ABSTRACT. The 10,000 nucleotide RNA genome of Rous sarcoma virus (RSV) was found to contain appro... more ABSTRACT. The 10,000 nucleotide RNA genome of Rous sarcoma virus (RSV) was found to contain approximately twelve N 6 -methyladenosine residues. Localization of these residues relative to the 3′ poly(A) end of the viral 30–40S RNA revealed that most of the N 6 -methyladenosine was distributed throughout the 3′ third of the RNA, where the src gene has been mapped. At the 5′ end, RSV RNA has methylated nucleosides in the m 7 GpppG m pCp capping structure. This cap has been detected as part of a large RNase T 1 -resistant oligonucleotide sequence. A possible sequence has been established for the first 25 nucleotides at the 5′ end of Prague B RSV 30–40S RNA.
In eukaryotic cells, an mRNA bearing a premature termination codon (PTC) or an abnormally long 3 ... more In eukaryotic cells, an mRNA bearing a premature termination codon (PTC) or an abnormally long 3 untranslated region (UTR) is often degraded by the nonsense-mediated mRNA decay (NMD) pathway. Despite the presence of a 5-to 7-kb 3 UTR, unspliced retroviral RNA escapes this degradation. We previously identified the Rous sarcoma virus (RSV) stability element (RSE), an RNA element downstream of the gag natural translation termination codon that prevents degradation of the unspliced viral RNA. Insertion of this element downstream of a PTC in the RSV gag gene also inhibits NMD. Using partial RNase digestion and selective 2-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry, we determined the secondary structure of this element. Incorporating RNase and SHAPE data into structural prediction programs definitively shows that the RSE contains an AU-rich stretch of about 30 single-stranded nucleotides near the 5 end and two substantial stem-loop structures. The overall secondary structure of the RSE appears to be conserved among 20 different avian retroviruses. The structural aspects of this element will serve as a tool in the future design of cis mutants in addressing the mechanism of stabilization.
A feature common to the life cycles of all retroviruses is the incomplete splicing of the primary... more A feature common to the life cycles of all retroviruses is the incomplete splicing of the primary viral RNA transcript. While splicing of a subset of the primary transcripts is necessary to generate subgenomic mRNAs (including env mRNA and additional mRNAs in the more complex retroviruses), a major fraction of the RNA transcripts are transported to the cytoplasm in an unspliced form, where they serve both as mRNA and as genomic RNA (Coffin, 1990; Stoltzfus, 1989).
Avian leukosis viruses (ALVs) that induce rapid B-cell lymphomas integrate into the c-myb gene an... more Avian leukosis viruses (ALVs) that induce rapid B-cell lymphomas integrate into the c-myb gene and produce an ALV-myb read-through RNA, which is spliced to produce a truncated Myb protein. The genetic determinants of such recombinant ALVs have been mapped to a 42-nt deletion within the gag gene. This deletion increases splicing efficiency since it is located within a negative regulator of splicing. We propose that the deletion leads to increased production of Myb protein by increasing splicing of an ALV-myb pre-mRNA.
The RNA genomes of simple retroviruses encode three genes (gag, pol, and env) which are required ... more The RNA genomes of simple retroviruses encode three genes (gag, pol, and env) which are required for replication. In addition, there are at least three well-defined cis-acting structures which regulate important aspects of the viral life cycle. The packaging signal at the 5Ј end of the RNA tags the genomic RNA for specific encapsidation into assembling virus. Since viral Env proteins are translated from spliced mRNAs, cis-acting splicing signals ensure that the proper ratio of spliced and unspliced viral RNAs is present in the infected cell. Finally, cis-acting elements at the 3Ј end of the genome promote the export of unspliced RNAs from the nucleus for translation and encapsidation. 1997 Academic Press
Human internal exons have an average size of 147 nt, and most are <300 nt. This small size is tho... more Human internal exons have an average size of 147 nt, and most are <300 nt. This small size is thought to facilitate exon definition. A small number of large internal exons have been identified and shown to be alternatively spliced. We identified 1115 internal exons >1000 nt in the human genome; these were found in 5% of all protein-coding genes, and most were expressed and translated. Surprisingly, 40% of these were expressed at levels similar to the flanking exons, suggesting they were constitutively spliced. While all of the large exons had strong splice sites, the constitutively spliced large exons had a higher ratio of splicing enhancers/silencers and were more conserved across mammals than the alternatively spliced large exons. We asked if large exons contain specific sequences that promote splicing and identified 38 sequences enriched in the large exons relative to small exons. The consensus sequence is C-rich with a central invariant CA dinucleotide. Mutation of these sequences in a candidate large exon indicated that these are important for recognition of large exons by the splicing machinery. We propose that these sequences are large exon splicing enhancers (LESEs).
Avian leukosis virus (ALV) induces B-cell lymphoma and other neoplasms in chickens by integrating... more Avian leukosis virus (ALV) induces B-cell lymphoma and other neoplasms in chickens by integrating within or near cancer genes and perturbing their expression. Four genes-MYC, MYB, Mir-155, and TERT-have previously been identified as common integration sites in these virus-induced lymphomas and are thought to play a causal role in tumorigenesis. In this study, we employ high-throughput sequencing to identify additional genes driving tumorigenesis in ALV-induced B-cell lymphomas. In addition to the four genes implicated previously, we identify other genes as common integration sites, including TNFRSF1A, MEF2C, CTDSPL, TAB2, RUNX1, MLL5, CXorf57, and BACH2. We also analyze the genome-wide ALV integration landscape in vivo and find increased frequency of ALV integration near transcriptional start sites and within transcripts. Previous work has shown ALV prefers a weak consensus sequence for integration in cultured human cells. We confirm this consensus sequence for ALV integration in vivo in the chicken genome. IMPORTANCE Avian leukosis virus induces B-cell lymphomas in chickens. Earlier studies showed that ALV can induce tumors through insertional mutagenesis, and several genes have been implicated in the development of these tumors. In this study, we use high-throughput sequencing to reveal the genome-wide ALV integration landscape in ALV-induced B-cell lymphomas. We find elevated levels of ALV integration near transcription start sites and use common integration site analysis to greatly expand the number of genes implicated in the development of these tumors. Interestingly, we identify several genes targeted by viral insertions that have not been previously shown to be involved in cancer.
The direct repeat (DR) sequences flanking the src gene in Rous sarcoma virus are essential posttr... more The direct repeat (DR) sequences flanking the src gene in Rous sarcoma virus are essential posttranscriptional control elements; at least one copy of this sequence is necessary for cytoplasmic accumulation of unspliced viral RNA. These sequences promote Rev-independent human immunodeficiency virus type 1 expression, suggesting they act as constitutive transport elements (CTEs). To determine which regions of this sequence are critical for CTE function, mutations in the downstream DR were generated and tested in a viral deletion construct lacking src and the upstream DR. Two single-point mutations and three different clustered mutations caused substantial reductions in reverse transcriptase activity, Gag protein levels, and unspliced viral RNA in the cytoplasm. Three conserved regions of the CTE, including nucleotides 8844 to 8847, 8862 to 8864, and 8868 to 8870, were most sensitive to inactivation by mutagenesis.
A difference in affinity for a Nonidet P-40-insoluble cellular matrix was observed between the pr... more A difference in affinity for a Nonidet P-40-insoluble cellular matrix was observed between the products of the viral and cellular src genes. It has previously been demonstrated that pp6Ov-src is associated with a detergent-insoluble matrix containing the cellular cytoskeleton (J. G. Burr, G. Dreyfuss, S. Penman, and J. M. Buchanan, Proc. Natl. Acad. Sci. USA 77:3484-3488, 1980). We observed a similar association of the transforming proteins of Fujinami sarcoma virus (P130gagfps) and Yamaguchi 73 avian sarcoma virus
Eukaryotic cells target mRNAs to the nonsense-mediated mRNA decay (NMD) pathway when translation ... more Eukaryotic cells target mRNAs to the nonsense-mediated mRNA decay (NMD) pathway when translation terminates within the coding region. In mammalian cells, this is presumably due to a downstream signal deposited during pre-mRNA splicing. In contrast, unspliced retroviral RNA undergoes NMD in chicken cells when premature termination codons (PTCs) are present in the gag gene. Surprisingly, deletion of a 401-nt 3¢ UTR sequence immediately downstream of the normal gag termination codon caused this termination event to be recognized as premature. We termed this 3¢ UTR region the Rous sarcoma virus (RSV) stability element (RSE). The RSE also stabilized the viral RNA when placed immediately downstream of a PTC in the gag gene. Deletion analysis of the RSE indicated a smaller functional element. We conclude that this 3¢ UTR sequence stabilizes termination codons in the RSV RNA, and termination codons not associated with such an RSE sequence undergo NMD.
Transformation of chicken cells by Fujinami sarcoma virus (FSV), PRC II or Y73 (three independent... more Transformation of chicken cells by Fujinami sarcoma virus (FSV), PRC II or Y73 (three independently isolated avian sarcoma viruses that are replication-defective and lack the Rous sarcoma virus src gene) resulted in significant elevation (4-13 fold) of phosphotyrosine levels in cellular protein. The gag-related proteins encoded by these avian sarcoma viruses (ASVs) were all associated with tyrosine-specific protein kinase activity when assayed in immune complexes and were phosphorylated at both tyrosine and serine residues in vivo. Both the phosphotyrosine level in protein of FSVinfected cells and the protein kinase activity assayed in immune complexes containing the FSV protein P140 were temperature-sensitive. The presumed transforming proteins of these ASVs were compared with those of Rous sarcoma virus (RSV), Abelson murine leukemia virus and the Snyder-Theilen and Gardner-Arnstein strains of feline sarcoma virus (FeSV), which have previously been associated with tyrosine-specific protein kinase activity. FSV and PRC II proteins were shown to be structurally related to one another and to the FeSV proteins by tryptic peptide mapping and by immunological studies. No homology was observed, however, between the transforming proteins of RSV, Y73, Abelson murine leukemia virus and the FSV/PRC II/ FeSV class, suggesting there may be at least four classes of retroviruses whose transformation mechanisms involve aberrant phosphorylation of cellular protein at tyrosine residues.
ABSTRACT Most replication-competent retroviruses cause cancer only after a long latent period, by... more ABSTRACT Most replication-competent retroviruses cause cancer only after a long latent period, by insertional mutagenesis of the host genome, usually resulting in activation of a cellular proto-oncogene. In contrast, acute-transforming retroviruses, which have transduced host proto-oncogenes, cause rapid tumor formation and death. In both cases, some of the same genes are overexpressed and/or mutated, including genes involved in mitogenic signaling, cell cycle control, and cell survival. We are near the 100th anniversaries of the discoveries of both leukemia (1908) and sarcoma (1910) viruses in birds. In honor of this important milestone, this review will be focused mainly on studies of oncogensis by avian viruses, which paved the way for studies with many other oncogenic retroviruses. Rous sarcoma virus (RSV) causes rapid oncogenesis by high level expression of an activated src gene tyrosine kinase. The host src gene was transduced by the virus without its C-terminal negative regulatory domain. In contrast, avian leukosis virus (ALV) induces B-cell lymphomas by insertional mutagenesis after the provirus integrates into the host genome. In addition to activation of classical proto-oncogenes, proviral insertions can activate cellular micro-RNAs called oncomiRs. The precursor of miR-155, which is upregulated in many human tumors, was first identified as a common ALV B-cell integration cluster (bic) in metastatic, long-latency lymphomas. Targets of miR-155 repression include tumor-suppressor genes, providing a novel mechanism for their inactivation in ALV-induced tumors. Telomerase reverse transcriptase is also activated by enhancer insertion in many ALV-induced lymphomas, providing a good model system for study of telomerase-dependent tumors. KeywordsRous sarcoma virus-Avian leukosis virus-Src-Oncogene-Insertional mutagenesis-miR-155-Bic-TERT
The avian leukosis virus ⌬LR-9 causes a high frequency of B-cell lymphomas within weeks after inj... more The avian leukosis virus ⌬LR-9 causes a high frequency of B-cell lymphomas within weeks after injection into 10-day-old chicken embryos. These lymphomas result from proviral integrations into the oncogene c-myb. In contrast, LR-9, which lacks the 42-nucleotide gag gene deletion of ⌬LR-9, does not cause a high frequency of c-myb-associated short-latency lymphomas. Although viral replication rates and spliced env mRNA levels were found to be similar for both viruses, ⌬LR-9 exhibited an increase in readthrough transcription compared to LR-9. The ⌬LR-9 deletion is located in the region of the gag gene corresponding to the matrix (MA) protein as well as in the negative regulator of splicing (NRS) element. To test whether disruption of the NRS or of the MA protein was responsible for inducing short-latency lymphomas, we generated viruses with NRS point mutations that maintained the wild-type Gag amino acid sequence. One of the mutant viruses induced an even higher incidence than ⌬LR-9 of short-latency lymphomas with viral integrations into c-myb. Thus, we propose that disruption of the NRS sequence promotes readthrough transcription and splicing to the downstream myb gene, causing overexpression of a slightly truncated Myb protein, which induces short-latency tumors.
Splicing of Rous sarcoma virus RNA is regulated in part by a cis-acting intronic RNA element call... more Splicing of Rous sarcoma virus RNA is regulated in part by a cis-acting intronic RNA element called the negative regulator of splicing (NRS). An NRS mutant affecting nt 916-923 disrupts U11 snRNP binding and reduces NRS activity (Gontarek et al., 1993, Genes & Dev 7:1926-1936). However, we observed that a U1 59 splice site-like sequence, which overlapped the U11 site, was also disrupted by this mutation. To determine whether the U1 or the U11 site was essential for NRS activity, we analyzed twelve additional mutants involving nt 915-926. All mutations that disrupted the potential base pairing between U1 snRNA and the NRS reduced NRS activity, including single point mutations at nt 915, 916, and 919. The point mutation at nt 919 was partially suppressed by a compensatory base change mutation in U1 snRNA. In contrast, a mutation which strengthened the potential base pairing between the U1 site and the NRS increased NRS activity. Surprisingly, mutations that specifically targeted the U11 59 splice site consensus sequence increased the levels of unspliced RNA, suggesting U11 binding plays an antagonistic role to NRS activity. We propose that U1 snRNP binding to the NRS inhibits splicing and is regulated by U11 snRNP binding to the overlapping sequence. Competition between U1 and U11 snRNPs would result in the appropriate balance of spliced to unspliced RNAs for optimal viral replication. Further, a virus mutated in the U1/U11 region of the NRS was found to have delayed replication.
cis-acting sequences of Rous sarcoma virus (RSV) RNA involved in control of the incomplete splici... more cis-acting sequences of Rous sarcoma virus (RSV) RNA involved in control of the incomplete splicing that is part of the retroviral life cycle have been studied. The 5' and two alternative 3' splice sites, as well as a negative regulator of splicing element in the intron, have been introduced into chimeric constructs, and their respective roles in splicing inhibition have been evaluated by transient transfection experiments. Although the RSV 5' splice site was used efficiently in these assays, substrates containing either the RSV env or the RSV src 3' splice site were not spliced completely, resulting in 40 to 50% unspliced RNA. Addition of the negative regulator of splicing element to substrates containing RSV 3' splice sites resulted in greater inhibition of splicing (70 to 80% unspliced RNA), suggesting that the two elements function independently and additively. Deletion of sequences more than 70 nucleotides upstream of the src 3' splice site resulted in efficient splicing at this site, suggesting that inefficient usage is not inherent in this splice site but is instead due to sequences upstream of it. Insertion of these upstream sequences into the intron of a heterologous pre-mRNA resulted in partial inhibition of its splicing. In addition, secondary structure interactions were predicted to occur between the src 3' splice site and the inhibitory sequences upstream of it. Thus, RSV splicing control involves both intronic sequences and 3' splice sites, with different mechanisms involved in the underutilization of the env and src splice acceptor sites.
A c/s-acting negative regulator of splicing (NRS) within the gag gene of RSV is involved in contr... more A c/s-acting negative regulator of splicing (NRS) within the gag gene of RSV is involved in control of the relative levels of spliced and unspliced viral mRNAs. Insertion of the NRS into the intron of an adenovirus pre-mRNA resulted in inhibition of splicing in vitro before the first cleavage step. Analyses of spliceosome assembly with this substrate showed that it formed large RNP complexes that did not migrate like mature spliceosomes on native gels. Affinity selection of the RNP complexes formed on NRS-containing pre-mRNAs showed an association with Ull and U12 snRNPs, as well as with the spliceosomal snRNPs. Immunoprecipitation with antisera specific for U1 and U2 snRNPS showed binding of both snRNPs to NRS RNA. A 7-nucleotide missense mutation in the NRS that prevented binding of Ull and U12 snRNPs impaired NRS activity in vivo, suggesting a functional role for Ull and U12 snRNPS in the inhibition of splicing mediated by the RSV NRS RNA.
All retroviruses need mechanisms for nucleocytoplasmic export of their unspliced RNA and for main... more All retroviruses need mechanisms for nucleocytoplasmic export of their unspliced RNA and for maintenance of this RNA in the cytoplasm, where it is either translated to produce Gag and Pol proteins or packaged into viral particles. The complex retroviruses encode Rev or Rex regulatory proteins, which interact with cis-acting viral sequences to promote cytoplasmic expression of incompletely spliced viral RNAs. Since the simple retroviruses do not encode regulatory proteins, we proposed that they might contain cis-acting sequences that could interact with cellular Rev-like proteins. To test this possibility, we initially looked for a cis-acting sequence in avian retroviruses that could substitute for Rev and the Rev response element in human immunodeficiency virus type 1 expression constructs. A cis-acting element in the 3 untranslated region of Rous sarcoma virus (RSV) RNA was found to promote Rev-independent expression of human immunodeficiency virus type 1 Gag proteins. This element was mapped between RSV nucleotides 8770 and 8925 and includes one copy of the direct repeat (DR) sequences flanking the RSV src gene; similar activity was observed for the upstream DR. To address the function of this element in RSV, both copies of the DR sequence were deleted. Subsequently, each DR sequence was inserted separately back into this deleted construct. While the viral construct lacking both DR sequences failed to replicate, constructs containing either the upstream or downstream DR replicated well. In the absence of both DRs, Gag protein levels were severely diminished and cytoplasmic levels of unspliced viral RNA were significantly reduced; replacement of either DR sequence led to normal levels of Gag protein and cytoplasmic unspliced RNA.
For simple retroviruses, such as the Rous sarcoma virus (RSV), post-transcriptional control eleme... more For simple retroviruses, such as the Rous sarcoma virus (RSV), post-transcriptional control elements regulate viral RNA splicing, export, stability, and packaging into virions. These RNA sequences interact with cellular host proteins to regulate and facilitate productive viral infections. One such element, known as the RSV stability element (RSE), is required for maintaining stability of the full-length unspliced RNA. This viral RNA serves as the mRNA for the Gag and Pol proteins and also as the genome packaged in progeny virions. When the RSE is deleted from the viral RNA, the unspliced RNA becomes unstable and is degraded in a Upf1-dependent manner. Current evidence suggests that the RSE inhibits recognition of the viral gag termination codon by the nonsense-mediated mRNA decay (NMD) pathway. We believe that the RSE acts as an insulator to NMD, thereby preventing at least one of the required functional steps that target an mRNA for degradation. Here, we discuss the history of the RSE and the current model of how the RSE is interacting with cellular NMD factors.
ABSTRACT. The 10,000 nucleotide RNA genome of Rous sarcoma virus (RSV) was found to contain appro... more ABSTRACT. The 10,000 nucleotide RNA genome of Rous sarcoma virus (RSV) was found to contain approximately twelve N 6 -methyladenosine residues. Localization of these residues relative to the 3′ poly(A) end of the viral 30–40S RNA revealed that most of the N 6 -methyladenosine was distributed throughout the 3′ third of the RNA, where the src gene has been mapped. At the 5′ end, RSV RNA has methylated nucleosides in the m 7 GpppG m pCp capping structure. This cap has been detected as part of a large RNase T 1 -resistant oligonucleotide sequence. A possible sequence has been established for the first 25 nucleotides at the 5′ end of Prague B RSV 30–40S RNA.
In eukaryotic cells, an mRNA bearing a premature termination codon (PTC) or an abnormally long 3 ... more In eukaryotic cells, an mRNA bearing a premature termination codon (PTC) or an abnormally long 3 untranslated region (UTR) is often degraded by the nonsense-mediated mRNA decay (NMD) pathway. Despite the presence of a 5-to 7-kb 3 UTR, unspliced retroviral RNA escapes this degradation. We previously identified the Rous sarcoma virus (RSV) stability element (RSE), an RNA element downstream of the gag natural translation termination codon that prevents degradation of the unspliced viral RNA. Insertion of this element downstream of a PTC in the RSV gag gene also inhibits NMD. Using partial RNase digestion and selective 2-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry, we determined the secondary structure of this element. Incorporating RNase and SHAPE data into structural prediction programs definitively shows that the RSE contains an AU-rich stretch of about 30 single-stranded nucleotides near the 5 end and two substantial stem-loop structures. The overall secondary structure of the RSE appears to be conserved among 20 different avian retroviruses. The structural aspects of this element will serve as a tool in the future design of cis mutants in addressing the mechanism of stabilization.
A feature common to the life cycles of all retroviruses is the incomplete splicing of the primary... more A feature common to the life cycles of all retroviruses is the incomplete splicing of the primary viral RNA transcript. While splicing of a subset of the primary transcripts is necessary to generate subgenomic mRNAs (including env mRNA and additional mRNAs in the more complex retroviruses), a major fraction of the RNA transcripts are transported to the cytoplasm in an unspliced form, where they serve both as mRNA and as genomic RNA (Coffin, 1990; Stoltzfus, 1989).
Avian leukosis viruses (ALVs) that induce rapid B-cell lymphomas integrate into the c-myb gene an... more Avian leukosis viruses (ALVs) that induce rapid B-cell lymphomas integrate into the c-myb gene and produce an ALV-myb read-through RNA, which is spliced to produce a truncated Myb protein. The genetic determinants of such recombinant ALVs have been mapped to a 42-nt deletion within the gag gene. This deletion increases splicing efficiency since it is located within a negative regulator of splicing. We propose that the deletion leads to increased production of Myb protein by increasing splicing of an ALV-myb pre-mRNA.
The RNA genomes of simple retroviruses encode three genes (gag, pol, and env) which are required ... more The RNA genomes of simple retroviruses encode three genes (gag, pol, and env) which are required for replication. In addition, there are at least three well-defined cis-acting structures which regulate important aspects of the viral life cycle. The packaging signal at the 5Ј end of the RNA tags the genomic RNA for specific encapsidation into assembling virus. Since viral Env proteins are translated from spliced mRNAs, cis-acting splicing signals ensure that the proper ratio of spliced and unspliced viral RNAs is present in the infected cell. Finally, cis-acting elements at the 3Ј end of the genome promote the export of unspliced RNAs from the nucleus for translation and encapsidation. 1997 Academic Press
Human internal exons have an average size of 147 nt, and most are <300 nt. This small size is tho... more Human internal exons have an average size of 147 nt, and most are <300 nt. This small size is thought to facilitate exon definition. A small number of large internal exons have been identified and shown to be alternatively spliced. We identified 1115 internal exons >1000 nt in the human genome; these were found in 5% of all protein-coding genes, and most were expressed and translated. Surprisingly, 40% of these were expressed at levels similar to the flanking exons, suggesting they were constitutively spliced. While all of the large exons had strong splice sites, the constitutively spliced large exons had a higher ratio of splicing enhancers/silencers and were more conserved across mammals than the alternatively spliced large exons. We asked if large exons contain specific sequences that promote splicing and identified 38 sequences enriched in the large exons relative to small exons. The consensus sequence is C-rich with a central invariant CA dinucleotide. Mutation of these sequences in a candidate large exon indicated that these are important for recognition of large exons by the splicing machinery. We propose that these sequences are large exon splicing enhancers (LESEs).
Avian leukosis virus (ALV) induces B-cell lymphoma and other neoplasms in chickens by integrating... more Avian leukosis virus (ALV) induces B-cell lymphoma and other neoplasms in chickens by integrating within or near cancer genes and perturbing their expression. Four genes-MYC, MYB, Mir-155, and TERT-have previously been identified as common integration sites in these virus-induced lymphomas and are thought to play a causal role in tumorigenesis. In this study, we employ high-throughput sequencing to identify additional genes driving tumorigenesis in ALV-induced B-cell lymphomas. In addition to the four genes implicated previously, we identify other genes as common integration sites, including TNFRSF1A, MEF2C, CTDSPL, TAB2, RUNX1, MLL5, CXorf57, and BACH2. We also analyze the genome-wide ALV integration landscape in vivo and find increased frequency of ALV integration near transcriptional start sites and within transcripts. Previous work has shown ALV prefers a weak consensus sequence for integration in cultured human cells. We confirm this consensus sequence for ALV integration in vivo in the chicken genome. IMPORTANCE Avian leukosis virus induces B-cell lymphomas in chickens. Earlier studies showed that ALV can induce tumors through insertional mutagenesis, and several genes have been implicated in the development of these tumors. In this study, we use high-throughput sequencing to reveal the genome-wide ALV integration landscape in ALV-induced B-cell lymphomas. We find elevated levels of ALV integration near transcription start sites and use common integration site analysis to greatly expand the number of genes implicated in the development of these tumors. Interestingly, we identify several genes targeted by viral insertions that have not been previously shown to be involved in cancer.
The direct repeat (DR) sequences flanking the src gene in Rous sarcoma virus are essential posttr... more The direct repeat (DR) sequences flanking the src gene in Rous sarcoma virus are essential posttranscriptional control elements; at least one copy of this sequence is necessary for cytoplasmic accumulation of unspliced viral RNA. These sequences promote Rev-independent human immunodeficiency virus type 1 expression, suggesting they act as constitutive transport elements (CTEs). To determine which regions of this sequence are critical for CTE function, mutations in the downstream DR were generated and tested in a viral deletion construct lacking src and the upstream DR. Two single-point mutations and three different clustered mutations caused substantial reductions in reverse transcriptase activity, Gag protein levels, and unspliced viral RNA in the cytoplasm. Three conserved regions of the CTE, including nucleotides 8844 to 8847, 8862 to 8864, and 8868 to 8870, were most sensitive to inactivation by mutagenesis.
A difference in affinity for a Nonidet P-40-insoluble cellular matrix was observed between the pr... more A difference in affinity for a Nonidet P-40-insoluble cellular matrix was observed between the products of the viral and cellular src genes. It has previously been demonstrated that pp6Ov-src is associated with a detergent-insoluble matrix containing the cellular cytoskeleton (J. G. Burr, G. Dreyfuss, S. Penman, and J. M. Buchanan, Proc. Natl. Acad. Sci. USA 77:3484-3488, 1980). We observed a similar association of the transforming proteins of Fujinami sarcoma virus (P130gagfps) and Yamaguchi 73 avian sarcoma virus
Eukaryotic cells target mRNAs to the nonsense-mediated mRNA decay (NMD) pathway when translation ... more Eukaryotic cells target mRNAs to the nonsense-mediated mRNA decay (NMD) pathway when translation terminates within the coding region. In mammalian cells, this is presumably due to a downstream signal deposited during pre-mRNA splicing. In contrast, unspliced retroviral RNA undergoes NMD in chicken cells when premature termination codons (PTCs) are present in the gag gene. Surprisingly, deletion of a 401-nt 3¢ UTR sequence immediately downstream of the normal gag termination codon caused this termination event to be recognized as premature. We termed this 3¢ UTR region the Rous sarcoma virus (RSV) stability element (RSE). The RSE also stabilized the viral RNA when placed immediately downstream of a PTC in the gag gene. Deletion analysis of the RSE indicated a smaller functional element. We conclude that this 3¢ UTR sequence stabilizes termination codons in the RSV RNA, and termination codons not associated with such an RSE sequence undergo NMD.
Transformation of chicken cells by Fujinami sarcoma virus (FSV), PRC II or Y73 (three independent... more Transformation of chicken cells by Fujinami sarcoma virus (FSV), PRC II or Y73 (three independently isolated avian sarcoma viruses that are replication-defective and lack the Rous sarcoma virus src gene) resulted in significant elevation (4-13 fold) of phosphotyrosine levels in cellular protein. The gag-related proteins encoded by these avian sarcoma viruses (ASVs) were all associated with tyrosine-specific protein kinase activity when assayed in immune complexes and were phosphorylated at both tyrosine and serine residues in vivo. Both the phosphotyrosine level in protein of FSVinfected cells and the protein kinase activity assayed in immune complexes containing the FSV protein P140 were temperature-sensitive. The presumed transforming proteins of these ASVs were compared with those of Rous sarcoma virus (RSV), Abelson murine leukemia virus and the Snyder-Theilen and Gardner-Arnstein strains of feline sarcoma virus (FeSV), which have previously been associated with tyrosine-specific protein kinase activity. FSV and PRC II proteins were shown to be structurally related to one another and to the FeSV proteins by tryptic peptide mapping and by immunological studies. No homology was observed, however, between the transforming proteins of RSV, Y73, Abelson murine leukemia virus and the FSV/PRC II/ FeSV class, suggesting there may be at least four classes of retroviruses whose transformation mechanisms involve aberrant phosphorylation of cellular protein at tyrosine residues.
ABSTRACT Most replication-competent retroviruses cause cancer only after a long latent period, by... more ABSTRACT Most replication-competent retroviruses cause cancer only after a long latent period, by insertional mutagenesis of the host genome, usually resulting in activation of a cellular proto-oncogene. In contrast, acute-transforming retroviruses, which have transduced host proto-oncogenes, cause rapid tumor formation and death. In both cases, some of the same genes are overexpressed and/or mutated, including genes involved in mitogenic signaling, cell cycle control, and cell survival. We are near the 100th anniversaries of the discoveries of both leukemia (1908) and sarcoma (1910) viruses in birds. In honor of this important milestone, this review will be focused mainly on studies of oncogensis by avian viruses, which paved the way for studies with many other oncogenic retroviruses. Rous sarcoma virus (RSV) causes rapid oncogenesis by high level expression of an activated src gene tyrosine kinase. The host src gene was transduced by the virus without its C-terminal negative regulatory domain. In contrast, avian leukosis virus (ALV) induces B-cell lymphomas by insertional mutagenesis after the provirus integrates into the host genome. In addition to activation of classical proto-oncogenes, proviral insertions can activate cellular micro-RNAs called oncomiRs. The precursor of miR-155, which is upregulated in many human tumors, was first identified as a common ALV B-cell integration cluster (bic) in metastatic, long-latency lymphomas. Targets of miR-155 repression include tumor-suppressor genes, providing a novel mechanism for their inactivation in ALV-induced tumors. Telomerase reverse transcriptase is also activated by enhancer insertion in many ALV-induced lymphomas, providing a good model system for study of telomerase-dependent tumors. KeywordsRous sarcoma virus-Avian leukosis virus-Src-Oncogene-Insertional mutagenesis-miR-155-Bic-TERT
The avian leukosis virus ⌬LR-9 causes a high frequency of B-cell lymphomas within weeks after inj... more The avian leukosis virus ⌬LR-9 causes a high frequency of B-cell lymphomas within weeks after injection into 10-day-old chicken embryos. These lymphomas result from proviral integrations into the oncogene c-myb. In contrast, LR-9, which lacks the 42-nucleotide gag gene deletion of ⌬LR-9, does not cause a high frequency of c-myb-associated short-latency lymphomas. Although viral replication rates and spliced env mRNA levels were found to be similar for both viruses, ⌬LR-9 exhibited an increase in readthrough transcription compared to LR-9. The ⌬LR-9 deletion is located in the region of the gag gene corresponding to the matrix (MA) protein as well as in the negative regulator of splicing (NRS) element. To test whether disruption of the NRS or of the MA protein was responsible for inducing short-latency lymphomas, we generated viruses with NRS point mutations that maintained the wild-type Gag amino acid sequence. One of the mutant viruses induced an even higher incidence than ⌬LR-9 of short-latency lymphomas with viral integrations into c-myb. Thus, we propose that disruption of the NRS sequence promotes readthrough transcription and splicing to the downstream myb gene, causing overexpression of a slightly truncated Myb protein, which induces short-latency tumors.
Splicing of Rous sarcoma virus RNA is regulated in part by a cis-acting intronic RNA element call... more Splicing of Rous sarcoma virus RNA is regulated in part by a cis-acting intronic RNA element called the negative regulator of splicing (NRS). An NRS mutant affecting nt 916-923 disrupts U11 snRNP binding and reduces NRS activity (Gontarek et al., 1993, Genes & Dev 7:1926-1936). However, we observed that a U1 59 splice site-like sequence, which overlapped the U11 site, was also disrupted by this mutation. To determine whether the U1 or the U11 site was essential for NRS activity, we analyzed twelve additional mutants involving nt 915-926. All mutations that disrupted the potential base pairing between U1 snRNA and the NRS reduced NRS activity, including single point mutations at nt 915, 916, and 919. The point mutation at nt 919 was partially suppressed by a compensatory base change mutation in U1 snRNA. In contrast, a mutation which strengthened the potential base pairing between the U1 site and the NRS increased NRS activity. Surprisingly, mutations that specifically targeted the U11 59 splice site consensus sequence increased the levels of unspliced RNA, suggesting U11 binding plays an antagonistic role to NRS activity. We propose that U1 snRNP binding to the NRS inhibits splicing and is regulated by U11 snRNP binding to the overlapping sequence. Competition between U1 and U11 snRNPs would result in the appropriate balance of spliced to unspliced RNAs for optimal viral replication. Further, a virus mutated in the U1/U11 region of the NRS was found to have delayed replication.
cis-acting sequences of Rous sarcoma virus (RSV) RNA involved in control of the incomplete splici... more cis-acting sequences of Rous sarcoma virus (RSV) RNA involved in control of the incomplete splicing that is part of the retroviral life cycle have been studied. The 5' and two alternative 3' splice sites, as well as a negative regulator of splicing element in the intron, have been introduced into chimeric constructs, and their respective roles in splicing inhibition have been evaluated by transient transfection experiments. Although the RSV 5' splice site was used efficiently in these assays, substrates containing either the RSV env or the RSV src 3' splice site were not spliced completely, resulting in 40 to 50% unspliced RNA. Addition of the negative regulator of splicing element to substrates containing RSV 3' splice sites resulted in greater inhibition of splicing (70 to 80% unspliced RNA), suggesting that the two elements function independently and additively. Deletion of sequences more than 70 nucleotides upstream of the src 3' splice site resulted in efficient splicing at this site, suggesting that inefficient usage is not inherent in this splice site but is instead due to sequences upstream of it. Insertion of these upstream sequences into the intron of a heterologous pre-mRNA resulted in partial inhibition of its splicing. In addition, secondary structure interactions were predicted to occur between the src 3' splice site and the inhibitory sequences upstream of it. Thus, RSV splicing control involves both intronic sequences and 3' splice sites, with different mechanisms involved in the underutilization of the env and src splice acceptor sites.
A c/s-acting negative regulator of splicing (NRS) within the gag gene of RSV is involved in contr... more A c/s-acting negative regulator of splicing (NRS) within the gag gene of RSV is involved in control of the relative levels of spliced and unspliced viral mRNAs. Insertion of the NRS into the intron of an adenovirus pre-mRNA resulted in inhibition of splicing in vitro before the first cleavage step. Analyses of spliceosome assembly with this substrate showed that it formed large RNP complexes that did not migrate like mature spliceosomes on native gels. Affinity selection of the RNP complexes formed on NRS-containing pre-mRNAs showed an association with Ull and U12 snRNPs, as well as with the spliceosomal snRNPs. Immunoprecipitation with antisera specific for U1 and U2 snRNPS showed binding of both snRNPs to NRS RNA. A 7-nucleotide missense mutation in the NRS that prevented binding of Ull and U12 snRNPs impaired NRS activity in vivo, suggesting a functional role for Ull and U12 snRNPS in the inhibition of splicing mediated by the RSV NRS RNA.
All retroviruses need mechanisms for nucleocytoplasmic export of their unspliced RNA and for main... more All retroviruses need mechanisms for nucleocytoplasmic export of their unspliced RNA and for maintenance of this RNA in the cytoplasm, where it is either translated to produce Gag and Pol proteins or packaged into viral particles. The complex retroviruses encode Rev or Rex regulatory proteins, which interact with cis-acting viral sequences to promote cytoplasmic expression of incompletely spliced viral RNAs. Since the simple retroviruses do not encode regulatory proteins, we proposed that they might contain cis-acting sequences that could interact with cellular Rev-like proteins. To test this possibility, we initially looked for a cis-acting sequence in avian retroviruses that could substitute for Rev and the Rev response element in human immunodeficiency virus type 1 expression constructs. A cis-acting element in the 3 untranslated region of Rous sarcoma virus (RSV) RNA was found to promote Rev-independent expression of human immunodeficiency virus type 1 Gag proteins. This element was mapped between RSV nucleotides 8770 and 8925 and includes one copy of the direct repeat (DR) sequences flanking the RSV src gene; similar activity was observed for the upstream DR. To address the function of this element in RSV, both copies of the DR sequence were deleted. Subsequently, each DR sequence was inserted separately back into this deleted construct. While the viral construct lacking both DR sequences failed to replicate, constructs containing either the upstream or downstream DR replicated well. In the absence of both DRs, Gag protein levels were severely diminished and cytoplasmic levels of unspliced viral RNA were significantly reduced; replacement of either DR sequence led to normal levels of Gag protein and cytoplasmic unspliced RNA.
For simple retroviruses, such as the Rous sarcoma virus (RSV), post-transcriptional control eleme... more For simple retroviruses, such as the Rous sarcoma virus (RSV), post-transcriptional control elements regulate viral RNA splicing, export, stability, and packaging into virions. These RNA sequences interact with cellular host proteins to regulate and facilitate productive viral infections. One such element, known as the RSV stability element (RSE), is required for maintaining stability of the full-length unspliced RNA. This viral RNA serves as the mRNA for the Gag and Pol proteins and also as the genome packaged in progeny virions. When the RSE is deleted from the viral RNA, the unspliced RNA becomes unstable and is degraded in a Upf1-dependent manner. Current evidence suggests that the RSE inhibits recognition of the viral gag termination codon by the nonsense-mediated mRNA decay (NMD) pathway. We believe that the RSE acts as an insulator to NMD, thereby preventing at least one of the required functional steps that target an mRNA for degradation. Here, we discuss the history of the RSE and the current model of how the RSE is interacting with cellular NMD factors.
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Papers by Karen Beemon