PDF(Pubmed)
Abstract：
Unspliced HIV-1 RNAs function as messenger RNAs for Gag or Gag-Pol polyproteins and progeny genomes packaged into virus particles. Recently, it has been reported that fate of the RNAs might be primarily determined, depending on transcriptional initiation sites among three consecutive deoxyguanosine residues (GGG tract) downstream of TATA-box in the 5\' long terminal repeat (LTR). Although HIV-1 RNA transcription starts mostly from the first deoxyguanosine of the GGG tract and often from the second or third deoxyguanosine, RNAs beginning with one guanosine (G1-form RNAs), whose transcription initiates from the third deoxyguanosine, were predominant in HIV-1 particles. Despite selective packaging of G1-form RNAs into virus particles, its biological impact during viral replication remains to be determined. In this study, we revealed that G1-form RNAs are primarily selected as a template for provirus DNA rather than other RNAs. In competitions between HIV-1 and lentiviral vector transcripts in virus-producing cells, approximately 80% of infectious particles were found to generate provirus using HIV-1 transcripts, while lentiviral vector transcripts were conversely selected when we used HIV-1 mutants in which the third deoxyguanosine in the GGG tract was replaced with deoxythymidine or deoxycytidine (GGT or GGC mutants, respectively). In the other analyses of proviral sequences after infection with an HIV-1 mutant in which the GGG tract in 3\' LTR was replaced with TTT, most proviral sequences of the GGG-tract region in 5\' LTR were found to be TTG, which is reasonably generated using the G1-form transcripts. Our results indicate that the G1-form RNAs serve as a dominant genome to establish provirus DNA.IMPORTANCESince the promoter for transcribing HIV-1 RNA is unique, all viral elements including genomic RNA and viral proteins have to be generated by the unique transcripts through ingenious mechanisms including RNA splicing and frameshifting during protein translation. Previous studies suggested a new mechanism for diversification of HIV-1 RNA functions by heterogeneous transcriptional initiation site usage; HIV-1 RNAs whose transcription initiates from a certain nucleotide were predominant in virus particles. In this study, we established two methods to analyze heterogenous transcriptional initiation site usage by HIV-1 during viral infection and showed that RNAs beginning with one guanosine (G1-form RNAs), whose transcription initiates from the third deoxyguanosine of the GGG tract in 5\' LTR, were primarily selected as viral genome in infectious particles and thus are used as a template to generate provirus for continuous replication. This study provides insights into the mechanism for diversification of unspliced RNA functions and requisites of lentivirus infectivity.
摘要：
未剪接的HIV-1RNA充当Gag或Gag-Pol多蛋白的信使RNA和包装成病毒颗粒的后代基因组。最近,据报道，RNA的命运可能主要决定，取决于5'长末端重复序列（LTR）中TATA-box下游三个连续脱氧鸟苷残基（GGG道）之间的转录起始位点。尽管HIV-1RNA转录主要从GGG道的第一个脱氧鸟苷开始，通常从第二个或第三个脱氧鸟苷开始，以一个鸟苷（G1型RNA）开始的RNA，它的转录从第三个脱氧鸟苷开始，在HIV-1颗粒中占主导地位。尽管将G1型RNA选择性包装到病毒颗粒中，其在病毒复制过程中的生物学影响仍有待确定。在这项研究中,我们发现G1型RNA主要被选择作为前病毒DNA的模板，而不是其他RNA。在病毒产生细胞中HIV-1和慢病毒载体转录物之间的竞争中，使用HIV-1转录本，发现大约80％的感染性颗粒产生前病毒，而当我们使用HIV-1突变体时，慢病毒载体转录本的选择相反，其中GGG区中的第三个脱氧鸟苷被脱氧胸苷或脱氧胞苷（GGT或GGC突变体，分别)。在感染HIV-1突变体后的前病毒序列的其他分析中，其中3'LTR中的GGG束被TTT取代，发现5'LTR中GGG束区域的大多数前病毒序列是TTG，这是使用G1形式转录本合理产生的。我们的结果表明，G1型RNA作为建立前病毒DNA的显性基因组。重要性由于转录HIV-1RNA的启动子是独特的，包括基因组RNA和病毒蛋白质在内的所有病毒元件都必须由独特的转录本通过巧妙的机制产生，包括蛋白质翻译过程中的RNA剪接和移码。先前的研究表明，通过异质转录起始位点的使用，HIV-1RNA功能多样化的新机制；从某个核苷酸开始转录的HIV-1RNA在病毒颗粒中占主导地位。在这项研究中,我们建立了两种方法来分析HIV-1在病毒感染过程中的异源转录起始位点的使用，并显示以一个鸟苷（G1型RNA）开始的RNA，其转录在5'LTR中从GGG束的第三个脱氧鸟苷开始，主要选择为感染性颗粒中的病毒基因组，因此用作模板以产生持续复制的原病毒。这项研究提供了对未剪接RNA功能多样化机制和慢病毒感染性必要条件的见解。
