A sensitive real-time PCR based assay to estimate the impact of amino acid substitutions on the competitive replication fitness of human immunodeficiency virus type 1 in cell culture.
A sensitive real-time PCR based assay to estimate the impact of amino acid substitutions on the competitive replication fitness of human immunodeficiency virus type 1 in cell culture.
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基于敏感的实时PCR测定法，以估算氨基酸取代对细胞培养中人类免疫缺陷病毒1型的竞争复制适应性的影响。
DOI：
10.1016/j.jviromet.2012.10.016
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发表时间：
2013-04
影响因子：
3.1
通讯作者：
Mullins JI
中科院分区：
文献类型：
--
作者：
Liu Y;Holte S;Rao U;McClure J;Konopa P;Swain JV;Lanxon-Cookson E;Kim M;Chen L;Mullins JI
人类免疫缺陷病毒1型（HIV-1）突变的固定，如那些赋予耐药性和免疫逃逸，可导致复制适应性的变化。为了评估这些变化，开发了实时TaqMan PCR检测试验和数据分析的统计方法，以在细胞培养物中的竞争性病毒复制实验中灵敏地估计相对病毒适合度。以两种形式构建在HIV-1 NL 4 -3骨架中具有目的基因的嵌合病毒，vifA（NL 4 -3中的天然vif基因）和vifB（与vifA具有六个同义核苷酸差异的vif基因）。随后，将感兴趣的突变引入嵌合病毒的NL 4 -3VifA骨架中，并在细胞培养物中将突变体与具有NL 4 -3VifB骨架中的同基因病毒序列的嵌合体竞争。为了评估细微的适应性差异，对培养物上清液进行纵向取样，并在实时PCR测定中使用vifA和vifB特异性引物对病毒进行差异定量。基于指数净增长模型，确定每种病毒的增长率，区分两种病毒的突变的适应度成本表示为突变体和天然变体之间的净增长率差。使用该测定，在HIV-1 Gag和Env中的高度保守位点处定量八个氨基酸取代的适应性影响。
Fixation of mutations in human immunodeficiency virus type 1 (HIV-1), such as those conferring drug resistance and immune escape, can result in a change in replication fitness. To assess these changes, a real-time TaqMan PCR detection assay and statistical methods for data analysis were developed to estimate sensitively relative viral fitness in competitive viral replication experiments in cell culture. Chimeric viruses with the gene of interest in an HIV-1NL4-3 backbone were constructed in two forms, vifA (native vif gene in NL4-3) and vifB (vif gene with six synonymous nucleotide differences from vifA). Subsequently, mutations of interest were introduced into the chimeric viruses in NL4-3VifA backbones, and the mutants were competed against the chimera with the isogenic viral sequence in the NL4-3VifB backbone in cell culture. In order to assess subtle fitness differences, culture supernatants were sampled longitudinally, and the viruses differentially quantified using vifA- and vifB-specific primers in real-time PCR assays. Based on an exponential net growth model, the growth rate of each virus was determined and the fitness cost of the mutation(s) distinguishing the two viruses represented as the net growth rate difference between the mutant and the native variants. Using this assay, the fitness impact of eight amino acid substitutions was quantitated at highly conserved sites in HIV-1 Gag and Env.
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影响因子：
5.4
作者：
DIMITROV, DS;WILLEY, RL;MARTIN, MA
通讯作者：
MARTIN, MA
HIV-1组M保守元素疫苗。
影响因子：
6.7
作者：
Rolland M;Nickle DC;Mullins JI
通讯作者：
Mullins JI
DOI：
10.1073/pnas.0811713106
发表时间：
2009-03-31
影响因子：
11.1
作者：
Anastassopoulou, Cleo G.;Ketas, Thomas J.;Moore, John P.
通讯作者：
Moore, John P.
影响因子：
5.4
作者：
Brockman, Mark A.;Schneidewind, Arne;Allen, Todd M.
通讯作者：
Allen, Todd M.
影响因子：
4.1
作者：
Nijhuis, Monique;van Maarseveen, Noortje M.;Boucher, Charles A. B.
通讯作者：
Boucher, Charles A. B.