Grupo de Virología SRNL, Instituto Nacional de Salud, Avenida Calle 26 No, 51 - 20 ZONA 6 CAN, Bogotá, Colombia.
Virol J. 2010 Dec 20;7:369. doi: 10.1186/1743-422X-7-369.
HIV-1 can be inhibited by RNA interference in vitro through the expression of short hairpin RNAs (shRNAs) that target conserved genome sequences. In silico shRNA design for HIV has lacked a detailed study of virus variability constituting a possible breaking point in a clinical setting. We designed shRNAs against HIV-1 considering the variability observed in naïve and drug-resistant isolates available at public databases.
A Bioperl-based algorithm was developed to automatically scan multiple sequence alignments of HIV, while evaluating the possibility of identifying dominant and subdominant viral variants that could be used as efficient silencing molecules. Student t-test and Bonferroni Dunn correction test were used to assess statistical significance of our findings.
Our in silico approach identified the most common viral variants within highly conserved genome regions, with a calculated free energy of ≥ -6.6 kcal/mol. This is crucial for strand loading to RISC complex and for a predicted silencing efficiency score, which could be used in combination for achieving over 90% silencing. Resistant and naïve isolate variability revealed that the most frequent shRNA per region targets a maximum of 85% of viral sequences. Adding more divergent sequences maintained this percentage. Specific sequence features that have been found to be related with higher silencing efficiency were hardly accomplished in conserved regions, even when lower entropy values correlated with better scores. We identified a conserved region among most HIV-1 genomes, which meets as many sequence features for efficient silencing.
HIV-1 variability is an obstacle to achieving absolute silencing using shRNAs designed against a consensus sequence, mainly because there are many functional viral variants. Our shRNA cocktail could be truly effective at silencing dominant and subdominant naïve viral variants. Additionally, resistant isolates might be targeted under specific antiretroviral selective pressure, but in both cases these should be tested exhaustively prior to clinical use.
HIV-1 可以通过表达靶向保守基因组序列的短发夹 RNA（shRNA）在体外被 RNA 干扰抑制。针对 HIV 的 shRNA 设计在病毒变异性方面缺乏详细研究，这可能成为临床应用中的一个突破点。我们根据在公共数据库中获得的原始和耐药分离株的变异性设计了针对 HIV-1 的 shRNA。
开发了一种基于 Bioperl 的算法，用于自动扫描 HIV 的多序列比对，同时评估鉴定可能用作有效沉默分子的主导和次要病毒变体的可能性。使用学生 t 检验和 Bonferroni Dunn 校正检验来评估我们研究结果的统计学意义。
我们的计算方法确定了高度保守基因组区域内最常见的病毒变体，其自由能计算值≥-6.6 kcal/mol。这对于 strand loading 到 RISC 复合物和预测的沉默效率评分至关重要，这些评分可结合使用以实现超过 90%的沉默。耐药和原始分离株的变异性表明，每个区域最常见的 shRNA 最多可靶向 85%的病毒序列。添加更多的发散序列可保持此百分比。已经发现与更高沉默效率相关的特定序列特征在保守区域中几乎无法实现，即使较低的熵值与更好的评分相关。我们在大多数 HIV-1 基因组中鉴定了一个保守区域，该区域满足了高效沉默所需的许多序列特征。
使用针对共识序列设计的 shRNA 实现绝对沉默存在 HIV-1 变异性障碍，主要是因为存在许多功能病毒变体。我们的 shRNA 鸡尾酒可能在沉默主导和次要原始病毒变体方面真正有效。此外，在特定抗逆转录病毒选择压力下可能会靶向耐药分离株，但在这两种情况下，在临床使用前都应进行详尽的测试。
Zotero 插件
