Gärtner Kathleen, Wiktorowicz Tatiana, Park Jeonghae, Mergia Ayalew, Rethwilm Axel, Scheller Carsten
Universität Würzburg, Institut für Virologie und Immunbiologie, Versbacher Str 7, 97078 Würzburg, Germany.
Retrovirology. 2009 Apr 6;6:32. doi: 10.1186/1742-4690-6-32.
Foamy viruses (FVs) are the most genetically stable viruses of the retrovirus family. This is in contrast to the in vitro error rate found for recombinant FV reverse transcriptase (RT). To investigate the accuracy of FV genome copying in vivo we analyzed the occurrence of mutations in HEK 293T cell culture after a single round of reverse transcription using a replication-deficient vector system. Furthermore, the frequency of FV recombination by template switching (TS) and the cross-packaging ability of different FV strains were analyzed.
We initially sequenced 90,000 nucleotides and detected 39 mutations, corresponding to an in vivo error rate of approximately 4 x 10-4 per site per replication cycle. Surprisingly, all mutations were transitions from G to A, suggesting that APOBEC3 activity is the driving force for the majority of mutations detected in our experimental system. In line with this, we detected a late but significant APOBEC3G and 3F mRNA by quantitative PCR in the cells. We then analyzed 170,000 additional nucleotides from experiments in which we co-transfected the APOBEC3-interfering foamy viral bet gene and observed a significant 50% drop in G to A mutations, indicating that APOBEC activity indeed contributes substantially to the foamy viral replication error rate in vivo. However, even in the presence of Bet, 35 out of 37 substitutions were G to A, suggesting that residual APOBEC activity accounted for most of the observed mutations. If we subtract these APOBEC-like mutations from the total number of mutations, we calculate a maximal intrinsic in vivo error rate of 1.1 x 10-5 per site per replication. In addition to the point mutations, we detected one 49 bp deletion within the analyzed 260000 nucleotides.Analysis of the recombination frequency of FV vector genomes revealed a 27% probability for a template switching (TS) event within a 1 kilobase (kb) region. This corresponds to a 98% probability that FVs undergo at least one additional TS event per replication cycle. We also show that a given FV particle is able to cross-transfer a heterologous FV genome, although at reduced efficiency than the homologous vector.
Our results indicate that the copying of the FV genome is more accurate than previously thought. On the other hand recombination among FV genomes appears to be a frequent event.
泡沫病毒（FVs）是逆转录病毒家族中遗传稳定性最高的病毒。这与重组FV逆转录酶（RT）在体外的错误率形成对比。为了研究FV基因组在体内复制的准确性，我们使用复制缺陷型载体系统分析了一轮逆转录后HEK 293T细胞培养物中突变的发生情况。此外，还分析了FV通过模板转换（TS）进行重组的频率以及不同FV毒株的交叉包装能力。
我们最初对90,000个核苷酸进行了测序，检测到39个突变，这相当于每个复制周期每个位点的体内错误率约为4×10⁻⁴。令人惊讶的是，所有突变都是从G到A的转换，这表明APOBEC3活性是我们实验系统中检测到的大多数突变的驱动力。与此一致的是，我们通过定量PCR在细胞中检测到晚期但显著的APOBEC3G和3F mRNA。然后，我们分析了另外170,000个核苷酸，这些核苷酸来自共转染APOBEC3干扰性泡沫病毒bet基因的实验，结果观察到G到A突变显著下降了50%，这表明APOBEC活性确实对FV在体内的复制错误率有很大贡献。然而，即使在存在Bet的情况下，37个替换中有35个是从G到A，这表明残余的APOBEC活性是观察到的大多数突变的原因。如果我们从突变总数中减去这些类似APOBEC的突变，我们计算出每个复制位点的最大内在体内错误率为1.1×10⁻⁵。除了点突变外，我们在分析的260,000个核苷酸中检测到一个49 bp的缺失。对FV载体基因组重组频率的分析显示，在1千碱基（kb）区域内发生模板转换（TS）事件的概率为27%。这相当于FV在每个复制周期至少发生一次额外TS事件的概率为98%。我们还表明，给定的FV颗粒能够交叉转移异源FV基因组，尽管效率低于同源载体。
我们的结果表明，FV基因组的复制比以前认为的更准确。另一方面，FV基因组之间的重组似乎是一个频繁发生的事件。
