HRAS, EGFR, MET, and RON Genes Are Recurrently Activated by Provirus Insertion in Liver Tumors Induced by the Retrovirus Myeloblastosis-Associated Virus 2.

Myeloblastosis-associated virus 2 (MAV-2) is a highly tumorigenic simple avian retrovirus. Chickens infected in ovo with MAV-2 develop tumors in the kidneys, lungs, and liver with a short latency, less than 8 weeks. Here we report the results of molecular analyses of MAV-2-induced liver tumors that fall into three classes: hepatic hemangiosarcomas (HHSs), intrahepatic cholangiocarcinomas (ICCs), and hepatocellular carcinomas (HCCs). Comprehensive inverse PCR-based screening of 92 chicken liver tumors revealed that in ca. 86% of these tumors, MAV-2 provirus had integrated into one of four gene loci: HRAS, EGFR, MET, and RON Insertionally mutated genes correlated with tumor type: HRAS was hit in HHSs, MET in ICCs, RON mostly in ICCs, and EGFR mostly in HCCs. The provirus insertions led to the overexpression of the affected genes and, in the case of EGFR and RON, also to the truncation of exons encoding the extracellular ligand-binding domains of these transmembrane receptors. The structures of truncated EGFR and RON closely mimic the structures of oncogenic variants of these genes frequently found in human tumors (EGFRvIII and sfRON).IMPORTANCE These data describe the mechanisms of oncogenesis induced in chickens by the MAV-2 retrovirus. They also show that molecular processes converting cellular regulatory genes to cancer genes may be remarkably similar in chickens and humans. We suggest that the MAV-2 retrovirus-based model can complement experiments performed using mouse models and provide data that could translate to human medicine.

Avian myeoloblastosis virus (AMV): only one side of the coin.

For many years, scientists and suppliers have refered to AMV-RT as the reverse transcriptase produced by the Avian Myelobalstosis Virus. This manuscript briefly reviews the molecular basis for biological dependence of AMV for the envelope and RT proteins that are produced by its natural helper the Myeloblastosis Associated Virus (MAV). Because the wide use of the term <> obscures scientific facts, it is worthwhile to clarify this issue for the scientific community, especially for younger scientists who might not be aware of the functional relationships that exist between these two viruses.

A proteomic analysis of protein variations during differentiation of v-myb-transformed monoblasts.

v-myb oncogene of avian myeloblastosis virus (AMV) transforms myelomonocytic cells in vitro and induces acute monoblastic leukemia in vivo. The transforming effect of the v-myb can be suppressed using phorbol ester (TPA) or histone deacetylase inhibitor trichostatin A (TSA), the inducers of cell differentiation that are in clinical trials. In this study, we used proteomics-based approach to identify proteins with variable expression in differentiated BM2 cells. Proteome variations induced by TPA and TSA were compared to examine the mechanism of differentiation-promoting effects of these drugs. We found that expression of several proteins participating in cell cytoskeleton rearrangement, heat shock response, proteosynthesis and cell signaling was altered in TPA- or TSA-treated cells. We present here the first comparative proteome analysis of v-myb-transformed monoblasts BM2 focused on identification of proteins involved in their terminal differentiation.

CBP sensitizes v-myb-transformed monoblasts to differentiation inducers.

CREB-binding protein (CBP) regulates gene expression by binding to certain components of basal transcription machinery and by histone acetylation. In addition, it integrates various cellular signaling pathways through binding to multiple transcription factors, including the Myb proteins. We report in this study that CBP can partially suppress function of the v-Myb oncoprotein in leukemic cells. Although originally described as an activator of v-Myb function, we show that CBP can also act as a v-Myb suppressor. Ectopic expression of murine CBP in v-Myb-transformed chicken monoblasts reduced transcriptional activation abilities of the v-Myb protein and increased sensitivity to differentiation inducers such as phorbol ester or trichostatin A. In addition, exogenous CBP affected morphology of differentiated cells derived from BM2 monoblasts. These results indicate that cellular context is an important factor determining whether CBP will activate or suppress the protein it targets.

E26 leukemia virus converts primitive erythroid cells into cycling multilineage progenitors.

Acute chicken leukemia retroviruses, because of their capacity to readily transform hematopoietic cells in vitro, are ideal models to study the mechanisms governing the cell-type specificity of oncoproteins. Here we analyzed the transformation specificity of 2 acute chicken leukemia retroviruses, the Myb-Ets- encoding E26 virus and the ErbA/ErbB-encoding avian erythroblastosis virus (AEV). While cells transformed by E26 are multipotent (designated "MEP" cells), those transformed by AEV resemble erythroblasts. Using antibodies to separate subpopulations of precirculation yolk sac cells, both viruses were found to induce the proliferation of primitive erythroid progenitors within 2 days of infection. However, while AEV induced a block in differentiation of the cells, E26 induced a gradual shift in their phenotype and the acquisition of the potential for multilineage differentiation. These results suggest that the Myb-Ets oncoprotein of the E26 leukemia virus converts primitive erythroid cells into proliferating definitive-type multipotent hematopoietic progenitors.

The viral envelope is a major determinant for the induction of lymphoid and myeloid tumours by avian leukosis virus subgroups A and J, respectively.

Among the six envelope subgroups of avian leukosis virus (ALV) that infect chickens, subgroups A (ALV-A) and J (ALV-J) are the most pathogenic and widespread among commercial chicken populations. While ALV-A is predominantly associated with lymphoid leukosis (LL) and less frequently with erythroblastosis (EB), ALV-J mainly induces tumours of the myeloid lineage. In order to examine the basis for the lineage specificity of tumour induction by these two ALV subgroups, we constructed two chimeric viruses by substituting the env genes into the reciprocal proviral clones. The chimeric HPRS-103(A) virus carrying the subgroup A env gene is identical to ALV-J prototype virus HPRS-103 except for the env gene, and the chimeric RCAS(J) virus carrying the subgroup J env gene is identical to the parent replication-competent ALV-A vector RCAS except for the env gene. In experimentally inoculated chickens, HPRS-103(A) virus induced LL and EB similar to ALV-A isolates such as RAV-1, while RCAS(J) virus induced myeloid leukosis (ML) and EB, similar to ALV-J, suggesting that the env gene is the major determinant for the lineage-specific oncogenicity. There were genetic differences in susceptibility to tumour induction between line 0 and line 15(I) chickens, indicating that in addition to the env gene, other viral or host factors could also serve as determinants for oncogenicity. Induction of both LL and ML by the two chimeric viruses occurred through the activation of c-myc, while the EB tumours were induced by activation of the c-erbB oncogene.

Anatomy of the primase-alpha DNA polymerase reaction accomplished by nucleoprotein complexes harboring an extrachromosomal DNA identical with avian myeloblastosis virus core-bound DNA: influencing by carbonyldiphosphonate, mimosine and butylphenyl deoxyguanosine-5'-triphosphate.

Activities of the primase (Pr)-alpha DNA polymerase (pol) enzyme complex belonging to the naturally occurring reaction systems represented by special NP complexes harboring an extrachromosomal DNA identical with avian myeloblastosis virus (AMV) core-bound DNA (J. Riman, A. Sulová and K. Horská, Acta virol. 39, 149-159 (1995); J. Ríman and A. Sulová, Acta virol. 41, 181-192 (1997)) were studied in the absence and presence of carbonyldiphosphonate (COMDP), mimosine (MIMO), to it related ciclopirox olamine (CPX) and butylphenyl deoxyguanosine-5'-triphosphate (BuPdGTP). Reaction products radioactively labeled for RNA and DNA and synthesized with the common four ribonucleoside triphosphates (rNTPs) or rNTPs and deoxyribonucleoside triphosphates (dNTPs) in the reaction medium, were analyzed by polyacrylamide gel electrophoresis (PAGE) at denaturing conditions. It was shown that COMDP strongly activates the Pr and uncouples its activity from that of alpha DNA pol with accumulation of initiator (i) RNAs of the basic length. This phenomenon is not affected by BuPdGTP. MIMO, in contrast, stimulates both pol activities of this enzyme complex and preserves their mutual coupling. The effects of COMDP, MIMO and CPX seem to be modulated by concentration of the ambient dNTPs. Addition of dNTPs to rNTPs makes the effects of COMDP and MIMO mutually exclusive, suggesting that both these agents, though chemically quite different, are competing for one active site responsible for coupling these both pol activities into the one Pr-alpha DNA pol reaction.

Avian retrovirus DNA internal attachment site requirements for full-site integration in vitro.

Concerted integration of retrovirus DNA termini into the host chromosome in vivo requires specific interactions between the cis-acting attachment (att) sites at the viral termini and the viral integrase (IN) in trans. In this study, reconstruction experiments with purified avian myeloblastosis virus (AMV) IN and retrovirus-like donor substrates containing wild-type and mutant termini were performed to map the internal att DNA sequence requirements for concerted integration, here termed full-site integration. The avian retrovirus mutations were modeled after internal att site mutations studied at the in vivo level with human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). Systematic overlapping 4-bp deletions starting at nucleotide positions 7, 8, and 9 in the U3 terminus had a decreasing detrimental gradient effect on full-site integration, while more internal 4-bp deletions had little or no effect. This decreasing detrimental gradient effect was measured by the ability of mutant U3 ends to interact with wild-type U3 ends for full-site integration in trans. Modification of the highly conserved C at position 7 on the catalytic strand to either A or T resulted in the same severe decrease in full-site integration as the 4-bp deletion starting at this position. These studies suggest that nucleotide position 7 is crucial for interactions near the active site of IN for integration activity and for communication in trans between ends bound by IN for full-site integration. The ability of AMV IN to interact with internal att sequences to mediate full-site integration in vitro is similar to the internal att site requirements observed with MLV and HIV-1 in vivo and with their preintegration complexes in vitro.

Ets and retroviruses - transduction and activation of members of the Ets oncogene family in viral oncogenesis.

Studies of retroviral-induced oncogenesis in animal systems led to the initial discovery of viral oncogenes and their cellular homologs, and provided critical insights into their role in the neoplastic process. V-ets, the founding member of the ETS oncogene family, was originally identified as part of the fusion oncogene encoded by the avian acute leukemia virus E26 and subsequent analysis of virus induced leukemias led to the initial isolation of two other members of the ETS gene family. PU.1 was identified as a target of insertional activation in the majority of tumors induced by the murine Spleen Focus Forming virus (SFFV), while fli-1 proved to be the target of Friend murine leukemia virus (F-MuLV) in F-MuLV induced erythroleukemia, as well as that of the 10A1 and Graffi viruses. The common features of the erythroid and myeloid diseases induced by these viruses provided the initial demonstration that these and other members of the ETS family play important roles in hematopoietic development as well as disease. This review provides an overview of the role of ETS genes in retrovirally induced neoplasia, their possible mechanisms of action, and how these viral studies relate to current knowledge of the functions of these genes in hematopoiesis.

Relationship between plus strand DNA synthesis removal of downstream segments of RNA by human immunodeficiency virus, murine leukemia virus and avian myeloblastoma virus reverse transcriptases.

During retroviral reverse transcription the genomic RNA is degraded by the RNase H activity of reverse transcriptase (RT). Previous results suggest that after RNA-directed DNA synthesis, fragments of RNA remain annealed to the newly synthesized DNA [DeStefano et al.(1991) J. Biol.Chem. 266, 7423-7431]. These must be removed to allow synthesis of the second DNA strand. We measured the ability of HIV-, AMV- and MuLV-RT to coordinate DNA-dependent DNA synthesis and removal of downstream segments of RNA. The substrates employed were DNA templates having upstream DNA and downstream RNA primers. We found that none of the wild type RTs elongated the upstream DNA without simultaneous degradation of the RNA. Consistent with these results, HIV-, AMV- and MuLV-RT showed relatively higher affinity for RNA than for DNA oligonucleotides bound to a DNA template. Differences were observed in the RNA degradation and DNA extension patterns generated by the different RTs. AMV-RT degraded the RNA to segments 11-12 nt long, and readily elongated the upstream DNA to the end of the template. MuLV- and HIV-RT degraded the RNA primarily to segments 15-16 nt long. At low concentrations of the latter two RTs, the DNA primer stalled when it encountered the 5'-end of the RNA. In sufficient excess, all of the RTs elongated the upstream primer without stalling. Even though we were unable to detect displacement of the downstream RNA by the wild type RTs, MuLV- and HIV-RT lacking RNase H, were able to elongate the upstream DNA to the end of the template without degradation of the RNA. This suggests that degradation of downstream pieces of RNA is not absolutely required before synthesis of the plus strand DNA. The implications of these findings for viral replication are discussed.

Retroviral integration: in vitro host site selection by avian integrase.

Viral integrase catalyzes the integration of the linear viral DNA genome into the chromatin of the infected host cell, an essential step in the life cycle of retroviruses. The reaction produces a characteristic small duplication of host sequences at the site of integration, implying that there is a close juxtaposition of the viral DNA ends during a concerted integration event. We have used an in vitro assay to measure the concerted integration of virus-like plasmid DNA into naked lambda DNA catalyzed by virion purified avian integrase. In contrast to in vivo avian integration, which has strong fidelity for a 6-bp duplication, purified avian integrase in the context of this assay produced a distribution of duplication sizes, with the 6-bp size dominating. The metal cofactor Mg2+ induced increased fidelity for the 6-bp duplication relative to that with Mn2+. The immediate sequence of the host site may also influence duplication size in that we found sites that sustained multiple independent integration events producing the same duplication size. Additionally, for each set of cloned integration sites (5, 6, and 7 bp), a unique but similar symmetrical pattern of G/C and A/T sequence biases was found. Using duplex oligonucleotides as target substrates, we tested the significance of the 6-bp G/C and A/T pattern for site selection. In the context of this assay, which is likely dominated by the integration of only one viral end, the 6-bp pattern was not preferred. Instead, integration was predominantly into the 3' ends of the oligonucleotides. The combined results of the lambda and oligonucleotide assays indicated that although host site selection has properties in common with recognition of the viral DNA termini, the nonrandom sequence preferences seen for host site selection were not identical to the sequence requirements for long terminal repeat recognition.

Okazaki fragments, a constant component of avian myeloblastosis virus core-bound 7 S DNA.

We have shown that the unusual CsCl-buoyant density and velocity sedimentation properties of the isolated host 7 S DNA species associated with the core fraction of avian myeloblastosis virus (AMV) are made mainly by tight association of RNA pieces prevalently joined to the single-stranded portion of this material. It was shown indirectly on sedimentation patterns of [methyl-3H]thymidine and [14C]uridine double-labelled and glyoxylated total AMV DNA, and directly in phosphorylation experiments with T4 polynucleotide kinase performed on the single-stranded portion of AMV DNA that the RNA-DNA link in AMV DNA is of a covalent nature and that the 5'-terminal end of DNA at the RNA-DNA junction is occupied by all four common deoxyribonucleotides. This first evidence of the presence of Okazaki fragments in 7 S AMV DNA clearly indicates that this DNA does not represent a randomly fragmented host DNA included by chance into virions but special fragments of host DNA having the properties of DNA replicative structures with possible consequences for some viral function(s) including those involved in virus-cell interactions.

Avian myeloblastosis virus core-bound 7 S DNA, a collection of minute replicative host-cell DNA structures.

The early replicative nature of avian myeloblastosis virus core-bound 7 S DNA (AMV DNA), indicated by our preceding findings (Ríman et al., 1993), has been confirmed using various experimental approaches. It has been shown by agarose and polyacrylamide gel electrophoresis that this DNA represents actually a collection of molecules the size of which is strongly reminiscent of the minute early replicative structures found in DNA of sea urchin embryos (Baldari et al., 1978). With such a characteristic correspond, the sequence properties of the individual AMV DNA clones, the majority of which were found to be AT-rich with ARS-like motifs and stretches of A-residues carrying conformational requirements for bending. In comparative hybridization experiments, AMV DNA exhibited the highest homology with chicken leukaemic myeloblast scaffold-bound DNA. Compatible with high replicative activity of AMV DNA was also found its specific [methyl-3H]thymidine radioactivity. The constancy of the virus content of this DNA and its virus age-dependent cleavage changes taking place inside the virus core structure open the question of possible significance of this special host DNA for the reaction machinery represented by the retroviral nucleoprotein core complex.

Identification of a novel vertebrate homeobox gene expressed in haematopoietic cells.

This paper describes the characterisation of a novel chicken homeobox gene, Prh, whose encoded homeodomain sequence differs significantly from those of other factors which have been described. As expected, a portion of the encoded protein, containing the homeodomain, is capable of sequence-specific DNA-binding. Outside the homeodomain, Prh, possesses an N-terminal region extremely rich in proline residues and a C-terminal acidic portion, either of which may function as transcription regulatory domains. Since, among the chicken tissues tested, its transcription is restricted to haematopoietic cells, lung and liver, it may function in tissue-specific patterns of gene regulation. Human and murine Prh homologues have also been identified; so it is likely that such genes are a general feature of vertebrate genomes.

Definition of functional domains in P135gag-myb-ets and p48v-myb proteins required to maintain the response of neuroretina cells to basic fibroblast growth factor.

The v-myb- and v-ets-containing E26 retrovirus induces the proliferation of chicken neuroretina (CNR) cells in minimal medium. Proliferation of E26 CNR cells is strongly stimulated by basic fibroblast growth factor (bFGF). The v-myb-containing avian myeloblastosis virus also induces the proliferation of infected CNR cells stimulated by bFGF. Both E26 CNR and avian myeloblastosis virus CNR cells are able to form colonies in soft agar in the presence of bFGF. This suggests that the v-myb product, a nuclear sequence-specific DNA-binding protein which activates gene expression in transient transfection assays, plays a role in the proliferative response of the infected CNR cells. To determine the structure-function relationships of P135gag-myb-ets and p48v-myb, we have used deletion mutants expressed in retroviral vectors and have analyzed their effect on CNR cell proliferation as well as their effect on the CNR cell response to bFGF. We show that v-ets is not required for bFGF stimulation but increases the proliferation of CNR cells in minimal medium. In the v-myb mutants, the gag sequences derived from the helper virus increase the potency of the myb gene. The carboxyl-terminal domain required for the growth and transformation of myeloid cells and needed for maximal trans-activation in transient DNA transfection assays in fibroblasts was not required for the growth and bFGF response of CNR cells. In contrast, the domain encompassing amino acids 240 to 301 (containing part of the transcriptional activation domain of v-myb) was absolutely required for the response of CNR cells to bFGF and could be functionally replaced by the carboxyl-terminal transcriptional activation domain of the VP16 protein of herpes simplex virus.

A temperature-sensitive phenotype of avian myeloblastosis virus: determinants that influence the production of viral mRNAs.

The oncogene v-myb of avian myeloblastosis virus is expressed from an mRNA that arises by splicing of the viral genome. In previous work, we described a mutant strain of avian myeloblastosis virus (tsAMV) that elicits temperature-sensitive transformation and suggested that the mutation affects production of the mRNA for v-myb. We now report that the principal determinant of the biochemical phenotype of tsAMV is a point mutation located in a crucial region of the splice acceptor site for v-myb mRNA. The mutation reduces v-myb mRNA production but could account for the conditional phenotype only in combination with an independent effect of temperature on the splicing of both wild-type and mutant viral RNAs, which we also describe here. Our findings dramatize the manner in which retroviruses normally control the splicing of their RNAs and implicate the sequence of the splice acceptor site in the control.

Specific amino acid substitutions are not required for transformation by v-myb of avian myeloblastosis virus.

The protein product of the v-myb oncogene of avian myeloblastosis virus, p48v-myb, differs structurally in several ways from its normal cellular homolog, p75c-myb. We demonstrated that the 11 specific amino acid substitutions found in two independent molecular clones of this virus were not required for the transformation of myeloblasts by v-myb.

Serum-free conditions for the growth of avian granulocyte and monocyte clones and primary leukemic cells induced by AMV, and the apparent conversion of granulocytic progenitors into monocytic cells by a factor in chicken serum.

The supportive activity of chicken serum for the soft-agar growth of chicken granulocyte and monocyte clones could be replaced with defined ingredients [bovine serum albumin (BSA), conalbumin, selenium, linoleic acid and for routine work, a liquid soy lecithin preparation (59% phospholipids, 39% linoleic acid, and less than 2% of inositol and choline)]. The lecithin preparation could be replaced with L-alpha-phosphatidylcholine. The source of colony-stimulating factor was medium conditioned by fibroblasts cultured under protein-free conditions. AMV-induced leukemic cells could be cloned under identical conditions. In the presence of both chicken serum (10%) and the replacement ingredients, most of the proliferative clones produced were monocytic (84%). In the presence of serum alone, all of them were monocytic. Under serum-free conditions, all the clones produced were granulocytic when a day-3 conditioned medium (CM) was used; monocytic ones were also present when a day-6 CM was used. When the serum was serially diluted in the presence of the replacement ingredients, the number of proliferative monocytic clones progressively decreased while the number of proliferative granulocytic clones progressively increased and the kinetics of each were essentially the same, only opposite in direction. Moreover, the total number of proliferative clones did not change more than 33% at any dilution (or in the absence of serum). We postulate the existence in the chick system of a serum macrophage differentiation factor (M-DF) which converts early granulocytic progenitors (or exclusively diverts a common progenitor) into monocytic cells.

Isolation and characterization of a temperature-sensitive mutant of avian myeloblastosis virus.

A temperature-sensitive (ts) mutant, GA 907/7, was isolated after mutagen treatment of avian myeloblastosis virus. When bone marrow cells or secondary yolk sac macrophages were infected with GA 907/7, the expression of transformation was greatly reduced at 41 degrees C. The results of temperature-shift experiments suggest that in GA 907/7 the putative v-myb gene product is functional only at 35.5 degrees C. Moreover, when ts-induced transformed cells were shifted to 41 degrees C, a partial morphological conversion to macrophage-like cells was obtained, while the majority of the cells underwent senescence and lysis. No leukemia was obtained when GA 907/7 was injected in 1-day-old chickens. Finally, a continuous cell line releasing genetically stable mutant virus was obtained after transformation of secondary yolk sac cells.