V-erbA is found in the avian erythroblastosis virus, AEV-ES4 and -R strains, isolated in 1934 by Englebreth-Holm. AEV transduced two different oncogenes, v-erbA and v-erbB. V-erbB is a homologue of the epidermal growth factor receptor tyrosine kinase (see below).
The two erb genes are inserted between gag and env and are separated by an erbB intron sequence, which encodes the C-terminal four amino acids and termination codon of \-erbA. The 11 preceding Erb-A amino acids are encoded by env sequence. At the N-terminus a substitution of 254 amino acids encoded by the viral gag gene is also apparent. Giving rise to the protein product of v-ErbA, p75&a&~erbA, v-ErbA is derived from c-ErbA, the thyroid hormone receptor a (THRA1). V-ErbA is a mutated form of chicken THRA1. In addition to deletions resulting from transduction (nine amino acid N-terminal deletion and a 13 amino acid C-terminal deletion), V-ErbA has two point mutations in the DNA-binding domain and 11 in the hormone-binding domain. These changes result in the loss of hormone-binding capacity but do not abrogate sequence-specific DNA binding. The p75gag'erbA protein is localized to the nucleus. P75g*g-erbA is phosphorylated on Serl6/17 by protein kinase A or protein kinase C. Mutation of the Serl6/ 17 phosphorylation site releases the block of erythroid differentiation exerted by v-ErbA.
THRA1 is a zinc-finger transcription factor, which binds to the response element TCAGGTCAT-GACCTGA, repressing its promoter activity. V-ErbA suppresses transcription of several genes, including avian erythrocyte anion transporter (band III), carbonic anhydrase II and /?-globin genes. Transcriptional repression of erythrocyte-specific genes correlates with the biological properties of v-Erb-A and requires formation of heterodimeric complexes with retinoid X receptor (RXR).
V-ErbA is not tumorigenic but cooperates with v-Erb-B to cause avian erythroleukemia and fibrosarcomas. In vitro, v-ErbA blocks erythroid differentiation in cooperation with v-ErbB and other sarcoma-inducing oncogenes in transformation. V-ErbA stimulates chicken embryo fibroblast (CEF) growth and enhances the tumorigenicity of v-ErbB transformed fibroblasts.
The avian retrovirus E26, which carries the ets oncogene, was isolated in 1962 at the Bulgarian Academy of Sciences from a case of avian erythroblastosis. The E26 genome consists of deleted viral gag and env genes, a truncated version of myb, and ets (¿gag-v-myb-v-ets-óenv). The myb-ets junction was created by an aberrant splice between a cryptic donor in myb and the normal splice acceptor in ets exona. The result is the generation of five additional amino acids at this junction (HGTSE). Comparison of c-Ets
(ETS-1) with v-Ets reveals amino acid substitutions at Ala285 and Ile445, which are replaced by Val in v-Ets. The 13 C-terminal residues of c-Ets have been replaced by 16 amino acid residues in v-Ets, 13 of which are derived from an inverted Ets-1 sequence. This results in a contiguous Gag-Myb-Ets open reading frame encoding a protein of 135 kDa, pl35gag-myb-eU ^ ^ ^ ^ nudeus_
V-Ets is a member of a family of transcription factors that bind to DNA motifs containing (C/A)GGA (A/T) through a conserved 85 amino acid region called the Ets domain. V-Ets has less stringent target sequence requirements than c-Ets and binds to a broad spectrum of DNA sequence motifs, suggesting that v-Ets transforms cells by altering expression of tightly regulated genes with nonconsensus Ets binding sites.
The predominant disease induced by E26 in chickens is erythroblastosis. In vitro, both myeloid and erythroid cells can be transformed by E26, reflecting the fact that E26 also contains the myeloid-specific oncogene, v-Myb (see below). E26 transforms quail fibroblasts, NIH-3T3 cells, immature erythroid cells, and stimulates proliferation of CEF.
The fos oncogene is carried by the Finkel—Biskis— Jinkins (FBJ-MSV) and Finkel-Biskis-Reilly (FBR-MSV) murine osteogenic sarcoma viruses. Dr Miriam Finkel isolated both viruses from mice with ^Sr-induced osteosarcomas. A virus isolated from an avian nephroblastoma, NK24, also contains the fos gene. The genome structures of FBJ and FBR-MSV differ significantly. FBJ-MSV contains v-fos and has no additional viral structural gene information, whereas FBR-MSV v-fos is fused to gag and non-fos sequences from the fox gene.
FBJ-MSV has five amino acid changes compared to c-Fos. A 104 bp deletion shifts the reading frame resulting in a C-terminus with 49 novel amino acids. Comparison with c-Fos reveals that FBJ-MSV v-Fos has lost 24 amino acids at the N-terminus (replaced with 310 gag amino acids) and 98 C-terminal amino acids (replaced with eight amino acids from fox). There are two C-terminal in frame deletions of 13 and 9 amino acids. The NK24 virus expresses an unaltered Fos protein linked to the gag encoded sequence.
The FBJ-MSV Fos protein product is 39 kDa, whereas the FBR-MSV fusion protein is p75gag-fos'fox. Fos is a member of the helix—loop-helix/leucine zipper transcription factor superfamily. Each member of the family contains a basic domain, a leucine zipper, two homology boxes (HOB1 and HOB2) within a transac-
tivation domain, and a ira«s-repression domain. FBJ-MSV and FBR-MSV both lack the trans-repression domain at the C-terminus. Both FBJ and FBR-MSV v-Fos proteins are localized in the nucleus.
FBR-MSV v-Fos, but not FBJ v-Fos, is myristylated at the N-terminus. Both proteins are phosphorylated on serine and threonine residues but not to the same extent as c-Fos, as the predominant phosphorylation sites on c-Fos are within the C-terminal sequences deleted from v-Fos. A basic motif, KCR, is conserved in the Fos family and redox-regulation of the cysteine residue mediates DNA binding. All Fos proteins display DNA binding activity at a site (TGAC/ GTCA) termed either activating protein-1 site (AP-1) or TPA response element (TRE) and form heterodimers with Jun proteins. These function as positive or negative transcription regulators binding to DNA via the basic domain.
FBJ, NK24 and FBR-MSV transform fibroblasts in vitro. FBR immortalizes murine cells in culture, while FBJ does not. Transforming ability appears to correlate with the presence of a C-terminal transactivation domain.
Avian sarcoma virus 17 (ASV17), which contains V-Jun, was isolated from a chicken sarcoma in 1987; 220 viral Gag residues (pl9 and <5pl0) are joined in frame to 296 Jun-encoded amino acids. Comparison to c-Jun reveals a 27 amino acid deletion in the N-terminus of v-Jun and three nonconservative substitutions in the C-terminal half; two of which are in the DNA binding domain. The v-Jun protein product p65gas',un is localized in the nucleus and has several domains: the Al activator domain, two homology box regions, HOB1 and HOB2, the e region and 6 region (the region of 27 amino acids deleted in v-Jun), A2 activation domain, and a Pro/Gln-rich ancillary DNA-binding domain. The primary DNA-binding domain, a basic region and leucine zipper, are in the C-terminal 110 residues. This region and the activation domain are necessary for transformation by Jun. V-Jun forms heterodimers with members of the Fos family and Fos-related antigens (FRA1 and FRA2) and binds with high affinity to a TPA response element (TGAc/gTCA), TRE.
Oncogenic activation of c-Jun results from 27 amino acid deletion in the N-terminus. This region contains a MAP kinase site allowing for control of c-Jun activity by phosphorylation. V-Jun is not subject to control by phosphorylation. Two of the three nonconservative amino acid substitutions may be important functionally. A Ser222 to Phe mutation prevents the glycogen synthase kinase-3 phosphoryla tion of a negative regulatory site. Mutation of Cys248 to Ser disrupts the oxidation of Cys248, inactivating DNA binding.
V-Jun induces tumors when injected into the wing web of young chicks. In vitro, v-Jun transforms chicken embryo fibroblasts.
The v-maf oncogene was identified in an avian retrovirus, AS42, isolated from a spontaneous mus-culoaponeurotic fibrosarcoma of chicken. The v-maf gene is inserted into the viral genome in the gag gene and v-maf encodes a 42 kDa basic region/leucine zipper protein, which is localized in the nucleus. The coding region of v-maf has only two structural changes when compared to c-maf.
V-Maf and other Maf family members form homodimers and heterodimers with each other and Fos and Jun. The DNA target sequence to which Maf binds is termed the Maf response element (MARE). It is a 13 or 14 bp element that contains a core TRE or CRE palindrome.
In vitro v-Maf transforms avian fibroblasts. In vivo the virus induces musculoaponeurotic fibrosarcomas.
The v-myb oncogene is found in two acutely transforming avian retroviruses, avian myeloblastosis virus (AMV) and E26. AMV v-myb replaces 26 codons at the 3' end of pol and most of env. There are 6 gag-coded amino acids at the N-terminus and 11 env amino acids at the C-terminus. E26 v-myb is fused in frame with v-ets, see above. Comparison of AMV v-myb with c-myb revealed extensive 5' (71 N-terminal amino acids) and 3' (198 C-terminal amino acids) deletions and 11 point mutations. E26 v-Myb lacks 80 N-terminal and 278 C-terminal amino acids of c-Myb and has one point mutation.
The AMV v-Myb protein product is p45v myh. The E26 v-Myb protein product is pU5gag-myb-ets. Both proteins are nuclear. C-Myb contains a DNA-binding domain with three repeat regions, Rl, R2, R3, transactivation domain, leucine zipper, trans-repression domain and a conserved domain in the C-terminus. N-terminal deletions in AMV v-Myb remove most of the Rl. The C-terminal conserved region is also deleted. E26 v-Myb lacks almost all of Rl, the leucine zipper region, the MAPK/CDC2 site, and the conserved domain. All Myb related genes contain a Cys residue at position 65.
Phosphorylation of a casein kinase II site at the N-terminus decreases c-Myb DNA binding. This site is absent in both E26 and AMV v-Myb. A MAPK/ CDC2 site in the C-terminus of c-Myb is not phosphorylated in AMV v-Myb. V-Myb binds directly to double stranded DNA and regulates transcription through a consensus site YAACT/(C)/ GGYCA. V-Myb regulates the expression of mim-1 and c-Myb.
AMV induces myeloid leukemia in chickens. In vitro, AMV transforms macrophage precursors (monoblasts). E26 transforms fibroblasts, myeloid and, as a result of the presence of ets, erythroid cells.
The v-myc gene was first identified in avian myelo-cytomatosis virus, MC29, but was subsequently found in four other virus isolates, CMII, OKIO, MH2 and FH3. All the v-myc genes are highly homologous and contain only eight single nucleotide changes when compared with c-myc. MC29 contains deleted gag, 422 v-myc amino acids, no pol and a complete env gene. CMII contains deleted gag, 421 v-myc amino acids, and complete pol and env genes. OKIO contains all of gag, 426 v-myc amino acids, deleted pol and env genes.
p5gfigag-myc is a second Myc protein product translated from a subgenomic RNA, which contains a small piece of gag sequence at the N-terminal end. MH2 contains deleted gag and env genes and 417 v-myc amino acids and a second oncogene, mil, which is homologous to v-raf. FH3 contains deleted gag gene and 421 myc amino acids.
All v-myc genes are expressed as fusion proteins with viral structural information. MC29: niOSgag'my'; CMII: P90*gag-myc; OKIO: P200-
gag Spol-myc^ p58Sgag-myc. MH2; prfgag-myc. jj^.
Several functional domains have been defined in the Myc family. A central acidic region influences the transforming host range of v-Myc proteins. Two nuclear localization signals have been defined. The C-terminus contains elements essential for DNA binding, a basic region, a helix—loop-helix motif and a leucine zipper. All v-Myc proteins are localized to the nucleus except P200gag'6pol'myc, which is found in the cytoplasm and the nucleus. The Ser62 is phosphorylated by MAP kinases, lies in a highly-conserved proline-rich region and carries transcriptional activation capacity.
Myc forms a sequence specific (CACGTG) DNA binding heterodimer with a helix—loop-helix protein, Max. The leucine zipper and the helix—loop—helix region are required for heterodimer formation. Both positive and negative regulation of gene expression has been associated with Myc family members.
In vivo, MC29, CMII or FH3 cause myelocytoma-tosis. MC29, MH2 or OKIO cause liver and kidney carcinomas. In vitro, MC29, CMII, OKIO, MH2 or FH3 transform immature macrophages or fibroblasts.
Primary fibroblasts are immortalized by v-Myc but are rendered tumorigenic only when an activated Ras is also expressed.
V-rel, the oncogene in avian reticuloendotheliosis virus strain T (REV-T), was isolated from a turkey with lymphoid leukosis. V-Rel is a member of the NF-ftB family of transcription factors. Incorporation of c-rel into the viral genome resulted in truncation of gag, pol and env. Using the env initiation codon, v-rel begins with 12 env amino acids and ends with 18 amino acids which are out of frame with respect to env. Comparison of v-Rel and turkey c-Rel revealed that v-Rel has lost two N-terminal amino acids and 118 amino acids at the C-terminus. V-Rel has 14 amino acids changes, ten of which are nonconserva-tive, and three sites of deletion where a total of five amino acids are deleted.
The protein product of v-rel is p59v rel, which is localized to both the cytoplasm and nucleus of infected cells. At the N-terminal end of v-Rel is the Rel homology domain, a DNA-binding domain conserved in all members of the NF-acB family. This region also specifies nuclear localization and protein-protein interaction. C-Rel contains sequences in the C-terminus important for cytoplasmic retention and transcriptional activation which are deleted from v-Rel. In the cytoplasm, v-Rel exists in a complex with several other cellular proteins, including the inhibitor IkB. Dissociation of I/cB from this complex allows translocation of v-Rel to the nucleus. V-Rel forms heterodimers with other members of the NF-kB family and binds to NF-kB motifs (NGGNNA/ TTTCC). In most cells, v-Rel represses gene expression; however, in transformed avian cells v-Rel activates transcription of MHC class I, HMG 14b, NF-kB, macrophage inflammatory protein-1 and an interleukin (IL)-8 related gene.
In vivo, REV-T causes acute reticuloendotheliosis. In vitro, v-Rel transforms a bone marrow-derived dendritic cell precursor, spleen-derived lymphoid cells, and fibroblasts.
The qin oncogene is the cell-derived insert in the genome of the avian sarcoma virus, ASV31. V-Qin is fused to Gag sequences at the N-terminus and eight cell coded amino acids link the cellular qin coding domain with the viral gag domain. Comparison of v-Qin with chicken c-Qin demonstrated several differences between the two. There are two non-conservative amino acid substitutions in the Qin coding region, a truncation in the C-terminus of the viral protein due to a premature stop codon. V-Qin is a nuclear protein.
V-Qin contains two domains: a winged helix domain and a repression domain. Regions between residues 74-141 and 383-395 are required for transformation. Qin is a member of the winged helix transcription factor family which function as important regulators of embryonal development and tissue differentiation in vertebrates and invertebrates, regulating expression of a number of genes. It is most closely related to brain factor-1. Qin also functions as a transcriptional repressor.
In vivo ASV31 induces fibrosarcomas in chickens. In vitro, the virus transforms avian fibroblasts.
V-ski is the oncogene found in the Sloan-Kettering viruses, SKV. Inially two molecular weight forms of v-Ski proteins were described. A 125 kDa form, in which the ski sequence was inserted into the gag gene, and a 110 kDa form, which was derived from the former via deletion of gag sequence 3' to the ski gene resulting in a Gag-Ski-Pol fusion protein. Both proteins are located in the nucleus of infected cells. By comparison to the c-Ski protein, v-Ski is truncated at both the N- and C-terminus. Both v-Ski and c-Ski can transform fibroblasts and so these truncations do not seem to be essential for oncogenic activity.
Ski has several protein motifs, including two amphipathic helices which are required for transforming activity. Ski can both transform cells and induce differentiation of muscle cells. The exact function of ski is unknown. However it appears to be capable of acting as a repressor of retinoic acid-induced transcription and this may be important for hematopoietic cell transformation. It can also activate transcription and this may be important for its effects on differentiation.
In vivo ski can effect the differentiation of muscle cells. It can also cause fibrosarcomas and stem cell leukemia when coexpressed with the v-sea oncogene. In vitro it can transform fibroblasts and hematopoietic cells.
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