Silent Point Mutation in an Avian Retrovirus RNA Processing Element Promotes c- myb -Associated Short-Latency Lymphomas

Abstract

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.A defining characteristic of retroviruses is that they randomly integrate their reverse-transcribed genome into the genome of the host cell. Selection for a specific integration site can occur when a retrovirus integrates into a proto-oncogene, resulting in a clonal tumor. When an avian leukosis virus (ALV) is injected into 1-day-old chicks, it typically induces clonal B-cell lymphomas exhibiting proviral DNA integrated into the oncogene c-myc (13). These B-cell lymphomas result from the deregulated expression of c-myc and take several months to develop. However, if an ALV is injected into 10-to 12-day-old chicken embryos, approximately 14% of the hatched chickens develop short-latency B-cell lymphomas that cause death within weeks (29). Moreover, injection of chicken embryos with the recombinant ALV EU-8 (which has a 42-nucleotide [nt] deletion in the gag gene) leads to short-latency B-cell lymphomas in 40 to 80% of the chickens (16,32).Most of these short-latency lymphomas contain proviral DNA integrated into the first intron of c-myb (15, 29). Transcription initiates at the ALV promoter in the 5Ј long terminal repeat (LTR) and reads through the polyadenylation sequence in the 3Ј LTR to transcribe the downstream c-myb gene (see Fig. 1). Splicing then occurs from the 5Ј splice site in the 5Ј end of the viral gag gene to the 3Ј splice site of the c-myb second exon ( Fig. 1) (15). Translation of the ALV-myb hybrid RNA yields a Myb protein that is truncated at its N terminus by 20 amino acids (15). Truncated Myb, in contrast to full-length Myb, is highly oncogenic when expressed from retroviral vectors introduced into 12-day-old chicken embryos (15). Further, the transcriptional profile of the short-latency, c-myb-associated tumors is distinct from that of c...

Telomerase reverse transcriptase expression elevated by avian leukosis virus integration in B cell lymphomas

“…The chicken tumors were generated in a study described in ref. 13. Tumor genomic DNA was prepared by standard proteinase K digestion followed by phenol-chloroform extraction.…”

“…ALV induces B cell lymphomas associated with clonal proviral integrations into various proto-oncogenes, including myc and myb (8)(9)(10)(11)(12)14). To determine whether myb integration acts as an initiating event in tumorigenesis in these chickens, as it does with previously studied rapid-onset ALVinduced lymphomas (8,9,14), we examined the clonality of myb integrations in previously studied tumors (13). If the tumor is clonal for myb, we expect to see equal signal from both the rearranged chromosome and the WT chromosome.…”

“…When 10-day-old chicken embryos were infected with certain mutant strains of ALV, B cell lymphomas caused death in up to 75% of the chickens by 10 weeks after hatching (13). We performed inverse PCR on genomic DNA from these lymphomas and identified 28 unique viral integration sites in tumors from eight different birds.…”

Packaging and reverse transcription of snRNAs by retroviruses may generate pseudogenes

“…2C). Our lab previously characterized a cisacting NRS element within the gag gene of avian retroviruses that suppresses splicing to the src 3Ј splice site and promotes polyadenylation (Arrigo and Beemon 1988;McNally et al 1991;Hibbert et al 1999;O'Sullivan et al 2002;Polony et al 2003). The NRS contains a 5Ј splice site-like sequence that has 12 out of 13 nucleotides (nt) complementary to U6 snRNA (nt 33-45).…”

Two Regions Promote U11 Small Nuclear Ribonucleoprotein Particle Binding to a Retroviral Splicing Inhibitor Element (Negative Regulator of Splicing)

“…1) suggest the intriguing possibility that inhibition results from an inappropriate, non-productive U6 interaction. The importance of the NRS was shown with viral constructs harboring U1-site mutations, which produced viruses with mildly delayed replication kinetics (19), and by the increased incidence of lymphomas in infected chickens (33).…”