High Fidelity of Homologous Retroviral Recombination in Cell Culture

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Genetic variation continues to be a major obstacle in the development of therapies and vaccines against retroviral infections and contributes extensively to viral pathogenesis and persistence. Recombination is one mechanism that increases retroviral variation by shuffling mutations from different genomes. Recent studies suggest that recombination not only shuffles the mutations but also generates them at high rates during reverse transcription. In contrast to these recent studies, this investigation shows that recombination does not generate mutations during recombination. A spleen necrosis virus (SNV)-based homologous recombination system was used to test the hypothesis that retroviral recombination is a high-fidelity process during replication of the virus in cell culture. The system consisted of a pair of SNV vectors expressing two drug resistance genes. The vectors were constructed so that cells containing recombinant proviruses could be selected by a double drug-resistant phenotype. Restriction enzyme digestion and agarose gel electrophoresis were used to map the location of recombination within 182 proviruses. Sequencing and single-strand conformation polymorphism techniques were then used to check for mutations within the recombinant proviruses. Since no mutations were detected among the 182 recombinants that were analyzed, homologous recombination is a high-fidelity process for retroviruses in cell culture.



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