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

Goodrich, David W.; Duesberg, Peter H.

doi:10.1073/pnas.87.9.3604

Cited by 18 publications

(20 citation statements)

References 32 publications

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“…and the five following p-ras exons, providing a strong promoter for efficient transcription of p21ras mRNAs and the packaging signal (10,(28)(29)(30)(31). After addition of the 3' LTR via DNA (31) or RNA (32) recombination, a replicating progenitor virus formed that can induce malignant transformation. During subsequent replication, the transforming activity of the progenitor virus may have been enhanced by point mutations in codons 12 and 59.…”

Section: Resultsmentioning

confidence: 99%

Development of transforming function during transduction of proto-ras into Harvey sarcoma virus.

Läng¹,

Treinies²,

Duesberg³

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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Oncogenic retroviruses are generated by transduction of the coding region of a protooncogene and acquire genetic changes during subsequent replication. Critical genetic events which occurred during and after transduction of rat proto-ras-lHa into Harvey sarcoma virus were identified by evaluating the transforming activity of plausible synthetic progenitor proviruses encompassing the complete proto-ras genomic region with or without various 5' deletions. All progenitor proviruses induced phenotypic transformation of mouse NIH 3T3 cells, although with a 5-to 10-fold lower frequency than Harvey sarcoma provirus. Although no tumor formation was observed in vivo after inoculation in the absence of helper murine retrovirus, both wild-type and progenitor viruses inoculated in the presence of helper virus induced tumors in newborn BALB/c mice. No critical alterations of the p2l1 coding region and no deletion of 5' genomic elements were detected in a progenitor virus encompassing the complete proto-ras genomic region that had been isolated from tumors. However, one progenitor virus that included all proto-ras exons induced tumors with a decreased latency. This virus contained a mutation in codon 12 (glycine to valine), which had apparently been selected during tumorigenesis in vivo. During the genesis of Harvey sarcoma virus, critical steps conferring transforming function are therefore transduction of coding proto-ras exons and enhancement of their transforming function by specific amino acid changes in p2lrs.Balb sarcoma virus (BalbSV) and Harvey sarcoma virus (HaSV) induce erythroblastosis and fibrosarcomas in infected mice by an apparent single-step mechanism (1-3). The two viruses express oncogenic p2lras proteins mutated in amino acid 12 and amino acids 12 and 59, respectively, relative to the normal p2lras protein of rat proto-ras-lHa (p-ras_JHa). Previous studies showed that the transforming function of both viruses was enhanced but did not depend on the virus-specific codons described (4-10). The latency of malignant tumor induction in infected mice and the efficiency of phenotypic transformation in vitro were comparable for viral ras (v-ras)-and p-ras-expressing viruses in one study (5). In contrast, Velu et al. (7) reported a correlation between the lower frequency of focal transformation of mouse NIH 3T3 cells by p-ras-expressing viruses and the preferential induction of lymphoid versus erythroblastic tumors in vivo. Sequence comparison between genomic rat p-ras-l sequences and v-ras of HaSV revealed the presence of one noncoding exon (exon 1) and a putative second noncoding exon (exon ?) upstream of the v-ras coding region (4, 6). Putative p-ras promoter regions and both noncoding exons are part of a 3.6-kbp HindIII (-2600)-HindIII (1026) rat genomic DNA fragment (Fig. 1) MATERIALS AND METHODSConstruction of Progenitor Proviruses. The construction of wild-type HaS provirus (termed pA) and synthetic HaS proviruses pApraslX and pA6.2-pras was described previously (4-6). pA6.2-pras (Fig.

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Section: Resultsmentioning

confidence: 99%

Development of transforming function during transduction of proto-ras into Harvey sarcoma virus.

Läng¹,

Treinies²,

Duesberg³

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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“…And immunodeficiency is a typical consequence of leukemia (32). High virus replication favors transduction because transduction proceeds via integration of proviral DNA into cell DNA (37). Thus, the higher probability of transduction in animals with cancer would explain the relatively high incidence of oncogenic retroviruses with two distinct cell-derived sequences.…”

Section: Below)mentioning

confidence: 99%

“…Retroviruses transduce cellular sequences via accidental illegitimate recombination with the cell (37). Since all known retroviral transductants are oncogenic, the presence of a specific transduced sequence in a virus is typically assumed to have an oncogenic function (6).…”

Section: Below)mentioning

confidence: 99%

Avian erythroblastosis virus E26: only one (myb) of two cell-derived coding regions is necessary for oncogenicity.

Duesberg²

1994

Proc. Natl. Acad. Sci. U.S.A.

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The oncogene hypothesis postulates that mutated cellular genes, termed proto-onc genes, function as cancer genes because they are related to retroviral onc genes. However, in contrast to retroviral onc genes, mutated proto-onc genes from cancers are not sufficient for carcinogenesis. Therefore, it has been proposed that mutated proto-onc genes depend on other proto-onc genes for carcinogenesis. Since the oncogene of the avian leukemia virus E26 includes coding regions derived from two cellular proto-onc genes, proto-myb and proto-ets, this hybrid gene has been proposed to be a model for twogene-carcinogenesis. Here we set out to test this proposal. For this purpose myb and ets deletion mutants of cloned E26 provirus were prepared, and the corresponding viruses, produced by transfected primary chicken embryo cells, were tested for leukemogenicity in newborn chickens. It was found that an ets deletion mutant was just as leukemogenic as the wild-type virus and that a myb deletion mutant lacked leukemogenicity completely. To eliminate the possibility that our E26 myb deletion mutant failed to be leukemogenic because it failed to replicate, the virus was titered by a quantitative polymerase chain reaction (PCR) method. By this method, E26 from the plasma of infected chickens was first allowed to reversetranscribe viral RNA to cDNA in vitro, and then the cDNA concentration was determined from the lowest dilution that gave a positive signal after amplification of E26 cDNA by the PCR method. Virus titers of about 101 per ml were found for wild type and for myb and ets deletion mutants of E26. It is concluded that the ets region is not essential for carcinogenesis, and E26 derives transforming function from overexpression of its proto-myb coding region via the retroviral promoter. Thus, E26 is a single-hit carcinogen and, like all other oncogenic retroviruses, is not a model for two-genecarcinogenesis. Viral ets probably reflects a genetic accident that transduced sequences of proto-ets together with protomyb in generating E26.

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“…These oncogenes were found embedded in viral genomes, replacing part of the viral coding sequences and rendering most acute transforming retroviruses replication defective. Scattered evidence, including a drug-resistant patient isolate and vectors studied in cultured cells, suggests the possibility of transduction by human immunodeficiency virus type 1 (HIV-1) (43, 53), but the ability of HIV to perform the specific steps required for transduction has not been addressed experimentally.Several models have been proposed for oncogene transduction (4,15,16,19,29,38,41,44,50,54,56,61). Most suggest that the fusing of host and viral sequences resulted from a series of rare events that are intrinsic to the viral replication strategy (52).…”

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confidence: 99%

“…Several models have been proposed for oncogene transduction (4,15,16,19,29,38,41,44,50,54,56,61). Most suggest that the fusing of host and viral sequences resulted from a series of rare events that are intrinsic to the viral replication strategy (52).…”

mentioning

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Human Immunodeficiency Virus Type 1 Transductive Recombination Can Occur Frequently and in Proportion to Polyadenylation Signal Readthrough

Telesnitsky

2004

J Virol

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One model for retroviral transduction suggests that template switching between viral RNAs and polyadenylation readthrough sequences is responsible for the generation of acute transforming retroviruses. For this study, we examined reverse transcription products of human immunodeficiency virus (HIV)-based vectors designed to mimic postulated transduction intermediates. For maximization of the discontinuous mode of DNA synthesis proposed to generate transductants, sequences located between the vectors' two long terminal repeats (vector "body" sequences) and polyadenylation readthrough "tail" sequences were made highly homologous. Ten genetic markers were introduced to indicate which products had acquired tail sequences by a process we term transductive recombination. Marker segregation patterns for over 100 individual products were determined, and they revealed that more than half of the progeny proviruses were transductive recombinants. Although most crossovers occurred in regions of homology, about 5% were nonhomologous and some included insertions. Ratios of encapsidated readthrough and polyadenylated transcripts for vectors with wild-type and inactivated polyadenylation signals were compared, and transductive recombination frequencies were found to correlate with the readthrough transcript prevalence. In assays in which either vector body or tail could serve as a recombination donor, recombination between tail and body sequences was at least as frequent as body-body exchange. We propose that transductive recombination may contribute to natural HIV variation by providing a mechanism for the acquisition of nongenomic sequences.Retroviral transduction of cellular genes is well documented (5, 58) and was best characterized by the discovery of the cellular counterparts of the oncogenes in acute transforming retroviruses (49). These oncogenes were found embedded in viral genomes, replacing part of the viral coding sequences and rendering most acute transforming retroviruses replication defective. Scattered evidence, including a drug-resistant patient isolate and vectors studied in cultured cells, suggests the possibility of transduction by human immunodeficiency virus type 1 (HIV-1) (43, 53), but the ability of HIV to perform the specific steps required for transduction has not been addressed experimentally.Several models have been proposed for oncogene transduction (4,15,16,19,29,38,41,44,50,54,56,61). Most suggest that the fusing of host and viral sequences resulted from a series of rare events that are intrinsic to the viral replication strategy (52). One model for transduction is as follows. First, a retrovirus integrates just upstream of the sequence that will be transduced. Retroviral polyadenylation signals are leaky (18), and readthrough of such signals fuses sequences adjacent to the proviral insertion to the viral genomic RNA's 3Ј end. Such readthrough transcripts can be encapsidated and serve as templates for normal reverse transcription products whose synthesis bypasses the RNA readthrough 3Ј ends (19,55)...

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

Cited by 18 publications

References 32 publications

Development of transforming function during transduction of proto-ras into Harvey sarcoma virus.

Development of transforming function during transduction of proto-ras into Harvey sarcoma virus.

Avian erythroblastosis virus E26: only one (myb) of two cell-derived coding regions is necessary for oncogenicity.

Human Immunodeficiency Virus Type 1 Transductive Recombination Can Occur Frequently and in Proportion to Polyadenylation Signal Readthrough

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