Core-Binding Factor Influences the Disease Specificity of Moloney Murine Leukemia Virus

The core site in the Moloney murine leukemia virus (Moloney MLV) enhancer was previously shown to be an important determinant of the T-cell disease specificity of the virus. Mutation of the core site resulted in a significant shift in disease specificity of the Moloney virus from T-cell leukemia to erythroleukemia. We and others have since determined that a protein that binds the core site, one of the core-binding factors (CBF) is highly expressed in thymus and is essential for hematopoiesis. Here we test the hypothesis that CBF plays a critical role in mediating pathogenesis of Moloney MLV in vivo. We measured the affinity of CBF for most core sites found in MLV enhancers, introduced sites with different affinities for CBF into the Moloney MLV genome, and determined the effects of these sites on viral pathogenesis. We found a correlation between CBF affinity and the latent period of disease onset, in that Moloney MLVs with high-affinity CBF binding sites induced leukemia following a shorter latent period than viruses with lower-affinity sites. The T-cell disease specificity of Moloney MLV also appeared to correlate with the affinity of CBF for its binding site. The data support a role for CBF in determining the pathogenic properties of Moloney MLV.     

Nuclear factors that bind to the enhancer region of nondefective Friend murine leukemia virus.

Nondefective Friend murine leukemia virus (MuLV) causes erythroleukemia when injected into newborn NFS mice, while Moloney MuLV causes T-cell lymphoma. Exchange of the Friend virus enhancer region, a sequence of about 180 nucleotides including the direct repeat and a short 3'-adjacent segment, for the corresponding region in Moloney MuLV confers the ability to cause erythroid disease on Moloney MuLV. We have used the electrophoretic mobility shift assay and methylation interference analysis to identify cellular factors which bind to the Friend virus enhancer region and compared these with factors, previously identified, that bind to the Moloney virus direct repeat (N. A. Speck and D. Baltimore, Mol. Cell. Biol. 7:1101-1110, 1987). We identified five binding sites for sequence-specific DNA-binding proteins in the Friend virus enhancer region. While some binding sites are present in both the Moloney and Friend virus enhancers, both viruses contain unique sites not present in the other. Although none of the factors identified in this report which bind to these unique sites are present exclusively in T cells or erythroid cells, they bind to three regions of the enhancer shown by genetic analysis to encode disease specificity and thus are candidates to mediate the tissue-specific expression and distinct disease specificities encoded by these virus enhancer elements.     

Novel host range and cytopathic variant of ecotropic Friend murine leukemia virus

A variant ecotropic Friend murine leukemia virus, F-S MLV, is capable of inducing the formation of large multinucleated syncytia in Mus dunni cells. This cytopathicity resembles that of Spl574 MLV, a novel variant recently isolated from the spleen of a Mus spicilegus mouse neonatally inoculated with Moloney MLV. F-S MLV is an N-tropic Friend MLV that also has the unusual ability to infect hamster cells, which are normally resistant to mouse ecotropic MLVs. Syncytium induction by both F-S MLV and Spl574 is accompanied by the accumulation of large amounts of unintegrated viral DNA, a hallmark of pathogenic retroviruses, but not previously reported for mouse ecotropic gammaretroviruses. Sequencing and site-specific mutagenesis determined that the syncytium-inducing phenotype of F-S MLV can be attributed to a single amino acid substitution (S84A) in the VRA region of the viral env gene. This site corresponds to that of the single substitution previously shown to be responsible for the cytopathicity of Spl574, S82F. The S84A substitution in F-S MLV also contributes to the ability of this virus to infect hamster cells, but Spl574 MLV is unable to infect hamster cells. Because this serine residue is one of the critical amino acids that form the CAT-1 receptor binding site, and because M. dunni and hamster cells have variant CAT-1 receptors, these results suggest that syncytium formation as well as altered host range may be a consequence of altered interaction between virus and receptor.     

trans-dominant interference with virus infection at two different stages by a mutant envelope protein of Friend murine leukemia virus.

A dominant negative mutant Friend murine leukemia virus (FMLV) env gene was cloned from an immunoselected Friend erythroleukemia cell. The mutant env had a point mutation which resulted in a Cys-to-Arg substitution at the 361st amino acid in the FMLV envelope protein (Env). The mutant Env was retained in the endoplasmic reticulum (ER) and accumulated because of its slow degradation. The NIH 3T3 cells expressing the mutant env were resistant to ecotropic Moloney MLV (MoMLV) penetration, suggesting that the mutant Env traps the ecotropic MLV receptors in the ER. When the mutant env gene was transfected into and expressed in the cells persistently infected with MoMLV, the wild-type Env was trapped in the ER, and the MoMLV production was suppressed. Thus, the mutant Env accumulating in the ER trans-dominantly and efficiently interfered with the ecotropic MLV infection at both the early and the late stages.     

Distinct segments within the enhancer region collaborate to specify the type of leukemia induced by nondefective Friend and Moloney viruses.

The nondefective Moloney and Friend murine leukemia viruses induce T-cell lymphomas and erythroleukemias, respectively, after being injected into newborn NFS mice. In previous studies, we showed that the distinct disease specificities of the two viruses could be switched by exchanging a small segment, about 200 nucleotides in length, encompassing their enhancer regions. This segment included the direct repeat sequence and an adjacent GC-rich region of about 20 nucleotides defined in studies of Moloney murine sarcoma virus enhancer-promoter function (L. A. Laimins, P. Gruss, R. Pozzatti, and G. Khoury, J. Virol. 49:183-189, 1984). The direct repeats of Friend and Moloney viruses are identical in a central core sequence of 32 nucleotides but have sequence differences on either side of this core as well as in their GC-rich segments. To determine whether disease specificity resides in part or in all of the direct repeat and GC-rich region, we constructed recombinants between Friend and Moloney viruses within this segment and tested them for their disease-inducing phenotypes. We found that disease specificity, in particular the ability of Friend virus sequence to confer erythroleukemogenicity on Moloney virus, is encoded throughout the region in at least three separable segments: the 5' and 3' halves of the direct repeat and the GC-rich segment. When just one of these segments (either both 5' halves of the direct repeat, both 3' halves, or just the GC-rich segment) from Friend virus was substituted into a Moloney virus genome, it conferred only a negligible or low incidence of erythroleukemia (less than or equal to 5% to between 10 and 15%). Any two segments together were considerably more potent (35 to 95% erythroleukemia), with the most effective pair being the two halves of the direct repeat. Individual segments and pairs of segments were considerably more potent determinants when they were matched with a genome of the same origin. Thus, although sequences outside the enhancer region are minor determinants of disease specificity when the enhancer is derived entirely from either Friend or Moloney virus, they can play a significant role when the enhancer is of mixed origin. Some recombinant enhancers conferred a long latent period of disease induction. This was particularly striking when the 5' halves of each copy of the direct repeat sequence were derived from Moloney virus and the 3' halves were derived from Friend virus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification of core-binding factor, a protein that binds the conserved core site in murine leukemia virus enhancers.

The Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major genetic determinant of the thymic disease specificity of the Moloney virus genetically maps to two protein binding sites in the Moloney virus enhancer, the leukemia virus factor b site and the adjacent core site. Point mutations introduced into either of these sites significantly shifts the disease specificity of the Moloney virus from thymic leukemia to erythroleukemia (N. A. Speck, B. Renjifo, E. Golemis, T. Frederickson, J. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We have purified several polypeptides that bind to the core site in the Moloney virus enhancer. These proteins were purified from calf thymus nuclear extracts by selective pH denaturation, followed by chromatography on heparin-Sepharose, nonspecific double-stranded DNA-cellulose, and core oligonucleotide-coupled affinity columns. We have achieved greater than 13,000-fold purification of the core-binding factors (CBFs), with an overall yield of approximately 19%. Analysis of purified protein fractions by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis reveals more than 10 polypeptides. Each of the polypeptides was recovered from an SDS-polyacrylamide gel, and those in the molecular size range of 19 to 35 kDa were demonstrated to have core-binding activity. The purified CBFs were shown by DNase I footprint analyses to bind the core site in the Moloney virus enhancer specifically, and also to core motifs in the enhancers from a simian immunodeficiency virus, the immunoglobulin mu chain, and T-cell receptor gamma-chain genes.     

Sequence specificity of the core-binding factor.

The core-binding factor (CBF) binds the conserved core motif in mammalian type C retrovirus enhancers. We analyzed the phosphate contacts made by CBF on the Moloney murine leukemia virus enhancer by ethylation interference assay. The phosphate contacts span 9 bp centered around the consensus core site. To examine the sequence preferences for CBF binding, we employed the technique of selected and amplified binding sequence footprinting (T. K. Blackwell and H. Weintraub, Science 250:1104-1110, 1990). The consensus binding site for CBF defined by selected and amplified binding sequence footprinting is PyGPyG GTPy.     

Role of a third extracellular domain of an ecotropic receptor in Moloney murine leukemia virus infection

The murine ecotropic retroviral receptor has been demonstrated to function as a mouse cationic amino acid transporter 1 (mCAT1), and is comprised of multiple membranespanning domains. Feral mouse (Mus dunni) cells are not susceptible to infection by the ecotropic Moloney murine leukemia virus (MoMLV), although they can be infected by other ecotropic murine leukemia viruses, including Friend MLV and Rauscher MLV. The relative inability of MoMLV to replicate in M. dunni cells has been attributed to two amino acids (V214 and G236) located within the third extracellular loop of the M. dunni CAT1 receptor (dCAT1). Via the exchange of the third extracellular loop of the mCAT1 cDNA encoding receptor from the permissive mouse and the corresponding portion of cDNA encoding for the nonpermissive M. dunni receptor, we have identified the most critical amino acid residue, which is a glycine located at position 236 within the third extracellular loop of dCAT1. We also attempted to determine the role of the third extracellular loop of the M. dunni CAT1 receptor with regard to the formation of the syncytium. The relationship between dCAT1 and virus-induced syncytia was suggested initially by our previous identification of two MLV isolates (S82F in Moloney and S84A in Friend MLV), both of which are uniquely cytopathic in M. dunni cells. In an attempt to determine the relationship existing between dCAT1 and the virally-induced syncytia, we infected 293-dCAT1 or chimeric dCAT1 cells with the S82F pseudotype virus. The S82F pseudotype virus did not induce the formation of syncytia, but did show increased susceptibility to 293 cells expressing dCAT1. The results of our study indicate that S82F-induced syncytium formation may be the result of cell-cell fusion, but not virus-cell fusion.     

Host cell cathepsins potentiate Moloney murine leukemia virus infection

The roles of cellular proteases in Moloney murine leukemia virus (MLV) infection were investigated using MLV particles pseudotyped with vesicular stomatitis virus (VSV) G glycoprotein as a control for effects on core MLV particles versus effects specific to Moloney MLV envelope protein (Env). The broad-spectrum inhibitors cathepsin inhibitor III and E-64d gave comparable dose-dependent inhibition of Moloney MLV Env and VSV G pseudotypes, suggesting that the decrease did not involve the envelope protein. Whereas, CA-074 Me gave a biphasic response that differentiated between Moloney MLV Env and VSV G at low concentrations, at which the drug is highly selective for cathepsin B, but was similar for both glycoproteins at higher concentrations, at which CA-074 Me inhibits other cathepsins. Moloney MLV infection was lower on cathepsin B knockout fibroblasts than wild-type cells, whereas VSV G infection was not reduced on the B-/- cells. Taken together, these results support the notion that cathepsin B acts at an envelope-dependent step while another cathepsin acts at an envelope-independent step, such as uncoating or viral-DNA synthesis. Virus binding was not affected by CA-074 Me, whereas syncytium induction was inhibited in a dose-dependent manner, consistent with cathepsin B involvement in membrane fusion. Western blot analysis revealed specific cathepsin B cleavage of SU in vitro, while TM and CA remained intact. Infection could be enhanced by preincubation of Moloney MLV with cathepsin B, consistent with SU cleavage potentiating infection. These data suggested that during infection of NIH 3T3 cells, endocytosis brings Moloney MLV to early lysosomes, where the virus encounters cellular proteases, including cathepsin B, that cleave SU.     

Mechanism of leukemogenesis induced by mink cell focus-forming murine leukemia viruses.

The Friend or Moloney mink cell focus-forming (MCF) virus encodes a recombinant-type envelope glycoprotein, gp70, that is closely related to the membrane glycoprotein, gp55, of Friend spleen focus-forming virus (SFFV). We have shown previously that gp55 has the ability to activate cell growth by binding to the cellular receptor for erythropoietin. Here we show that gp70 encoded by either the Friend or Moloney MCF virus also binds to the erythropoietin receptor and that coexpression of the receptor and gp70 in an interleukin-3 (IL-3)-dependent cell line can activate IL-3-independent growth. Furthermore, when the cDNA for the human IL-2 receptor beta chain, which is related by sequence to the erythropoietin receptor, was introduced into this cell line, it became growth factor independent after infection either with SFFV or with one of the two MCF viruses but not with an ecotropic virus. Based on these observations, we propose a mechanism for the early stage of leukemogenesis induced by the MCF-type murine leukemia viruses.     

In vitro binding of purified murine ecotropic retrovirus envelope surface protein to its receptor, MCAT-1.

An amino-terminal portion of the Friend murine leukemia virus (MLV) envelope surface protein [SU, residues 1 to 236 [SU:(1-236)]] and its receptor, MCAT-1, were each purified from insect cells after expression by using recombinant baculoviruses. Friend SU:(1-236) bound specifically to Xenopus oocytes that expressed MCAT-1 with an affinity (Kd, 55 nM) similar to that of viral SU binding to permissive cells. Direct binding of Friend SU:(1-236) to purified MCAT-1 was observed in detergent and after reconstitution into liposomes. Analysis of binding demonstrated that MCAT-1 and Friend SU:(1-236) interact with a stoichiometry of near 1:1. These findings demonstrate that the amino-terminal domain from the SU of ecotropic murine retroviruses contains an MCAT-1 binding domain.     

Cloning and characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding factor.

Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major determinant of the thymic disease specificity of Moloney virus genetically maps to the conserved viral core motif in the Moloney virus enhancer. Point mutations introduced into the core site significantly shifted the disease specificity of the Moloney virus from thymic leukemia to erythroid leukemia (N.A. Speck, B. Renjifo, E. Golemis, T.N. Fredrickson, J.W. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We previously reported the purification of core-binding factors (CBF) from calf thymus nuclei (S. Wang and N.A. Speck, Mol. Cell. Biol. 12:89-102, 1992). CBF binds to core sites in murine leukemia virus and T-cell receptor enhancers. Affinity-purified CBF contains multiple polypeptides. In this study, we sequenced five tryptic peptides from two of the bovine CBF proteins and isolated three cDNA clones from a mouse thymus cDNA library encoding three of the tryptic peptides from the bovine proteins. The cDNA clones, which we call CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6, encode three highly related but distinct proteins with deduced molecular sizes of 22.0, 21.5, and 17.6 kDa that appear to be translated from multiply spliced mRNAs transcribed from the same gene. CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6 do not by themselves bind the core site. However, CBF beta p22.0 and CBF beta p21.5 form a complex with DNA-binding CBF alpha subunits and as a result decrease the rate of dissociation of the CBF protein-DNA complex. Association of the CBF beta subunits does not extend the phosphate contacts in the binding site. We propose that CBF beta is a non-DNA-binding subunit of CBF and does not contact DNA directly.