Sequence Analysis of In Vivo-Expressed HIV-1 Spliced RNAs Reveals the Usage of New and Unusual Splice Sites by Viruses of Different Subtypes

HIV-1 RNAs are generated through a complex splicing mechanism, resulting in a great diversity of transcripts, which are classified in three major categories: unspliced, singly spliced (SS), and doubly spliced (DS). Knowledge on HIV-1 RNA splicing in vivo and by non-subtype B viruses is scarce. Here we analyze HIV-1 RNA splice site usage in CD4+CD25+ lymphocytes from HIV-1-infected individuals through pyrosequencing. HIV-1 DS and SS RNAs were amplified by RT-PCR in 19 and 12 samples, respectively. 13,108 sequences from HIV-1 spliced RNAs, derived from viruses of five subtypes (A, B, C, F, G), were identified. In four samples, three of non-B subtypes, five 3’ splice sites (3’ss) mapping to unreported positions in the HIV-1 genome were identified. Two, designated A4i and A4j, were used in 22% and 25% of rev RNAs in two viruses of subtypes B and A, respectively. Given their close proximity (one or two nucleotides) to A4c and A4d, respectively, they could be viewed as variants of these sites. Three 3’ss, designated A7g, A7h, and A7i, located 20, 32, and 18 nucleotides downstream of A7, respectively, were identified in a subtype C (A7g, A7h) and a subtype G (A7i) viruses, each in around 2% of nef RNAs. The new splice sites or variants of splice sites were associated with the usual sequence features of 3’ss. Usage of unusual 3’ss A4d, A4e, A5a, A7a, and A7b was also detected. A4f, previously identified in two subtype C viruses, was preferentially used by rev RNAs of a subtype C virus. These results highlight the great diversity of in vivo splice site usage by HIV-1 RNAs. The fact that four of five newly identified splice sites or variants of splice sites were detected in non-subtype B viruses allows anticipating an even greater diversity of HIV-1 splice site usage than currently known.HIV-1 RNA通过复杂的剪接机制产生，形成具有高度多样性的转录本（transcripts），可分为三大类：未剪接转录本、单剪接（singly spliced, SS）转录本与双剪接（doubly spliced, DS）转录本。目前针对体内HIV-1 RNA剪接以及非B亚型病毒剪接的相关认知仍较为匮乏。 本研究通过焦磷酸测序（Pyrosequencing）技术，对HIV-1感染者CD4+CD25+淋巴细胞中的HIV-1 RNA剪接位点使用情况展开分析。研究分别在19份和12份样本中，通过逆转录聚合酶链式反应（Reverse Transcription Polymerase Chain Reaction, RT-PCR）扩增得到HIV-1 DS与SS RNA，最终共获得来自5个亚型（A、B、C、F、G）病毒的13108条HIV-1剪接RNA序列。 在4份样本（其中3份来自非B亚型病毒）中，共鉴定出5个位于HIV-1基因组此前未报道位置的3’剪接位点（3’ splice sites, 3’ss）。其中两个位点分别命名为A4i与A4j，在亚型B与亚型A的病毒中，分别有22%和25%的rev转录本（rev RNAs）使用该位点；由于二者分别与A4c、A4d位点间距仅1或2个核苷酸，可将其视为上述位点的变异体。 另有3个3’剪接位点分别命名为A7g、A7h与A7i，它们分别位于A7位点下游20、32与18个核苷酸处，在亚型C（A7g、A7h）与亚型G（A7i）病毒的nef转录本（nef RNAs）中，各自约占2%的比例。新发现的剪接位点或剪接位点变异体均具备3’ss的典型序列特征。 本研究同时检测到非常规3’ss A4d、A4e、A5a、A7a与A7b的使用情况。此前在两株亚型C病毒中发现的A4f位点，在一株亚型C病毒的rev转录本中呈现优先使用的特征。 上述结果凸显了体内HIV-1 RNA剪接位点使用的高度多样性。值得注意的是，5个新发现的剪接位点或剪接位点变异体中有4个来自非B亚型病毒，这提示HIV-1剪接位点的使用多样性可能远超目前已知的范围。