Transduction of cellular neo mRNA by retrovirus-mediated recombination

Abstract

Transduction of cellular oncogenes by retroviruses is thought to be a multistep process, involving transcriptional activation of a cellular gene by upstream proviral integration and joining of cellular DNA to retroviral transcriptional signals, followed by copackaging and recombination with a helper virus genome during reverse transcription. To examine the molecular mechanism of the reverse transcriptase-mediated recombination, we introduced into mouse fibroblast cells a variety of constructs in which the neo selectable marker was joined to flanking retroviruslike or cell-like sequences. After superinfection and copackaging with a replication-competent Mo-MuLVs"PF virus, the formation of recombinant neo transducing viruses was assessed in a second round of virus infection by the ability to confer G418 resistance to infected cells. Our results showed that recombinant neo proviruses were generated from neo RNA containing either a 5' or 3' retroviral end, implying that one recombination event with helper virus RNA was sufficient to incorporate the neo gene into proviral DNA. Recombination occurred with an apparent frequency of 10-4 to 10-5 per replication cycle in the absence of homology between the two recombining partners. This frequency, however, increased at least 100-fold if homology was provided at the site of recombination. Our results support the hypothesis that neo-transducing viruses arise via reverse transcriptase-mediated recombination of RNA rather than by recombination proceeding through DNA intermediates. Unexpectedly, removal of the retroviral packaging site 'W reduced the number of neo recombinants only slightly. Our data indicated that although RNAs lacking the 'I site are poorly packaged into virions, those RNAs that are included in the virions undergo frequent recombination, even if there is no selection for recombination. Many of the neo recombinants formed with the Jconstructs had undergone additional recombinations and often incorporated the site from the helper RNA.

cis-active structural motifs involved in specific encapsidation of Moloney murine leukemia virus RNA

“…Indeed, conformational arrangements involving the primer binding site and Psi domains have been observed (26), suggesting a possible interdependence of the two domains. Additionally, the effect of mutation AB at the level of the reverse transcription step is in agreement with some (35,36) but not all (37) previous studies.…”

“…While putative stem-loop structures in the Psi region could contribute to the function of the M-MuLV encapsidation signal, such an interpretation is complicated by the fact that other functions have been ascribed to this region. Notably, the M-MuLV 5Ј untranslated region also appears to influence the initiation of reverse transcription, ribosome binding, splicing, and viral RNA dimerization (8,9,29,32,35,36). Indeed, the dimer linkage structure, which maps to the 5Ј untranslated region, may mediate a dimerization event that is a prerequisite to RNA packaging, although the precise relationship between dimerization and encapsidation is not clear.…”

Unusually high frequency of reconstitution of long terminal repeats in U3-minus retrovirus vectors by DNA recombination or gene conversion

“…It was estimated that 3 to 5% of proviruses had undergone at least one template switch (6,27,39). DNA recombination has been proposed as a mechanism for the transduction of cellular genes by retroviruses (22,23).…”

Répression et activation des rétrovirus murins dans les cellules totipotentes

“…Une variante de ce schéma a été produite dans le passé pour expliquer la capture des oncogènes par les rétrovirus (notamment chez les aviaires), à ceci près qu'alors, c'est le promoteur virale du LTR 5' qui assure la capture de l'oncogène situé en 3' du site d'intégration (fig ure 4) [19]. Bien que ce nouveau type de capture de séquence 5' reste à démontrer plus précisément, sa vrai semblance est suggérée par certaines ont établi que la présence d'un seul L TR est suffisante pour que les évé nements de recombinaison entre les ARN coempaquetés dans la particule virale aient lieu [20]. Cela constitue rait une nouvelle indication de 1 'action des transposons (dont les rétrovirus ne sont qu 'un des exem ples) dans le maintien de la fluidité des génomes [21].…”