Summary of conserved local base-pairing interactions in HIV-1 PR and RT wild-type consensus sequences

The MFOLD thermodynamic folding algorithm, which predicts RNA structure based on minimum free energy on folding, was used to detect potential local base pairing interactions between RNA nucleotides in the PR-RT region. Mfold analysis was carried out using a sliding window scanning process (implemented in perl) to minimize any arbitrary effects of the region chosen for analysis. Stepwise scanning in a 5’ to 3’ direction with a ‘sliding window’ of fixed length was used to generate subsequences within the region of the HIV genome under analysis (codons 1-99 of PR and 1-240 of RT) and 500bp of flanking sequence either side (derived from subtype consensus sequences available from the Los Alamos HIV Sequence database). A step size of 20bp was used in combination with a range of window sizes (300, 600, 900 and 1200bp). Base pairing interactions within predicted structures generated using this approach were then analysed. The frequency of base-paring between pairs was calculated across each of the windows in which the pair occurred. When a range of structures was predicted for a sequence under analysis, the average frequency of pairs across each of the predicted structures was scored. Base pairing interactions that were consistently predicted across all windows and structures (>90%) were considered robust. With MFOLD, all sequences were folded at 37 degrees Celsius using default parameters.

本研究采用基于折叠最小自由能原理预测RNA二级结构的MFOLD热力学折叠算法（MFOLD thermodynamic folding algorithm），对PR-RT区域内RNA核苷酸间的潜在局部碱基配对相互作用进行检测。本次MFOLD分析采用滑动窗口扫描流程（通过Perl语言实现），以最小化因分析区域选取所带来的任意性偏差。本次分析采用固定长度的“滑动窗口”沿5’端至3’端方向逐步扫描，在待分析的HIV基因组区域（PR的1-99号密码子及RT的1-240号密码子）及其两侧各500bp的侧翼序列（源自洛斯阿拉莫斯HIV序列数据库提供的亚型共识序列）内生成待分析子序列。本次分析采用20bp的步长，搭配300、600、900及1200bp共4种窗口尺寸。随后对该方法生成的预测结构内的碱基配对相互作用展开分析。针对每一对发生碱基配对的核苷酸，计算其在所有包含该配对的窗口中的出现频率。若待分析序列可预测出多种结构，则对所有预测结构中各碱基对的平均出现频率进行打分。在所有窗口及预测结构中均被稳定检出（检出率>90%）的碱基配对相互作用被认定为稳健相互作用。使用MFOLD进行折叠时，所有序列均在37摄氏度下以默认参数完成折叠。