Akv Murine Leukemia Virus Research Articles

Leukemogenesis by murine leukemia viruses: lessons for koala retrovirus (KoRV)

Murine leukemia viruses (MuLVs) are the prototypical gammaretroviruses, and they have been extensively studied with regard to how they cause disease. Leukemogenesis by two MuLVs is reviewed here: the endogenous Akv MuLV of AKR mice, and exogenous Moloney MuLV. Important features of MuLV leukemogenesis include the in vivo generation of envelope recombinants (MCFs) through recombination with endogenous MuLVs, and induction of preleukemic changes typified by splenic hyperplasia secondary to bone marrow defects. Studies of MuLV leukemogenesis help to frame virological questions about how koala retrovirus (KoRV) may induce neoplastic or other diseases in koalas. Fan, Hung. 2014. Leukemogenesis by murine leukemia viruses: lessons for koala retrovirus (KoRV). In The Koala and its Retroviruses: Implications for Sustainability and Survival, ed. Geoffrey W. Pye, Rebecca N. Johnson and Alex D. Greenwood. Technical Reports of the Australian Museum, Online 24: 83–88. The discovery of koala retrovirus (KoRV) in free-ranging and captive koalas (Phascolarctos cinereus) has been viewed with concern and interest. The primary concern is that KoRV-associated disease such as neoplasms, while yet to be conclusively proven to be KoRV caused, could increase the threats to survival of these animals. In the scientific community there is interest for several reasons: KoRV may be associated with lymphoma in koalas, it appears to be recently introduced into this species, and endogenization is an ongoing process. KoRV infection in koalas may provide an opportunity to study introduction and spread of a gammaretrovirus into a new host species and its accompanying effects. This process has happened in other species, notably mice, but in the more distant past, so some of the processes can only be deduced. At the same time, information learned from the relationship of murine gammaretroviruses and their hosts may provide lessons for understanding the potential relationships of KoRV and disease in koalas. The recent discovery of a second KoRV (KoRV-B) that may be associated with leukemogenicity (Xu et al., 2013) has similarities to oncogenesis in murine leukemia viruses (MuLVs). Leukemogenesis by MuLVs will be summarized here and possible implications to KoRV pathogenesis will be pointed out. Murine leukemia viruses MuLVs were first discovered in inbred mouse strains that had high incidences of leukemia. These studies resulted in isolation of several MuLV strains that cause leukemias of different hematopoietic lineages. For instance Moloney MuLV (M-MuLV) and Gross MuLV induce T-lymphoma, while Friend (F-MuLV) and Rauscher MuLV (R-MuLV) induce erythroleukemia and myeloid leukemia (Fan, 1997). These are the predominant MuLVs used in studies of MuLV leukemogenesis. They are prototypical retroviruses of the gammaretrovirus family. MuLVs can be classified into types based on their envelope proteins and the kinds of cells that they infect, determined by the cell surface proteins that they bind. The leukemogenic MuLVs are mostly ecotropic; they infect cells of mice and rats, but they do not infect most

Antisense Transcription in Gammaretroviruses as a Mechanism of Insertional Activation of Host Genes

Transcription of retroviruses is initiated at the U3-R region boundary in the integrated provirus and continues unidirectionally to produce genomic and mRNA products of positive polarity. Several studies have recently demonstrated the existence of naturally occurring protein-encoding transcripts of negative polarity in complex retroviruses. We report here on the identification of transcripts of negative polarity in simple murine leukemia virus (MLV). In T-cell and B-cell lymphomas induced by SL3-3 and Akv MLV, antisense transcripts initiated in the U3 region of the proviral 5' long terminal repeat (LTR) and continued into the cellular proto-oncogenes Jdp2 and Bach2 to create chimeric transcripts consisting of viral and host sequence. The phenomenon was validated in vivo using a knock-in mouse model homozygous for a single LTR at a position known to activate Nras in B-cell lymphomas. A 5' rapid amplification of cDNA ends (RACE) analysis indicated a broad spectrum of initiation sites within the U3 region of the 5' LTR. Our data show for the first time transcriptional activity of negative polarity initiating in the U3 region of simple retroviruses and suggest a novel mechanism of insertional activation of host genes. Elucidation of the nature and potential regulatory role of 5' LTR antisense transcription will be relevant to the design of therapeutic vectors and may contribute to the increasing recognition of pervasive eukaryotic transcription.

The AKV Murine Leukemia Virus Is Restricted and Hypermutated by Mouse APOBEC3

APOBEC3 proteins are potent restriction factors against retroviral infection in primates. This restriction is accompanied by hypermutations in the retroviral genome that are attributable to the cytidine deaminase activity of the APOBEC3 proteins. Studies of nucleotide sequence diversity among endogenous gammaretroviruses suggest that the evolution of endogenous retroelements could have been shaped by the mutagenic cytidine deaminase activity of APOBEC3. In mice, however, APOBEC3 appears to restrict exogenous murine retroviruses in the absence of detectable levels of deamination. AKV is an endogenous retrovirus that is involved in causing a high incidence of thymic lymphoma in AKR mice. A comparative analysis of several mouse strains revealed a relatively low level of APOBEC3 expression in AKR mice. Here we show that endogenous mouse APOBEC3 restricts AKV infection and that this restriction likely reflects polymorphisms affecting APOBEC3 abundance rather than differences in the APOBEC3 isoforms expressed. We also observe that restriction of AKV by APOBEC3 is accompanied by G-->A hypermutations in the viral genome. Our findings demonstrate that APOBEC3 acts as a restriction factor in rodents affecting the strain tropism of AKV, and they provide good support for the proposal that APOBEC3-mediated hypermutation contributed to the evolution of endogenous rodent retroviral genomes.

The Zinc Finger SET Domain Gene Prdm14 Is Overexpressed in Lymphoblastic Lymphomas with Retroviral Insertions at Evi32

BackgroundAKXD recombinant inbred strains of mice have proven to be very useful in the identification of potential oncogenes and tumor suppressors involved in the development of lymphoid and myeloid malignancies. In these tumors, the hematopoietic insertion of an active AKV murine leukemia virus (MuLV) is associated with the onset of disease. Common sites of retroviral insertion (CIS) identify genes causally associated with the development or initiation of lymphoma.MethodologyIn the present study, we analyzed a previously uncharacterized CIS, Ecotropic Viral Integration Site 32 (Evi32), which is located on mouse chromosome 1. We analyzed candidate genes in the region to identify those involved in Evi32 mediated oncogenesis.ResultsHere we show that proviral insertion at Evi32 correlates with significant overexpression of a putative transcription factor, PR-domain containing 14 (Prdm14). Tumors with insertions at Evi32 are consistently lymphoid in nature. Prdm14 is normally expressed early in embryonic development with the highest expression in undifferentiated embryonic stem (ES) cells. This study implicates Prdm14 as a proto-oncogene involved in lymphoblastic lymphoma formation.

Impairment of alternative splice sites defining a novel gammaretroviral exon within gagmodifies the oncogenic properties of Akv murine leukemia virus

BackgroundMutations of an alternative splice donor site located within the gag region has previously been shown to broaden the pathogenic potential of the T-lymphomagenic gammaretrovirus Moloney murine leukemia virus, while the equivalent mutations in the erythroleukemia inducing Friend murine leukemia virus seem to have no influence on the disease-inducing potential of this virus. In the present study we investigate the splice pattern as well as the possible effects of mutating the alternative splice sites on the oncogenic properties of the B-lymphomagenic Akv murine leukemia virus.ResultsBy exon-trapping procedures we have identified a novel gammaretroviral exon, resulting from usage of alternative splice acceptor (SA') and splice donor (SD') sites located in the capsid region of gag of the B-cell lymphomagenic Akv murine leukemia virus. To analyze possible effects in vivo of this novel exon, three different alternative splice site mutant viruses, mutated in either the SA', in the SD', or in both sites, respectively, were constructed and injected into newborn inbred NMRI mice. Most of the infected mice (about 90%) developed hematopoietic neoplasms within 250 days, and histological examination of the tumors showed that the introduced synonymous gag mutations have a significant influence on the phenotype of the induced tumors, changing the distribution of the different types as well as generating tumors of additional specificities such as de novo diffuse large B cell lymphoma (DLBCL) and histiocytic sarcoma. Interestingly, a broader spectrum of diagnoses was made from the two single splice-site mutants than from as well the wild-type as the double splice-site mutant. Both single- and double-spliced transcripts are produced in vivo using the SA' and/or the SD' sites, but the mechanisms underlying the observed effects on oncogenesis remain to be clarified. Likewise, analyses of provirus integration sites in tumor tissues, which identified 111 novel RISs (retroviral integration sites) and 35 novel CISs (common integration sites), did not clearly point to specific target genes or pathways to be associated with specific tumor diagnoses or individual viral mutants.ConclusionWe present here the first example of a doubly spliced transcript within the group of gammaretroviruses, and we show that mutation of the alternative splice sites that define this novel RNA product change the oncogenic potential of Akv murine leukemia virus.

Enhancer mutations of Akv murine leukemia virus inhibit the induction of mature B-cell lymphomas and shift disease specificity towards the more differentiated plasma cell stage

This study investigates the role of the proviral transcriptional enhancer for B-lymphoma induction by exogenous Akv murine leukemia virus. Infection of newborn inbred NMRI mice with Akv induced 35% plasma cell proliferations (PCPs) (consistent with plasmacytoma), 33% diffuse large B-cell lymphomas, 25% follicular B-cell lymphomas and few splenic marginal zone and small B-cell lymphomas. Deleting one copy of the 99-bp proviral enhancer sequence still allowed induction of multiple B-cell tumor types, although PCPs dominated (77%). Additional mutation of binding sites for the glucocorticoid receptor, Ets, Runx, or basic helix–loop–helix transcription factors in the proviral U3 region, however, shifted disease induction to almost exclusively PCPs, but had no major influence on tumor latency periods. Southern analysis of immunoglobulin rearrangements and ecotropic provirus integration patterns showed that many of the tumors/cell proliferations induced by each virus were polyclonal. Our results indicate that enhancer mutations weaken the ability of Akv to induce mature B-cell lymphomas prior to the plasma cell stage, whereas development of plasma cell proliferations is less dependent of viral enhancer strength.

A murine leukemia virus with Cre-LoxP excisible coding sequences allowing superinfection, transgene delivery, and generation of host genomic deletions

BackgroundTo generate a replication-competent retrovirus that could be conditionally inactivated, we flanked the viral genes of the Akv murine leukemia virus with LoxP sites. This provirus can delete its envelope gene by LoxP/Cre mediated recombination and thereby allow superinfection of Cre recombinase expressing cells.ResultsIn our studies, the virus repeatedly infected the cell and delivered multiple copies of the viral genome to the host genome; the superinfected cells expressed a viral transgene on average twenty times more than non-superinfected cells. The insertion of multiple LoxP sites into the cellular genome also led to genomic deletions, as demonstrated by comparative genome hybridization.ConclusionWe envision that this technology may be particularly valuable for delivering transgenes and/or causing deletions.

Mutational library analysis of selected amino acids in the receptor binding domain of envelope of Akv murine leukemia virus by conditionally replication competent bicistronic vectors

The envelope protein of retroviruses is responsible for viral entry into host cells. Here, we describe a mutational library approach to dissect functional domains of the envelope protein involving a retroviral vector, which expresses both the envelope protein of Akv murine leukemia virus (MLV) and the neomycin phosphotransferase II (Neo) selection marker from the same transcript. Envelope expression was achieved by inserting an internal ribosome entry site (IRES) between the neo and the env genes. We found the structure of the linker between the IRES element and env to be critical for sufficient envelope expression. This vector functions as a replication competent mini-virus in a culture of NIH 3T3 derived semi-packaging cells that express the viral Gag and Pol proteins. Titers comparable to those of wild type virus were achieved by this system. To test this vector system, we created a random mutational library of Arg 85 and Asp 86 in the first variable region of Akv envelope protein. Homologous amino acids to Asp 86 in Moloney and Friend murine leukemia viruses are thought to be directly involved in receptor binding. Subsequent selection of mutants capable of infecting murine NIH 3T3 cells indicated that the wild type aspartic acid or another hydrophilic residue at position 86 is an important determinant for envelope function.

Modulation of homo- and heterodimerization of Harvey sarcoma virus RNA by GACG tetraloops and point mutations in palindromic sequences.

Retroviruses harbour a diploid genome of two plus-strand RNAs linked non-covalently at the dimer linkage structure. Co-packaging of two parental RNAs is a prerequisite for recombination in retroviruses, but formation of heterodimers has not been demonstrated directly in vivo. Here, we explore elements in Harvey sarcoma virus (HaSV) RNA involved in homodimerization and heterodimerization with RNA of Moloney (Mo) and Akv murine leukemia viruses (MLV). By an in vitro assay, we found that HaSV dimerization specificity could be modulated by mutations in a decanucleotide palindrome (Pal) probably folded into a kissing-loop. Autocomplementary and non-autocomplementary sequences introduced into the putative loop directed the specificity towards formation of homodimers and heterodimers, respectively. Two stem-loop (SL) structures, both exposing a GACG tetraloop, enhanced the formation of stable HaSV dimers.A similar decanucleotide palindrome has been implicated in homodimerization of MLVs. Heterodimers between HaSV RNA and Mo- or Akv MLV were unstable, but could be stabilized by introduction of two point mutations in the putative HaSV kissing-loop, creating exact complementarity with Mo/Akv MLV palindromes. Moreover, such changes increased the HaSV RNA affinity for the two MLV RNAs. Similar to HaSV RNA homodimers, formation of heterodimers with Mo- or Akv MLV RNAs was induced by the presence of GACG loops. On the basis of these results, we propose that palindromic sequences act as variable determinants of specificity and GACG tetraloops as conserved determinants in the formation of homodimers and heterodimers of gamma-retrovirus retroviral RNAs in vivo. The complementarity of loop sequences in the packaging signal upstream of the GACG tetraloops might therefore determine homo- and heterodimerization specificity and recombination activity of these viruses.

Murine leukemia virus proviral insertions between the N-ras and unr genes in B-cell lymphoma DNA affect the expression of N-ras only.

Akv1-99, a variant of Akv murine leukemia virus, induces B-cell lymphomas with nearly 100% incidence and a mean latency period of 12 months after injection into newborn NMRI mice. PCR amplification and sequence analyses of DNA flanking integrated proviruses revealed proviral insertion into the N-ras/unr (upstream of N-ras) locus in 2 out of 13 B-cell lymphomas, both of which appeared clonal by Southern blotting analysis. These two tumors showed increased expression levels of N-ras by Northern blotting, as did a third tumor shown by reverse transcriptase PCR to have a nonclonal provirus integration located in the same area. However, no significant changes in expression were observed when using a specific probe for the unr gene. All proviruses were integrated in the same transcriptional orientation as unr and N-ras genes. By promoter insertion, the two Akv1-99 proviruses integrated between exon -1 and exon 1 of N-ras gave rise to two different spliced products, whereas the provirus integrated into unr used only an exon skipping pattern. The absence of mutations of the N-ras codons 12, 13, 18, and 61 suggests that activation of the proto-oncogene is exclusively due to overexpression by retroviral promoter insertion, and furthermore, Northern blot analyses indicate that the expression of unr is unaffected by N-ras overexpression even in the case where the unr gene itself is the target of proviral insertion. Thus, altogether our findings indicate that overexpression of N-ras plays a role in development of murine leukemia virus-induced B-cell lymphomas, leaving the expression of the tightly linked unr gene unaltered.

Selection of functional tRNA primers and primer binding site sequences from a retroviral combinatorial library: identification of new functional tRNA primers in murine leukemia virus replication.

Retroviral reverse transcription is initiated from a cellular tRNA molecule and all known exogenous isolates of murine leukemia virus utilise a tRNA(Pro)molecule. While several studies suggest flexibility in murine leukemia virus primer utilisation, studies on human immunodeficiency virus and avian retro-viruses have revealed evidence of molecular adapt-ation towards the specific tRNA isoacceptor used as replication primer. In this study, murine leukemia virus tRNA utilisation is investigated by in vivo screening of a retroviral vector combinatorial library with randomised primer binding sites. While most of the selected primer binding sites are complementary to the 3'-end of tRNA((Pro)), we also retrieved PBS sequences matching four other tRNA molecules and demonstrate that Akv murine leukemia virus vectors may efficiently replicate using tRNA(Arg(CCU)), tRNA(Phe(GAA))and a hitherto unknown human tRNA(Ser(CGA)).

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.

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.

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.

Identification of a new viral protein containing CAp30 and NCp10 sequences in murine and feline leukemia retroviruses

Because Pr65gag is in part located in the nucleus and contains a putative bipartite nuclear targeting signal, we investigated the cellular location and structure of P55gag, a gag-encoded polyprotein known to lack the nucleocapsid (NC) protein NCp10. P55gag was found to be restricted to the cytoplasm of Moloney murine leukemia virus-infected cells. Of interest, P55gag was produced in cells infected by a viral protease deletion mutant and by a recombinant murine sarcoma virus known to lack the protease gene. Surprisingly, our structural and immunological studies indicated that P55gag also lacks carboxy-terminal residues of CAp30. During the course of studying P55gag, we detected a new viral protein within purified virus particles that contained NCp10 tryptic peptide sequences and a CAp30 tryptic peptide lacking in P55gag. This viral protein, which we have named nucleocapsid-related protein (NCRP), also contained antigenic epitopes present in CAp30 and NCp10. P55gag- and NCRP-like proteins were also observed in AKV murine leukemia virus and feline leukemia virus systems. The precise site of cleavage within Pr65gag that produces P55gag and NCRP is unknown but lies upstream of the CAp30-NCp10 junction within the carboxy-terminal domain of CAp30. The existence of a form of NCp10 containing carboxy-terminal CAp30 sequences raises interesting possibilities about its functional role in genomic RNA packaging and/or viral RNA dimerization.

Basic helix-loop-helix proteins in murine type C retrovirus transcriptional regulation

E boxes, recognition sequences for basic helix-loop-helix (bHLH) transcription factors, are detected in the enhancer and promoter regions of several murine type C retroviruses. Here we show that ALF1, a member of bHLH protein family of transcription factors, in vitro binds with differing affinities to distinct E-box sequences found in the U3 regulatory regions of Friend, Moloney, SL3-3, and Akv murine leukemia viruses (MLVs) as well as Friend spleen focus-forming virus (SFFV). In NIH 3T3 fibroblasts, ALF1 overexpression elevated transcription from the U3 region of Moloney MLV and the complete long terminal repeat regions of Friend SFFV, Akv MLV, and SL3-3 MLV but neither from the U3 region nor from the complete long terminal repeat of Friend MLV. Introduction of mutations in the Akv MLV E boxes showed the E-box cis elements to be required for the function of ALF1 as a transcription factor. ALF1 and the glucocorticoid receptor, with overlapping DNA binding sequences, did not act synergistically with respect to transcriptional trans activation of expression from the Akv MLV promoter-enhancer region. We conclude that ALF1 in vivo may be an important transcription regulator for Akv, SL3-3, and Moloney MLVs as well as for Friend SFFV.

Lack of correlation between basal expression levels and susceptibility to transcriptional shutdown among single-gene murine leukemia virus vector proviruses

Integrated retroviruses or retroviral vectors may be transcriptionally inactive although their promoter-enhancer regions are able to direct transcription in the host cell. We have used single-gene retroviral vectors with a long terminal repeat-directed neo marker gene to determine if the level of transcription relates to the susceptibility of a provirus to inactivation. We used two isogenic vectors, carrying long terminal repeats with a strong and a weak transcriptional enhancer derived from SL3-3 and Akv murine leukemia viruses, respectively. Nonselected cell clones of the murine lymphoid cell line L691 with single integrated vector proviruses exhibiting a 20-fold range of initial expression levels were studied. The basal expression level of a given cell clone with a single provirus did not show any pattern of correlation with the long-term stability of expression, as monitored for periods up to 150 days. Our results thus indicate that the inactivation mechanism operates independently of the initial transcriptional activity of the provirus.

A correlation between dexamethasone inducibility and basal expression levels of retroviral vector proviruses.

Identical transcription units inserted at different positions of mammalian chromosomes may vary widely in transcriptional activity. We have used a set of ten cell clones with random unselected single integrations of retroviral vectors to study such position effects. The vector used carries a neo gene driven by the Akv murine leukemia virus long terminal repeat that has only a weak promoter-enhancer activity in the target cell, the lymphoid cell line L691. Under transient expression conditions, the strength of the Akv promoter-enhancer in the L691 cells is increased by dexamethasone. In cell clones with single vector integrations, a correlation is observed between the non-induced expression levels and the degree of dexamethasone induction. The strongest relative induction is found for the integrated vectors with the lowest non-induced expression levels and approaches the inducibility under transient expression. These results indicate that expression levels are composed of distinct contributions from the integrated vector and from the site of integration and are best explained in terms of a model in which the sites of chromosomal integration exert variable positive enhancer effects upon vector transcription.

Molecular cloning of osteoma-inducing replication-competent murine leukemia viruses from the RFB osteoma virus stock.

We report the molecular cloning of two replication-competent osteoma-inducing murine leukemia viruses from the RFB osteoma virus stock (M. P. Finkel, C. A. Reilly, Jr., B. O. Biskis, and I. L. Greco, p. 353-366, in C. H. G. Price and F. G. M. Ross, ed., Bone--Certain Aspects of Neoplasia, 1973). Like the original RFB osteoma virus stock, viruses derived from the molecular RFB clones induced multiple osteomas in mice of the CBA/Ca strain. The cloned RFB viruses were indistinguishable by restriction enzyme analysis and by nucleotide sequence analysis of their long-terminal-repeat regions and showed close relatedness to the Akv murine leukemia virus.

Murine helix-loop-helix transcriptional activator proteins binding to the E-box motif of the Akv murine leukemia virus enhancer identified by cDNA cloning.

The enhancer region of Akv murine leukemia virus contains the sequence motif ACAGATGG. This sequence is homologous to the E-box motif originally defined as a regulatory element in the enhancers of immunoglobulin mu and kappa genes. We have used double-stranded oligonucleotide probes, corresponding to the E box of the murine leukemia virus Akv, to screen a randomly primed lambda gt11 cDNA expression library made from mouse NIH 3T3 fibroblast RNA. We have identified seven lambda clones expressing DNA-binding proteins representing two different genes termed ALF1 and ALF2. The results of sequencing ALF2 cDNA suggests that we have recovered the gene for the basic-helix-loop-helix transcription factor A1, the murine analog of the human transcription factor E47. The cDNA sequence of ALF1 codes for a new member of the basic-helix-loop-helix protein family. Two splice variants of ALF1 cDNA have been found, differing by a 72-bp insertion, coding for putative proteins of 682 and 706 amino acids. The two ALF1 mRNAs are expressed at various levels in mouse tissues. In vitro DNA binding assays, using prokaryotically expressed ALF1 proteins, demonstrated specific binding of the ALF1 proteins to the Akv murine leukemia virus E-box motif ACAGATGG. Expression in NIH 3T3 fibroblasts of GAL4-ALF1 chimeric protein stimulated expression from a minimal promoter linked to a GAL4 binding site, indicating the existence of a transcriptional activator domain in ALF1.