In-depth analysis of G-to-A hypermutation rate in HIV-1 env DNA induced by endogenous APOBEC3 proteins using massively parallel sequencing
In-depth analysis of G-to-A hypermutation rate in HIV-1 env DNA induced by endogenous APOBEC3 proteins using massively parallel sequencing
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DOI：
10.1016/j.jviromet.2010.11.016
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发表时间：
2011-02-01
影响因子：
3.1
通讯作者：
Metzner, Karin J.
中科院分区：
文献类型：
--
作者：
Knoepfel, Stefanie A.;Di Giallonardo, Francesca;Metzner, Karin J.
当辅助病毒蛋白Vif缺失或无功能时，一些APOBEC3蛋白导致HIV-1 DNA中G-to-A超突变。到目前为止，克隆和测序已经完成了对G-to-A超突变的研究。这是耗时和劳动密集型的，特别是在体内研究的背景下，其中超突变序列的数量可能非常低。因此，使用454焦磷酸测序FLX系统开发了一种大规模并行测序方案，用于深入分析G-to-A超突变。使用HIV-1 NL4-3 Delta vif在T细胞系和pbmc中感染两轮后扩增了部分HIV-1 env并对其进行焦磷酸测序。我们采用了具体的标准来应对主要的技术挑战：(1)包含超突变序列，(2)HIV-1 env的高基因组多样性，以及(3)排除由焦磷酸测序引起的包含移码错误的序列。总共获得了超过14万个序列。1.3-6.5%的鸟嘌呤突变为腺嘌呤，最常发生在GG二核苷酸背景下，这是APOBEC3G的首选脱胺位点。非g -to- a突变仅发生在低频率(
Some APOBEC3 proteins cause G-to-A hypermutation in HIV-1 DNA when the accessory viral protein Vif is absent or non-functional. So far, cloning and sequencing has been performed to study G-to-A hypermutation. This is time-consuming and labour-intensive especially in the context of in vivo investigations where the number of hypermutated sequences can be very low. Thus, a massively parallel sequencing protocol has been developed for in-depth analysis of G-to-A hypermutation using the 454 pyrosequencing FLX system. Part of HIV-1 env was amplified and pyrosequenced after two rounds of infection in T cell lines and PBMCs using HIV-1 NL4-3 Delta vif. Specific criteria were applied to cope with major technical challenges: (1) the inclusion of hypermutated sequences, (2) the high genome diversity of HIV-1 env, and (3) the exclusion of sequences containing frameshift errors caused by pyrosequencing. In total, more than 140,000 sequences were obtained. 1.3-6.5% of guanines were mutated to adenine, most frequently in the GG dinucleotide context, the preferred deamination site of APOBEC3G. Non-G-to-A mutations occurred only in low frequencies (