Retroviral transduction of oncogenic sequences involves viral DNA instead of RNA (original) (raw)
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Development of transforming function during transduction of proto-ras into Harvey sarcoma virus
Proceedings of the National Academy of Sciences, 1994
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
Evidence that retroviral transduction is mediated by DNA not by RNA
Proceedings of the National Academy of Sciences, 1990
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' Abbreviations: HaSV, Harvey sarcoma virus; MoV, Moloney virus; LTR, long terminal repeat; ffu, focus-forming unit(s); pfu, plaqueforming unit(s).
Retroviral recombination during reverse transcription
Proceedings of the National Academy of Sciences, 1990
After mixed infection, up to half of related retroviruses are recombinants. During infection, retroviral RNA genomes are first converted to complementary DNA (cDNA) and then to double-stranded DNA. Thus recombination could occur during reverse transcription, by RNA template switching, or after reverse transcription, by breakage and reunion of DNA. It has not been possible to distinguish between these two potential mechanisms of recombination because both single-stranded cDNA and double-stranded proviral DNA exist in infected cells during the eclipse period. Therefore we have analyzed for recombinant molecules among cDNA products transcribed in vitro from RNA of disrupted vinons. Since recombinants from aflelic parents can only be distinguished from parental genomes by point mutations, we have examined the cDNAs from virions with distinct genetic structures for recombinant-specific size and sequence markers. The parents share a common internal allele that allows homology-directed recombination, but each contains specific flanking sequences. One parent is a synthetically altered Harvey murine sarcoma virus RNA that lacks a retroviral 3' terminus but carries a Moloney murine retrovirus-derived envelope gene (env) fragment 3' of its transforming ras gene. The other parent is intact Moloney virus. Using a Harvey-specific 5' primer and a Moloney-specific 3' primer, we have found recombinant cDNAs with the polymerase chain reaction, proving directly that retroviruses can recombine during reverse transcription unassisted by cellular enzymes, probably by template switching during cDNA synthesis. The recombinants that were obtained in vitro were identical with those obtained in parallel experiments in vivo.
Retroviral oncogenes: a historical primer
Nature Reviews Cancer, 2012
Retroviruses are the original source of oncogenes. The discovery and characterization of these genes were made possible by the introduction of quantitative cell biological and molecular techniques for the study of tumor viruses. Key features of all retroviral oncogenes were first identified in src, the oncogene of Rous sarcoma virus. These include non-involvement in viral replication, coding for a single protein, and cellular origin. The myc, ras and erbB oncogenes quickly followed src, and these together with pi3k are now recognized as critical driving forces in human cancer.
Proceedings of the National Academy of Sciences, 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 recombinant virus/mutation enhances transforming function/recombinant cancer gene hypothesis) 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
Journal of Virology
The ras genes of BALB and Harvey sarcoma viruses contain point mutations in codon 12 or codons 12 and 59, relative to proto-ras from normal animal and human cells. By in vitro recombination between cloned rat proto-ras and cloned BALB and Harvey sarcoma proviruses, we constructed recombinant proviruses with normal proto-ras-coding regions. These recombinant proviruses transformed mouse 3T3 cells upon transfection. However, when the transforming efficiencies of proviral DNAs were compared after transfection with helper provirus, recombinant proviruses were 2 to 30 times less efficient than the corresponding wild-type proviruses. Recombinant sarcoma viruses isolated from cells transformed by cloned proviral DNA contained the expected normal ras-coding region. They transformed rat embryo cells and induced erythroblastosis and sarcomas in newborn mice as efficiently as wild-type viruses did. We conclude that conversion of normal proto-ras genes to viral ras genes depends on truncation of normal proto-ras regulatory elements and substitution by retroviral (long terminal repeat) promoters and that the transforming function of long terminal repeat-ras genes is enhanced by point mutations.
Journal of Virology, 1989
The ras genes of BALB and Harvey sarcoma viruses contain point mutations in codon 12 or codons 12 and 59, relative to proto-ras from normal animal and human cells. By in vitro recombination between cloned rat proto-ras and cloned BALB and Harvey sarcoma proviruses, we constructed recombinant proviruses with normal proto-ras-coding regions. These recombinant proviruses transformed mouse 3T3 cells upon transfection. However, when the transforming efficiencies of proviral DNAs were compared after transfection with helper provirus, recombinant proviruses were 2 to 30 times less efficient than the corresponding wild-type proviruses. Recombinant sarcoma viruses isolated from cells transformed by cloned proviral DNA contained the expected normal ras-coding region. They transformed rat embryo cells and induced erythroblastosis and sarcomas in newborn mice as efficiently as wild-type viruses did. We conclude that conversion of normal proto-ras genes to viral ras genes depends on truncation of normal proto-ras regulatory elements and substitution by retroviral (long terminal repeat) promoters and that the transforming function of long terminal repeat-ras genes is enhanced by point mutations.
Proceedings of the National Academy of Sciences, 1981
Harvey murine sarcoma virus (Ha-MuSV) is a mouse-rat recombinant retrovirus that encodes a protein designated p21, required for virally induced transformation. Using a radiolabeled DNA fragment from the p21 coding region, we have detected homologous DNA sequences in the normal DNA of rats and of several other vertebrate species. Moreover, many tested cells from these species contain low levels of a p21 protein that is highly related to viral 21. Now we report two independent fragments from normal rat DNA containing sequences (sarc) homologous to the Ha-MuSV transforming region that were cloned in the bacteriophage vector Charon 4A. Sarc sequences in the one fragment are completely colinear with the viral sequences and share apparently all restriction endonuclease sites. Sarc sequences in the second fragment have several sets of intervening sequences and lack some restriction endonuclease sites found in the viral transforming region. Despite the presence of these intervening sequences in the second sarc fragment, we have been able to ligate this sarc fragment to the long terminal repeat sequence of Ha-MuSV and to induce cellular transformation and high levels of p21 expression upon transfection of this DNA to NIH 3T3 mouse cells. These results suggest that elevated levels of p21, normally expressed at low levels in a variety of cells, can induce cellular transformation.
Activation of the cellular src gene by transducing retrovirus
Molecular and Cellular Biology, 1986
Newly isolated strains of avian sarcoma virus, S1 and S2, were shown to have the transduced cellular src gene as their viral transforming gene (Yamagishi et al., Virology 137:266-275, 1984). In this work, the S1 and S2 genomes were molecularly cloned, and the junction sequences between the viral genomes and the c-src genes and the complete nucleotide sequences of the v-src genes transduced in these viruses were determined. Data on the junction sequences suggested that 5' recombination had occurred between the 5'-noncoding region of c-src and the 5' region of the gag sequence encoding p19 in both viruses and that 3' recombination had occurred in the last coding exon of c-src with either the middle portion of the env sequence encoding gp85 for S1 or the 3' portion of pol coding for reverse transcriptase for S2. Comparison of the amino acid sequences of the S1 and S2 src products deduced from the nucleotide sequences (pp62S1-src and pp62S2-src with that of c-src pro...
Proceedings of the National Academy of Sciences, 1985
The myeloproliferative sarcoma virus not only transforms fibroblasts but also causes extensive expansion of the hematopoietic stem cell compartment on infection of adult mice. Similar to the Moloney sarcoma virus, it carries the mos oncogene. Moloney sarcoma virus, however, does not induce myeloproliferation and leukemia in adult mice. The difference between the two viruses was explored by using their molecularly cloned genomes and the cellular mos oncogene to construct recombinant genomes. It was shown that the U3 region of the viral long terminal repeat (LTR) has a decisive function in determining the target cell specificity of the myeloproliferative sarcoma virus. Any mos gene, whether of cellular or viral origin, is sufficient in conjunction with the proper LTR to induce myeloproliferation. Our results indicate that the pathogenicity of acutely transforming viruses is determined not only by the oncogene but also by sequences in the viral LTR.