the primer binding site-modified viruses induce T-cell lymphomas similar to those induced by the wild-type virus in terms of frequencies of genomic rearrangements within the T-cell receptor beta-chain, the immunoglobulin kappa light chain, and the c-myc locus. Whereas none of the mutants were found to revert to tRNAPro primer utilization, in two tumors resulting from the injection of the SL3-3 Lys3 mutant the primer binding site was altered to match that of a new primer species, tRNA1,2Lys. In addition, recombination with endogenous viruses resulting in the generation of recombinant viruses carrying a glutamine primer binding site was detected in the majority of the tumors induced by the SL3-3 Lys3 mutant as well as in two tumors induced by wild-type SL3-3 and the SL3-3 Arg1,2 mutant.

3T3 Cells↗
PubMed1998

RNA levels of human retrovirus receptors Pit1 and Pit2 do not correlate with infectibility by three retroviral vector pseudotypes.

The gibbon ape leukemia virus (GaLV) and the amphotropic murine leukemia virus (A-MuLV) infect human cells via specific receptors, Pit1 and Pit2, respectively. mRNA levels of these receptors were determined by Northern analysis and for Pit2 in addition by quantitative RT-PCR. Pit1 and Pit2 were expressed in different amounts in human tissues and cell lines; Pit1-specific mRNA was generally more abundant than Pit2 mRNA. No correlation was found between Pit1 and Pit2 RNA levels and infectibility by GaLV and A-MuLV pseudotyped vectors, respectively. GaLV and A-MuLV revealed a partial reciprocal interference. MuLV-10A1 can utilize both Pit1 and Pit2 for entry into cells but could not infect any of the 14 human cell lines more efficiently than A-MuLV or GaLV. Interference assays suggested that MuLV-10A1 has a higher affinity for and infected most cells predominantly by Pit2. However, at least in one cell line it used Pit1 more efficiently for entry. We conclude that (1) Pit1 and Pit2 mRNA levels in human cells are not indicative of the infectibility by GaLV and A-MuLV pseudotypes, respectively; (2) A-MuLV can infect target cells as efficiently as can GaLV, although Pit2 RNA is less abundant than Pit1 RNA; (3) factor(s) in addition to the presence of Pit1 and Pit2 are involved in retroviral infection; and (4) MuLV-10A1 pseudotype does not infect human cells more efficiently than do A-MuLV and GaLV pseudotypes.

Base Sequence↗
PubMed1998

Extended minus-strand DNA as template for R-U5-mediated second-strand transfer in recombinational rescue of primer binding site-modified retroviral vectors.

We have previously demonstrated recombinational rescue of primer binding site (PBS)-impaired Akv murine leukemia virus-based vectors involving initial priming on endogenous viral sequences and template switching during cDNA synthesis to obtain PBS complementarity in second-strand transfer of reverse transcription (Mikkelsen et al., J. Virol. 70:1439-1447, 1996). By use of the same forced recombination system, we have now found recombinant proviruses of different structures, suggesting that PBS knockout vectors may be rescued through initial priming on endogenous virus RNA, read-through of the mutated PBS during minus-strand synthesis, and subsequent second-strand transfer mediated by the R-U5 complementarity of the plus strand and the extended minus-strand DNA acceptor template. Mechanisms for R-U5-mediated second-strand transfer and its possible role in retrovirus replication and evolution are discussed.

3T3 Cells↗
PubMed1998

Single amino acid insertion in loop 4 confers amphotropic murine leukemia virus receptor function upon murine Pit1.

Pit1 is the human receptor for gibbon ape leukemia virus (GALV) and feline leukemia virus subgroup B (FeLV-B), while the related human protein Pit2 is a receptor for amphotropic murine leukemia virus (A-MuLV). The A-MuLV-related isolate 10A1 can utilize both Pit1 and Pit2 as receptors. A stretch of amino acids named region A was identified in Pit1 (residues 550 to 558 in loop 4) as critical for GALV and FeLV-B receptor function. We have here investigated the role of region A in A-MuLV and 10A1 entry. Insertion of a single amino acid in region A of mouse Pit1 resulted in a functional A-MuLV receptor, showing that region A plays a role in A-MuLV infection. Moreover, the downregulation of 10A1 receptor function by changes in region A of human Pit1 indicates that this region is also involved in 10A1 entry. Therefore, region A seems to play a role in infection by all viruses utilizing Pit1 and/or Pit2 as receptors.

3T3 Cells↗
PubMed1998

B-Cell lymphoma induction by akv murine leukemia viruses harboring one or both copies of the tandem repeat in the U3 enhancer.

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.

3T3 Cells↗
PubMed1998

Recombination in the 5' leader of murine leukemia virus is accurate and influenced by sequence identity with a strong bias toward the kissing-loop dimerization region.

Retroviral recombination occurs frequently during reverse transcription of the dimeric RNA genome. By a forced recombination approach based on the transduction of Akv murine leukemia virus vectors harboring a primer binding site knockout mutation and the entire 5' untranslated region, we studied recombination between two closely related naturally occurring retroviral sequences. On the basis of 24 independent template switching events within a 481-nucleotide target sequence containing multiple sequence identity windows, we found that shifting from vector RNA to an endogenous retroviral RNA template during minus-strand DNA synthesis occurred within defined areas of the genome and did not lead to misincorporations at the crossover site. The nonrandom distribution of recombination sites did not reflect a bias for specific sites due to selection at the level of marker gene expression. We address whether template switching is affected by the length of sequence identity, by palindromic sequences, and/or by putative stem-loop structures. Sixteen of 24 sites of recombination colocalized with the kissing-loop dimerization region, and we propose that RNA-RNA interactions between palindromic sequences facilitate template switching. We discuss the putative role of the dimerization domain in the overall structure of the reverse-transcribed RNA dimer and note that related mechanisms of template switching may be found in remote RNA viruses.

3T3 Cells↗
PubMed1997

Mapping of a major osteomagenic determinant of murine leukemia virus RFB-14 to non-long terminal repeat sequences.

Certain isolates of murine leukemia viruses (MuLVs) have, apart from a leukemogenic potential, the capability of inducing diseases of nonhematopoietic tissues in susceptible strains of mice. We have reported on the molecular cloning of a bone-tumorigenic virus, RFB-14 MuLV, which was found to induce benign bone tumors, osteomas, with 100% incidence in mice of the CBA/Ca strain (L. Pedersen, W. Behnisch, J. Schmidt, A. Luz, F. S. Pedersen, V. Erfle, and P. G. Strauss, J. Virol. 66:6186-6190, 1992). In order to analyze the bone tumor-inducing phenotype of RFB-14 MuLV, we have studied the pathogenic potential of recombinant viruses between RFB-14 and the nonosteomagenic, highly leukemogenic SL3-3 MuLV. The recombinants were constructed so as to reveal whether a major determinant of osteomagenicity maps to sequences within or outside the long terminal repeats (LTR). Our data show that a major determinant of the osteoma-inducing potential of RFB-14 MuLV maps to the non-LTR region of the genome. Furthermore, we demonstrate that a strong determinant of leukemogenicity is harbored by the non-LTR region of SL3-3 MuLV.

3T3 Cells↗
PubMed1997

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.

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.

Animals↗
PubMed1997

An SL3-3 murine leukemia virus enhancer variant more pathogenic than the wild type obtained by assisted molecular evolution in vivo.

SL3-3 is a highly T-lymphomagenic murine retrovirus in which the transcriptional enhancer is a major oncogenic determinant. Here, we describe an SL3-3 enhancer variant that induced T-cell lymphomas in all inoculated mice with a shorter latency period than wild-type SL3-3. The enhancer repeat region of this variant contains two deletions encompassing the nuclear factor 1 binding sites in addition to an additional intact enhancer repeat element. Tumors induced by this variant were T-cell lymphomas, as indicated by T-cell receptor rearrangements, and contained the input provirus enhancer regions. The variant was the result of mutation of specific transcription factor binding sites in the viral enhancer, isolation of rare second-site enhancer variants from the resulting induced tumors, and subsequent restoration of the original first-site mutations of one such variant. We have termed this process assisted molecular evolution.

3T3 Cells↗
PubMed1997

Complementation of a primer binding site-impaired murine leukemia virus-derived retroviral vector by a genetically engineered tRNA-like primer.

Reverse transcription of retroviral genomes is primed by a tRNA annealed to an 18-nucleotide primer binding site. Here, we present a primer complementation system to study molecular interaction of the replication machinery with the primer and primer binding site in vivo. Introduction of eight base substitutions into the primer binding site of a murine leukemia virus-based vector allowed efficient RNA encapsidation but resulted in severely reduced vector replication capacity. Replication was restored upon complementation with a synthetic gene designed to encode a complementary tRNA-like primer, but not with a noncomplementary tRNA-like molecule. The engineered primer was shown to be involved in both the initiation of first-strand synthesis and second-strand transfer. These results provide an in vivo demonstration that the retroviral replication machinery may recognize sequence complementarity rather than actual primer binding site and 3' primer sequences. Use of mutated primer binding site vectors replicating via engineered primers may add additional control features to retroviral gene transfer technology.

Animals↗
PubMed1997

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.

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.

Base Sequence↗
PubMed1997

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

Murine retrovirus SL3-3 is highly T lymphomagenic. Its pathogenic properties are determined by the transcriptional enhancer of the U3 repeat region which shows preferential activity in T cells. Within the U3 repeats, the major determinant of T-cell specificity has been mapped to binding sites for the AML1 transcription factor family (also known as the core binding factor [CBF], polyomavirus enhancer binding protein 2 [PEBP2], and SL3-3 enhancer factor 1 [SEF-1]). SL3-3 viruses with AML1 site mutations have lost a major determinant of T-cell-specific enhancer function but have been found to retain a lymphomagenic potential, although disease induction is slower than for the SL3-3 wild type. To compare the specificities and mechanisms of disease induction of wild-type and mutant viruses, we have examined lymphomas induced by mutant viruses harboring transversions of three consecutive base pairs critical to AML1 site function (B. Hallberg, J. Schmidt, A. Luz, F. S. Pedersen, and T. Grundström. J. Virol. 65:4177-4181, 1991). Our results show that the mutated AML1 sites are genetically stable during lymphomagenesis and that ecotropic provirus numbers in DNA of tumors induced by wild-type and mutant viruses fall within the same range. Moreover, proviruses were found to be integrated at the c-myc locus in similar proportions of wild-type and mutant SL3-3-induced tumors, and the mutated AML1 sites of proviruses at c-myc are unaltered. In some cases, however, including one c-myc-integrated provirus, a single-base pair change was detected in a second, weaker AML1 binding site. By DNA rearrangement analysis of the T-cell receptor beta-locus, tumors induced by the AML1 site mutants are found to be of the T-cell type. Thus, although the AML1 site mutants have weakened T-cell-specific enhancers they are T-lymphomagenic, and wild-type- and mutant-virus-induced tumor DNAs are similar with respect to the number of overall ecotropic and c-myc-integrated clonal proviruses. The SL3-3 wild-type and AML1 site mutant viruses may therefore induce disease by similar mechanisms.

Animals↗