High fidelity of homologous retroviral recombination in cell culture

Abstract

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.

Fifteen to Twenty Percent of HIV Substitution Mutations Are Associated with Recombination

“…Statistics were calculated by comparing the original unpermuted data with the generated null distribution. recombination and mutation are not associated, although previous studies addressing this question have led to conflicting results (28)(29)(30). In this study, we have employed a novel HIV marker system and a high-throughput pyrosequencing system to simultaneously measure mutation and recombination rates directly on an authentic HIV-1 genome.…”

“…Three previous studies have attempted to evaluate the potential linkages between recombination and mutation in retroviruses (28)(29)(30). In the first two studies, 65,000 and 4,100 bp were sequenced in a spleen necrosis virus (SNV) vector and HIV genomes (with 2 and 0.1 anticipated mutation events), yet 0 and 2 mutations were detected, respectively, leading the authors of those studies to conclude that any association was absent or uncertain (28,29).…”

“…Whether retroviral mutation is linked with recombination has been a subject of debate for decades, and numerous studies have measured HIV mutation and retroviral recombination rates in vitro and in cell culture (2,(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). Nevertheless, the question of whether mutation and recombination are associated has not been resolved directly due to numerous inherent technical difficulties.…”

“…Others have taken advantage of retroviral vector or reporter systems, in which the expressions of reporter genes (such as fluorescent proteins, cell surface markers, and/or antibiotic resistance genes) were used to select cells containing recombined viral genomes for recombination and mutation analyses (28)(29)(30)(31)(35)(36)(37)(38)(39). As coexpression of reporter gene products in a single cell is needed to identify recombination events, these systems generally use a low multiplicity of infection (MOI) to avoid dual infection introducing experimental bias (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39). However, as these systems require expression of the gene products, natural mutation events or recombination events that lead to nonproductive expression of reporter genes will be unaccounted for.…”

“…It would have been advantageous if recombination and mutation rates could be estimated directly from the newly synthesized and/or integrated viral cDNA that is independent of the MOI. Furthermore, as those studies depend on PCR to amplify specific genomic sequences for recombination and/or mutation analyses (28)(29)(30), the potential of PCRinduced recombination and mutation must be controlled for to limit overestimation of these two retroviral biological processes (40).…”