RESUMEN
The distinguishing feature of self-inactivating (SIN) retroviral vectors is the deletion of the enhancer/promoter sequences in the U3 region of the 3' long terminal repeat. This design is used to overcome transcriptional interference and prevent downstream transcription from the 3' long terminal repeat. SIN vectors were derived from a number of different retroviruses. Studies of SIN vectors show that extensive U3 deletions in HIV-based vectors do not alter viral titers or the in vitro and in vivo properties of the vectors. However, deletion of the U3 sequences in γ- and α-retroviruses correlates with defects in 3' RNA processing and reduces viral titers by >10-fold. Here, we studied the steps in the retroviral life cycle that are affected by the deletion of sequences in the 3' U3 region of Moloney murine leukemia virus-derived retroviral vectors. The results show that the amounts of both full-length and internal RNA transcripts of U3-minus vectors are reduced in the nuclei of transfected cells, an effect that is probably due to a general defect in 3' RNA processing. Furthermore, full-length RNA transcripts were also defective in terms of nuclear export. This defect was complemented by transferring the U3 region to another position within the retroviral vector, indicating that the U3 region contains position-independent cis-acting sequences that are required for the transport of full-length viral transcripts. The results also suggest that the leader region of Moloney murine leukemia virus contains inhibitory/regulatory sequences, which prevent export and mediate nuclear retention of full-length viral RNA.
Asunto(s)
Virus de la Leucemia Murina de Moloney/genética , Secuencias Repetidas Terminales , Transporte Activo de Núcleo Celular , Animales , Elementos de Facilitación Genéticos , Expresión Génica , Vectores Genéticos , Ratones , Virus de la Leucemia Murina de Moloney/metabolismo , Células 3T3 NIH , Regiones Promotoras Genéticas , Procesamiento Postranscripcional del ARN , ARN Viral/genética , ARN Viral/metabolismo , Eliminación de Secuencia , Transfección , Replicación ViralRESUMEN
The spectrum and features of neurological disorders have been changed due to the Chernobyl catastrophe in the Republic of Belarus. More recently neurologists in Belarus have noted a significant increase in the frequency of myasthenia gravis (MG) with concomitant rise in the thymomas. There is some evidence suggesting that retroviruses play a key role in the development and pathogenesis of autoimmune diseases. This study analyzed thymomas from 45 MG patients from the Republic of Belarus by using PCR and primers for two regions of FV--gag and bel-2 genes. The results showed that none of the varied thymuses from the 45 MG patients contained FV genome. No relationship can be confirmed between FV and this disease and the results suggest that no pathological link between FV and MG exists.
Asunto(s)
Genoma Viral , Miastenia Gravis/complicaciones , Virus Espumoso de los Simios/genética , Timoma/complicaciones , Timoma/virología , ADN Viral/análisis , Proteínas de Unión al ADN/genética , Productos del Gen gag/genética , Humanos , Miastenia Gravis/diagnóstico , Reacción en Cadena de la Polimerasa , República de Belarús , Timoma/diagnóstico , Transactivadores/genéticaRESUMEN
BACKGROUND: Retroviruses have a diploid genome and recombine at high frequency. Recombinant proviruses can be generated when two genetically different RNA genomes are packaged into the same retroviral particle. It was shown in several studies that recombinant proviruses could be generated in each round of HIV-1 replication, whereas the recombination rates of SNV and Mo-MuLV are 5 to 10-fold lower. The reason for these differences is not clear. One possibility is that these retroviruses may differ in their ability to copackage genomic RNAs produced at different chromosomal loci. RESULTS: To investigate whether there is a difference in the efficiency of heterodimer formation when two proviruses have the same or different chromosomal locations, we introduced two different Mo-MuLV-based retroviral vectors into the packaging cell line using either the cotransfection or sequential transfection procedure. The comparative study has shown that the frequency of recombination increased about four-fold when the cotransfection procedure was used. This difference was not associated with possible recombination of retroviral vectors during or after cotransfection and the ratios of retroviral virion RNAs were the same for two variants of transfection. CONCLUSIONS: The results of this study indicate that a mechanism exists to enable the preferential copackaging of Mo-MuLV genomic RNA molecules that are transcribed on the same DNA template. The properties of Mo-MuLV genomic RNAs transport, processing or dimerization might be responsible for this preference. The data presented in this report can be useful when designing methods to study different aspects of replication and recombination of a diploid retroviral genome.