Structure, origin, and transforming activity of feline leukemia virus-myc recombinant provirus FTT.

A myc-containing recombinant feline leukemia provirus, designated FTT, was molecularly cloned from the cat T-cell lymphoma line F422. Its transforming activity, as well as the nucleotide sequence of the 3' 2.7 kilobases of FTT, including v-myc, was determined. The predicted v-myc protein differs from feline c-myc by three amino acid changes and is truncated by two amino acids at the carboxyl terminus. Comparison with feline leukemia virus (FeLV), feline c-myc, and other FeLV proviruses indicates that recombination junctions involved in the generation of FeLV-onc viruses occur at preferred locations within the virus. They usually follow or occur within the sequence ACCCC at 5' junctions and may result from homologous recombination between sequences of marked purine-pyrimidine strand bias, especially at 3' junctions. Some recombination sites also resemble recombinase recognition sequences utilized in immunoglobulin and T-cell receptor variable-region joining. Transfection of primary rat embryo fibroblasts and subsequent in vivo analysis revealed that morphologic and tumorigenic transformation require cotransfection of FTT with human EJ-ras DNA; neither gene alone is sufficient. FTT v-myc is expressed in these transformed rat cells as a 3.0-kilobase subgenomic RNA; however, in contrast to the depressed level of c-myc expression in v-myc-involved feline tumors, steady-state levels of rat c-myc RNA and protein are apparently unaltered.     

Nucleotide sequence of the gag gene and gag-pol junction of feline leukemia virus.

The nucleotide sequence of the gag gene of feline leukemia virus and its flanking sequences were determined and compared with the corresponding sequences of two strains of feline sarcoma virus and with that of the Moloney strain of murine leukemia virus. A high degree of nucleotide sequence homology between the feline leukemia virus and murine leukemia virus gag genes was observed, suggesting that retroviruses of domestic cats and laboratory mice have a common, proximal evolutionary progenitor. The predicted structure of the complete feline leukemia virus gag gene precursor suggests that the translation of nonglycosylated and glycosylated gag gene polypeptides is initiated at two different AUG codons. These initiator codons fall in the same reading frame and are separated by a 222-base-pair segment which encodes an amino terminal signal peptide. The nucleotide sequence predicts the order of amino acids in each of the individual gag-coded proteins (p15, p12, p30, p10), all of which derive from the gag gene precursor. Stable stem-and-loop secondary structures are proposed for two regions of viral RNA. The first falls within sequences at the 5' end of the viral genome, together with adjacent palindromic sequences which may play a role in dimer linkage of RNA subunits. The second includes coding sequences at the gag-pol junction and is proposed to be involved in translation of the pol gene product. Sequence analysis of the latter region shows that the gag and pol genes are translated in different reading frames. Classical consensus splice donor and acceptor sequences could not be localized to regions which would permit synthesis of the expected gag-pol precursor protein. Alternatively, we suggest that the pol gene product (RNA-dependent DNA polymerase) could be translated by a frameshift suppressing mechanism which could involve cleavage modification of stems and loops in a manner similar to that observed in tRNA processing.     

Nucleic acid sequence and oncogenic properties of the HZ2 feline sarcoma virus v-abl insert.

Hardy-Zuckerman 2 feline sarcoma virus (HZ2-FeSV), isolated from a multicentric feline fibrosarcoma is a replication-defective acute transforming feline retrovirus which originated by transduction of feline c-abl sequences with feline leukemia virus (FeLV) and is known to encode a 110-kilodalton gag-abl fusion protein with tyrosine-specific protein kinase activity (P. Besmer, W. D. Hardy, E. E. Zuckerman, P. J. Bergold, L. Lederman, and H. W. Snyder, Nature (London) 303:825-828, 1983). The nucleotide sequence of the abl segment in the HZ2-FeSV genome was determined and compared with the murine and human v-abl and c-abl sequences. The predicted transforming protein consists of 344 amino acids (aa) of FeLV gag origin, 439 aa of abl origin, and at least 200 aa of FeLV pol origin (p110gag-abl-pol). The 1,317-base-pair HZ2-FeSV v-abl segment (fv-abl) corresponds to 5' abl sequences which include the region known to specify the protein kinase domain. The 5' 189 base pairs of fv-abl correspond to 5' c-abl sequences not contained in Abelson murine leukemia virus (MuLV) v-abl. The mouse c-abl exon which contains these segments was identified, and its nucleotide sequence was determined. Comparison of the predicted amino acid sequence of fv-abl with those of Abelson MuLV v-abl and c-abl revealed five aa differences. The 5' junction between FeLV and abl was found to involve a preferred region in FeLV gag p30 (P. Besmer, J. E. Murphy, P. C. George, F. H. Qiu, P. J. Bergold, L. Lederman, H. W. Snyder, D. Brodeur, E. E. Zuckerman, and W. D. Hardy, Nature (London) 320:415-421, 1986). A six-base homology exists at the recombination site between the parental FeLV and the c-abl sequences. The 3' junction between fv-abl and FeLV pol predicts an in-frame fusion of fv-abl and FeLV pol. A transformed cell line containing a truncated gag-abl-pol protein, p85, that lacks most of the FeLV pol sequences was obtained by transfection of NIH 3T3 mouse cells. This result implies that the pol sequences of the p110gag-abl-pol protein are dispensable for fibroblast transformation. To assess whether the fv-abl segment specifies the unique biological properties of HZ2-FeSV, we constructed a Moloney MuLV-based version of HZ2-FeSV, Mo-MuLV(fv-abl), in which the fv-abl sequences were contained in a genetic context similar to that in HZ2-FeSV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular cloning of proviral DNA and structural analysis of the transduced myc oncogene of avian oncovirus CMII.

Molecularly cloned proviral DNA of avian oncogenic retrovirus CMII was isolated by screening a genomic library of a CMII-transformed quail cell line with a myc-specific probe. On a 10.4-kilobase EcoRI fragment, the cloned DNA contained 4.4 kilobases of CMII proviral sequences extending from the 5' long terminal repeat to the EcoRI site within the partial (delta) complement of the env gene. The gene order of CMII proviral DNA is 5'-delta gag-v-myc-delta pol-delta env-3'. All three structural genes are partially deleted: the gag gene at the 3' end, the env gene at the 5' end, and the pol gene at both ends. The delta gag (0.83 kilobases)-v-myc (1.50 kilobases) sequences encode the p90gag-myc transforming protein of CMII. In comparison with the p110gag-myc protein of acute leukemia virus MC29, p90gag-myc lacks amino acids corresponding to additional 516 bases of gag sequences and 12 bases of 5' v-myc sequences present in the MC29 genome. Nucleotide sequence analysis of CMII proviral DNA at the delta gag-v-myc and the v-myc-delta pol junctions revealed significant homologies between avian retroviral structural genes and the cellular oncogene c-myc precisely at the positions corresponding to the gene junctions in CMII. Furthermore, the delta gag-v-myc junction in CMII corresponds to sequence elements in gag and C-myc that are possible splicing signals. The data suggest that transduction of cellular oncogenes may involve RNA splicing and recombination with homologous sequences on retroviral vectors. Different sequence elements of both the retroviral vectors and the c-myc gene recombined during genesis of highly oncogenic retroviruses CMII, MC29, or MH2.     

The amino-terminal domain of the v-fms oncogene product includes a functional signal peptide that directs synthesis of a transforming glycoprotein in the absence of feline leukemia virus gag sequences.

The nucleotide sequence of a 5' segment of the human genomic c-fms proto-oncogene suggested that recombination between feline leukemia virus and feline c-fms sequences might have occurred in a region encoding the 5' untranslated portion of c-fms mRNA. The polyprotein precursor gP180gag-fms encoded by the McDonough strain of feline sarcoma virus was therefore predicted to contain 34 v-fms-coded amino acids derived from sequences of the c-fms gene that are not ordinarily translated from the proto-oncogene mRNA. The (gP180gag-fms) polyprotein was cotranslationally cleaved near the gag-fms junction to remove its gag gene-coded portion. Determination of the amino-terminal sequence of the resulting v-fms-coded glycoprotein, gp120v-fms, showed that the site of proteolysis corresponded to a predicted signal peptidase cleavage site within the c-fms gene product. Together, these analyses suggested that the linked gag sequences may not be necessary for expression of a biologically active v-fms gene product. The gag-fms sequences of feline sarcoma virus strain McDonough and the v-fms sequences alone were inserted into a murine retroviral vector containing a neomycin resistance gene. Both constructs were biologically active when transfected into NIH 3T3 cells and produced morphologically transformed foci at equivalent efficiencies. When transfected into a cell line (psi 2) expressing complementary viral gene functions, G418-resistant (Neor) cells containing either of these vector DNAs produced high titers of transforming viruses. Analysis of proteins produced in cells containing the vector lacking gag gene sequences showed that gP180gag-fms was not synthesized, whereas normal levels of both immature gp120v-fms and mature gp140v-fms were detected. The glycoprotein was efficiently transported to the cell surface, and it retained wild-type tyrosine kinase activity. We conclude that a cryptic hydrophobic signal peptide sequence in v-fms was unmasked by gag deletion, thereby allowing the correct orientation and transport of the v-fms gene product within membranous organelles. It seems likely that the proteolytic cleavage of gP180gag-fms is mediated by signal peptidase and that the amino termini of gp140v-fms and the c-fms gene product are identical.     

The v-ski oncogene encodes a truncated set of c-ski coding exons with limited sequence and structural relatedness to v-myc.

The nucleotide sequence of a biologically active v-ski gene from a cloned proviral segment shows that ski is a 1,312-base sequence embedded in the p19 region of the avian leukosis virus gag gene. The v-ski sequence contains a single open translational reading frame that encodes a polypeptide with a molecular mass of 49,000 daltons. The predicted amino acid sequence includes nuclear localization motifs that have been identified in other nuclear oncoproteins. It also contains a proline-rich region and a set of cysteine and histidine residues that could constitute a metal-binding domain. Two regions of the amino acid sequences of v-ski and v-myc are related, and the two proteins exhibit similar distributions of hydrophobic and hydrophilic amino acids. Cloned segments of the chicken c-ski proto-oncogene totaling 65 kilobases have been analyzed, and regions related to v-ski have been sequenced. The results indicate that v-ski is derived from at least five coding exons of c-ski, that it is correctly spliced, and that it is missing c-ski coding sequences at both its 5' and 3' ends. The c-ski and avian leukosis virus sequences that overlap the 5' virus/v-ski junction in Sloan-Kettering virus contain an 18-of-20-base sequence match that presumably played a role in the transduction of ski by facilitating virus/c-ski recombination.     

Two unrelated cell-derived sequences in the genome of avian leukemia and carcinoma inducing retrovirus MH2.

Molecularly cloned proviral DNA of avian replication-defective retrovirus Mill Hill No. 2 (MH2) was analyzed. The MH2 provirus measures 5.5 kb including two long terminal repeats (LTR), and contains a partial complement of the structural gene gag, 1.5 kb in size, near the 5' terminus, and a 1.3-kb segment of the v-myc transforming gene near the 3' terminus. These v-myc sequences are closely related to the v-myc transforming gene of avian acute leukemia virus MC29, and to the cellular chicken gene c-myc. The gag and myc domains on the MH2 provirus are separated by unique sequences, 1.3 kb in size and termed v-mil, which are unrelated to v-myc, or to other oncogenes or structural genes of the avian leukemia-sarcoma group of retroviruses. Normal chicken DNA contains sequences closely related to v-mil, termed c-mil. Analyses of chicken c-mil clones isolated from a recombinant DNA library of the chicken genome reveal that c-mil is a single genetic locus with a complex split gene structure. In the MH2 genome, v-mil is expressed via genome-sized mRNA as a gag-related hybrid protein, p100gag-mil, while v-myc is apparently expressed via subgenomic mRNA independently from major coding regions of structural genes. The presence in the MH2 genome of two unrelated cell-derived sequences and their independent expression may be significant for the oncogenic specificities of this virus.     

Molecular cloning of Snyder-Theilen feline leukemia and sarcoma viruses: comparative studies of feline sarcoma virus with its natural helper virus and with Moloney murine sarcoma virus

Extrachromosomal DNA obtained from mink cells acutely infected with the Snyder-Theilen (ST) strain of feline sarcoma virus (feline leukemia virus) [FeSV(FeLV)] was fractionated electrophoretically, and samples enriched for FeLV and FeSV linear intermediates were digested with EcoRI and cloned in lambda phage. Hybrid phages were isolated containing either FeSV or FeLV DNA "inserts" and were characterized by restriction enzyme analysis, R-looping with purified 26 to 32S viral RNA, and heteroduplex formation. The recombinant phages (designated lambda FeSV and lambda FeLV) contain all of the genetic information represented in FeSV and FeLV RNA genomes but lack one extended terminally redundant sequence of 750 bases which appears once at each end of parental linear DNA intermediates. Restriction enzyme and heteroduplex analyses confirmed that sequences unique to FeSV (src sequences) are located at the center of the FeSV genome and are approximately 1.5 kilobase pairs in length. With respect to the 5'-3' orientation of genes in viral RNA, the order of genes in the FeSV genome is 5'-gag-src-env-c region-3'; only 0.9 kilobase pairs of gag and 0.6 kilobase pairs of env-derived FeLV sequences are represented in ST FeSV. Heteroduplex analyses between lambda FeSV or lambda FeLV DNA and Moloney murine sarcoma virus DNA (strain m1) were performed under conditions of reduced stringency to demonstrate limited regions of base pair homology. Two such regions were identified: the first occurs at the extreme 5' end of the leukemia and both sarcoma viral genomes, whereas the second corresponds to a 5' segment of leukemia virus "env" sequences conserved in both sarcoma viruses. The latter sequences are localized at the 3' end of FeSV src and at the 5' end of murine sarcoma virus src and could possibly correspond to regions of helper virus genomes that are required for retroviral transforming functions.     

Nucleotide sequence of AKV murine leukemia virus.

AKV is an endogenous, ecotropic murine leukemia virus that serves as one of the parents of the recombinant; oncogenic mink cell focus-forming viruses that arise in preleukemic AKR mice. I report the 8,374-nucleotide-long sequence of AKV, as determined from the infectious molecular clone AKR-623. The 5'-leader sequence of AKV extends to nucleotide 639, after which lies a long open reading frame encoding the gag and pol gene products. The reading frame is interrupted by a single amber codon separating the gag and pol genes. The pol gene overlaps the env gene within the 3' region of the AKV genome. The nucleotide sequence of the 5' region of AKV reveals the following features. (i) The 5'-leader sequence lacks any AUG codon to initiate translation of gPr80gag, suggesting that gPr80gag is not required for the replication of AKV. (ii) A short portion of the leader region diverges in sequence from the closely related Moloney murine leukemia virus and appears to be related to a sequence highly repeated in eucaryotic genomes. (iii) As in Moloney murine leukemia virus, there is a potential RNA secondary structure flanking the amber codon that separates the gag and pol genes. This structure might function as a regulatory protein binding site that controls the relative levels of synthesis of the gag and pol precursors. The nucleotide sequence of the 3' region of AKV is compared with sequences reported previously from both infectious and noninfectious molecular clones of AKV.     

Avian oncovirus MH2: molecular cloning of proviral DNA and structural analysis of viral RNA and protein

Viral RNA, molecularly cloned proviral DNA, and virus-specific protein of avian retrovirus MH2 were analyzed. The complexity and sequence conservation of the transformation-specific v-myc sequences of MH2 RNA were compared with those of the other members of the MC29 subgroup of acute leukemia viruses, MC29, CMII, and OK10, and with chicken cellular c-myc sequences. All T1 oligonucleotides mapping within the 1.3-kilobase coding region of MC29 v-myc have homologous counterparts in the RNAs of all MC29 subgroup viruses and in c-myc. These counterparts are either identical in composition or altered by single point mutations. Hence, the 47,000-dalton carboxy-terminal sequences of the transforming proteins of these viruses and of the cellular gene product are probably highly conserved but may contain single amino acid substitutions. T1 oligonucleotide mapping of MH2 RNA indicated that the MH2 v-myc sequences map close to the 3' end of viral RNA. A genomic library of an MH2-transformed quail cell line was prepared by using the Charon 4A vector system. By screening with an myc-specific probe, a clone containing the entire MH2 provirus (lambda MH2-1) was isolated. Digestion of cloned DNA with KpnI yielded a 5.1-kilobase fragment hybridizing to both gag- and myc-specific probes. Further restriction mapping of lambda MH2-1 DNA showed that about 1.6 kilobases of the gag gene are present near the 5' end of proviral DNA, and the conserved part of v-myc, i.e., 1.3 kilobases, is present near the 3' end of proviral DNA. These two domains are separated by a segment of at least 1 kilobase of different genetic origin, including additional unique sequences unrelated to virion genes. Tryptic peptide analysis of the gag-related protein of MH2, p100, revealed gag-specific peptides and several unique methionine-containing peptides. One of the latter is possibly shared with the polymerase precursor protein Pr180gag-pol, but no myc-specific peptides, defined for the MC29 protein p110gag-myc, appear to be present in MH2 p100. The data on viral RNA, proviral DNA, and protein of MH2 reveal a unique genetic structure for this virus of the MC29 subgroup and suggest that its v-myc gene is not expressed as a gag-related protein.     

The murine gene encoding the highly conserved Sm B protein contains a nonfunctional alternative 3' splice site.

The cDNA and partial genomic nucleotide (nt) sequences were derived for the mouse Sm B polypeptide and compared to the cDNA and genomic sequences encoding human Sm B. The deduced amino acid (aa) sequences from the mouse and human genes are identical with the exception of a single conserved aa substitution, accounting for the ability of anti-Sm antibodies to recognize the Sm polypeptides from a broad range of species. The genomic sequence of mouse B gene is similar to the human B genomic locus that extends from exon 6 to exon 7. These loci include conservation of both 3' alternative splice sites and putative branch points required to process B and B' mRNAs in human cells. However, the nt sequence downstream from the putative distal 3' splice junction and single nt flanking the 3' splice site consensus sequence, differ between mouse and human B. This results in a murine mRNA with a different predicted secondary structure around the distal 3' splice site when compared to humans. Thus, secondary structural constraints in the mRNA or changes in the exon sequence might prevent recognition of this alternative splice site to form B' mRNA in murine tissues.     

Provirus of M7 baboon endogenous virus: nucleotide sequence of the gag-pol region

A 3,023-base nucleotide sequence of the M7 baboon endogenous virus genome, spanning the 5' noncoding region as well as the entire gag gene and part of the pol gene, is reported. Within the 562-base 5' noncoding region, a 21-base sequence complementary to the OH terminus of tRNApro is located immediately downstream from the long terminal repeat. Amino acid sequences were deduced from the 1,596 nucleotides comprising the gag gene, and the four structural gag polypeptides, p12, p15, p30, and p10, appeared to be coded contiguously. Only one termination codon interrupted the M7 gag and pol genes. The data suggest that 55 additional amino acids may be attached to the NH2 terminus of the gag precursor protein. However, such a sequence was not detected in virions or in virus-infected cells. With the exception of the p15 region, nucleotide and amino acid sequences of the gag and pol regions of M7 virus exhibited strong homologies to those of Moloney leukemia virus.     

Nucleotide sequence and genome organization of canine parvovirus.

The genome of a canine parvovirus isolate strain (CPV-N) was cloned, and the DNA sequence was determined. The entire genome, including ends, was 5,323 nucleotides in length. The terminal repeat at the 3' end of the genome shared similar structural characteristics but limited homology with the rodent parvoviruses. The 5' terminal repeat was not detected in any of the clones. Instead, a region of DNA starting near the capsid gene stop codon and extending 248 base pairs into the coding region had been duplicated and inserted 75 base pairs downstream from the poly(A) addition site. Consensus sequences for the 5' donor and 3' acceptor sites as well as promotors and poly(A) addition sites were identified and compared with the available information on related parvoviruses. The genomic organization of CPV-N is similar to that of feline parvovirus (FPV) in that there are two major open reading frames (668 and 722 amino acids) in the plus strand (mRNA polarity). Both coding domains are in the same frame, and no significant open reading frames were apparent in any of the other frames of both minus and plus DNA strands. The nucleotide and amino acid homologies of the capsid genes between CPV-N and FPV were 98 and 99%, respectively. In contrast, the nucleotide and amino acid homologies of the capsid genes for CPV-N and CPV-b (S. Rhode III, J. Virol. 54:630-633, 1985) were 95 and 98%, respectively. These results indicate that very few nucleotide or amino acid changes differentiate the antigenic and host range specificity of FPV and CPV.     

Activation of cryptic splice sites in murine sarcoma virus-124 mutants.

We have examined splice site activation in relation to intron structure in murine sarcoma virus (MuSV)-124 RNA. MuSV-124 contains inactive murine leukemia virus env gene splice sites (termed 5' env and 3' env) as well as cryptic sites in the gag and v-mos genes (termed 5' gag and 3' mos) which are activated for thermosensitive splicing by a 1,487-base intronic deletion in the MuSV-124 derived MuSVts110 retrovirus. To determine conditions permissive for splice site activation, we examined MuSV-124 mutants deleted in the 1,919-base intron bounded by the 5' gag and 3' mos sites. Several of these deletions activated thermosensitive splicing either at the same sites used in MuSVts110 or in a previously unreported temperature-sensitive splice event between the 5' gag and 3' env sites. These data suggested that the thermosensitive splicing phenotype characteristic of MuSVts110 required neither a specialized intron nor selection of a particular 3' splice site. The 3' env and 3' mos sites were found to compete for splicing to the 5' gag site; the more upstream 3' env site was exclusively used in MuSV-124 mutants containing both sites, whereas selection of the 3' mos site required removal of the 3' env site. Branchpoint sequences were found to have a potential regulatory role in thermosensitive splicing. Insertion of a beta-globin branchpoint sequence in a splicing-inactive MuSV-124 mutant activated efficient nonthermosensitive splicing at the 3' mos site, whereas a mutated branchpoint activated less efficient but thermosensitive splicing.     

Nucleotide sequences of feline sarcoma virus long terminal repeats and 5' leaders show extensive homology to those of other mammalian retroviruses.

The nucleotide sequences of the Gardner-Arnstein feline sarcoma virus (FeSV) long terminal repeat and the adjacent leader sequences 5' to the viral gag gene were determined. These were compared with homologous portions of Synder-Theilen FeSV and with previously published sequences for Moloney murine sarcoma virus and simian sarcoma virus proviral DNA. More than 75% of the residues in the FeSV R and U5 regions were homologous to sequences within the same regions of the other viral long terminal repeats. Unexpectedly, alignment of the FeSV sequences with those of the Moloney murine sarcoma and simian sarcoma viruses showed similar extents of homology within U3. The homologous U3 regions included the inverted repeats, a single set of putative enhancer sequences, corresponding to a "72-base-pair" repeat, and sequences, including the CAT and TATA boxes, characteristic of eucaryotic promotors. The 5' leader sequences of both FeSV strains included a binding site for prolyl tRNA and a putative splice donor sequence. In addition, the FeSV leader contained a long open reading frame which was adjacent to and in phase with the ATG codon at the 5' end of the FeSV gag gene. The open reading frame could code for a signal peptide of about 7.4 kilodaltons. Our results support the concept that the virogenic portions of both FeSV and simian sarcoma virus were ancestrally derived from viruses of rodent origin, with conservation of regulatory sequences as well as the viral structural genes.