RESUMO
Gene-transfer vectors based on lentiviruses are distinguished by their ability to transduce non-dividing cells. The HIV-1 proteins Matrix, Vpr and Integrase have been implicated in the nuclear import of the viral genome in non-dividing cells. Here we show that a sequence within pol is also required in cis. It contains structural elements previously associated with the progress of reverse transcription in target cells. We restored these elements in cis within late-generation lentiviral vectors. The new vector transduced to a much higher efficiency several types of human primary cells, when both growing and growth-arrested, including haematopoietic stem cells assayed by long-term repopulation of NOD/SCID mice. On in vivo administration into SCID mice, the vector induced higher plasma levels of human clotting factor IX (F.IX) than non-modified vector. Our results indicate that nuclear translocation of the genome is a rate-limiting step in lentiviral infection of both dividing and non-dividing cells, and that it depends on protein and nucleic acid sequence determinants. Full rescue of this step in lentivirus-based vectors improves performance for gene-therapy applications.
Assuntos
Núcleo Celular/genética , Produtos do Gene pol/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , HIV-1/genética , Transdução Genética/genética , Animais , Sequência de Bases , Divisão Celular , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Fator IX/análise , Fator IX/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Produtos do Gene pol/fisiologia , Genes Virais/genética , Genes Virais/fisiologia , HIV-1/fisiologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Linfócitos/citologia , Linfócitos/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos SCID , Dados de Sequência Molecular , RNA Viral/genética , RNA Viral/metabolismo , Integração ViralRESUMO
Scatter Factors control a complex genetic program known as 'invasive growth'. HGF (Scatter factor 1) and MSP (Scatter Factor 2) bind to tyrosine kinase receptors encoded by the proto-oncogenes MET and RON. Using the appropriate 'kinase inactive' mutant receptors, we show that ligand-induced activation of Met results in transphosphorylation of Ron, and vice versa. Transphosphorylation is direct, as it occurs in Met or Ron receptors lacking the docking sites for signal transducers. Phosphate groups are transferred to the tyrosine phosphorylation sites responsible both for kinase up-regulation (Met: Y1234/Y1235 and Ron: Y1238/Y1239) and for generation of signal transducer docking sites (Met: Y1349/Y1356 and Ron Y1353/Y1360). The transphosphorylation specifically takes place for the receptor subfamily, as it is not observed between Met or Ron and ErbB1, ErbB2 or TrkA. Cross-linking experiments show that non-covalent Met-Ron complexes are present on the cell surface, before ligand-induced dimerization. Co-expression of a kinase inactive Ron receptor with naturally-occurring oncogenic Met mutants suppresses the transforming phenotype, suggesting a dominant negative role for the inefficient kinase partner. These data show that, while specific for their ligands, scatter factor receptors cross-talk and cooperate in intracellular signaling.
