Cancer Genes by Illegitimate Recombination a

Retroviral transduction of cellular nucleic acid sequences requires illegitimate RNA or DNA recombination. To test a model that postulates transduction via efficient illegitimate recombination during reverse transcription of viral and cellular RNAs, we have measured the ability of Harvey sarcoma viruses (HaSVs) with artificial 3' termini to recover a retroviral 3' terminus from helper Moloney virus (MoV) by illegitimate and homologous recombination. For this purpose, mouse NIH 3T3 cells were transformed with Harvey proviruses and then superinfected with MoV. The proviruses lacked the 3' long terminal repeat and an untranscribed region of the 5' long terminal repeat to prevent virus regeneration from input provirus. Only 0-11 focus-forming units of HaSV were generated upon MoV superinfection of 3 x 106 cells transformed by Harvey proviruses with MoV-unrelated termini. This low frequency is consistent with illegitimate DNA recombination via random Moloney provirus integration 3' of the transforming viral ras gene in the 10'-kilobase mouse genome. When portions of murine viral envelope (env) genes were attached 3' of ras, 102-105 focus-forming units of HaSV were generated, depending on the extent of homology with env of MoV. These recombinants all contained HaSV-specific sequences 5' and MoV-specific sequences 3' of the common env homology. They were probably generated by recombination during reverse transcription rather than by recombination among either input or secondary proviruses, since (i) the yield of recombinants was reduced by a factor of 10 when the env sequence was flanked by splice signals and (ii) HaSV RNAs without retroviral 3' termini would be inadequate templates for provirus synthesis. We conclude that there is no efficient illegitimate recombination in retroviruses. In view of known precedents of illegitimate DNA recombination, the structure of known viral onc genes, and our evidence for illegitimate DNA recombination via provirus integration, we favor the DNA model of transduction over the RNA model. Retroviral transduction of oncogenic sequences requires either RNA-or DNA-mediated illegitimate recombination. Based on an experimental system that appeared to involve "6surprisingly efficient" illegitimate recombination by RNA template switching during reverse transcription (1), the RNA model is currently favored (1-11). This model holds that a retrovirus would integrate upstream of a proto-onc gene, spontaneously lose its 3' terminus, or fail to terminate during transcription (9), and produce a 5' viral-3' proto-onc mRNA. In a second step, the cellular 3' region of this hybrid RNA would recombine with the nonhomologous 3' region of an intact (helper) retrovirus during reverse transcription in order to obtain a retroviral 3' terminus. A competing DNA model holds that all steps of retroviral transduction are mediated by illegitimate primary DNA recombination (12).The experimental system on which the RNA model is based showed efficient regeneration of Harvey murine sarcoma virus (HaSV) from ce...

Section: Methodsmentioning

confidence: 99%

Evidence that retroviral transduction is mediated by DNA not by RNA.

Goodrich

Duesberg²

1990

“…Recently, we developed a test for the transforming function of LTR-promoted proto-myc genes from viral lymphomas in quail embryo cells (21). On the basis of this functional test we show here that proto-myc genes promoted by defective as well as intact proviruses are sufficient to transform primary quail cells, like retroviruses with transforming myc genes (39,40). Thus, secondary clonal events appear to be necessary for tumorigenesis but not for transformation.…”

mentioning

confidence: 76%

“…However, this seems particularly implausible for myc genes because the viral equivalents of LTR-proto-myc genes directly transform not only fibroblasts and epithelial cells, but also lymphocytes to cause acute leukemias (2). We speculate that single-hit transformation is relevant not only to rare (see below) carcinogenic mutations caused by retroviruses without transforming genes but also to cancers with clonal chromosomal abnormalities generated by non-viral mutagens (1,39,40). Discrepancies Between the Probabilities of Cell Transformation by Provirus Insertion and Tumorigenesis.…”

Section: Transforming Function Ofmentioning

confidence: 99%

“…DISCUSSION Retrovirus-Promoted Proto-myc Recombinants Are Sufficient for Transformation of Embryo Cells. Because a single copy of an LTR-proto-myc hybrid gene is sufficient to A C transform embryo cells, as is the case with the viral myc genes of MH2 and OK10 (21,33,39,40), and because the transforming efficiencies of LTR-proto-myc and proviral myc genes are the same and both types of genes encode nearly identical proteins (33), we conclude that transformation by LTR-proto-myc genes is a single-hit event. Thus, our results indicate that proto-myc genes promoted by defective and nondefective proviruses are autonomous, single-hit transforming genes in embryo cells.…”

Section: Transforming Function Ofmentioning

confidence: 99%

See 1 more Smart Citation

Avian proto-myc genes promoted by defective or nondefective retroviruses are single-hit transforming genes in primary cells.

Zhou¹,

Duesberg²

1989

Lymphomas of certain strains of chickens infected by retroviruses frequently contain recombinant transforming genes in which the promoter of the cellular proto-myc gene is replaced by that of a defective rather than an intact retrovirus. Here we ask whether the resulting hybrid genes are sufficient for tumorigenic transformation like viral myc genes. Further, we ask whether retroviruses must be defective in order to mutate proto-myc to a transforming gene or whether the defectiveness plays a transformation-independent function in tumorigenesis. For this purpose the defective provirus of proviral-proto-myc recombinants from lymphomas were repaired, or intact proviruses were recombined with proto-myc genes in vitro, and then compared to recombinant proto-myc genes with defective proviruses for transforming function in quail embryo fibroblasts. It was found that a single copy of a provirus-proto-myc recombinant gene with an intact provirus is sufficient to transform a quail embryo cell in vitro. Moreover, our analyses showed that multiple internal retroviral deletions [corrected] eliminate or inhibit provirus expression. The effect of these deletions [corrected] was detectable only because the inactive proviruses were linked to the selectable, transforming proto-myc gene marker. It is consistent with our results that proviral defectiveness of recombinant proto-myc genes is necessary in vivo for the clonal growth of a transformed cell into a tumor to escape antiviral immunity. The large discrepancy between the probabilities of provirus insertion and tumorigenesis is suggested to reflect the low probabilities of spontaneous deletion of the provirus and of rare, strain-specific defects of tumor-resistance genes of the host.

“…The proto-ras gene is the cellular precursor of the murine Harvey sarcoma virus (12). However, subsequent experiments showed that this observation was limited to some morphologically unstable, aneuploid rodent cell lines; i.e., primary rodent and human cells could not be transformed by proto-ras genes with point mutations (23,24).…”

mentioning

confidence: 99%

A retroviral promoter is sufficient to convert proto-src to a transforming gene that is distinct from the src gene of Rous sarcoma virus.

Zhou

Duesberg

1990

A retroviral promoter is sufficient to convert proto-src to a transforming gene that is distinct from the src gene of Rous sarcoma virus (proto-src Contributed by Peter H. Duesberg, August 31, 1990 ABSTRACT The src genes of four natural isolates of avian sarcoma viruses differ from cellular proto-src in two genetic substitutions: the promoter of the cellular gene is replaced by a retroviral counterpart, and at least six codons from the 3' terminus: are replaced by retroviral or heterologous cellderived elements. Since virus constructs with a complete protosrc coding region failed to transform avian cells but acquired transforming function by point mutations of various codons, it