Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Rd, Cambridge, CB2 1QP, UK.
Retrovirology. 2018 Jan 22;15(1):10. doi: 10.1186/s12977-018-0394-5.
The retrovirus murine leukemia virus (MuLV) has an 8.3 kb RNA genome with a simple 5'-gag-pol-env-3' architecture. Translation of the pol gene is dependent upon readthrough of the gag UAG stop codon; whereas the env gene is translated from spliced mRNA transcripts. Here, we report the first high resolution analysis of retrovirus gene expression through tandem ribosome profiling (RiboSeq) and RNA sequencing (RNASeq) of MuLV-infected cells.
Ribosome profiling of MuLV-infected cells was performed, using the translational inhibitors harringtonine and cycloheximide to distinguish initiating and elongating ribosomes, respectively. Meta-analyses of host cell gene expression demonstrated that the RiboSeq datasets specifically captured the footprints of translating ribosomes at high resolution. Direct measurement of ribosomal occupancy of the MuLV genomic RNA indicated that ~ 7% of ribosomes undergo gag stop codon readthrough to access the pol gene. Initiation of translation was found to occur at several additional sites within the 5' leaders of the gag and env transcripts, upstream of their respective annotated start codons.
These experiments reveal the existence of a number of previously uncharacterised, ribosomally occupied open reading frames within the MuLV genome, with possible regulatory consequences. In addition, we provide the first direct measurements of stop codon readthrough efficiency during cellular infection.
逆转录病毒鼠白血病病毒（MuLV）具有一个 8.3kb 的 RNA 基因组，具有简单的 5'-gag-pol-env-3' 结构。pol 基因的翻译依赖于对 gag UAG 终止密码子的通读；而 env 基因则从拼接的 mRNA 转录本翻译而来。在这里，我们通过串联核糖体分析（RiboSeq）和 MuLV 感染细胞的 RNA 测序（RNASeq），首次对逆转录病毒基因表达进行了高分辨率分析。
使用翻译抑制剂 harringtonine 和 cycloheximide 对 MuLV 感染的细胞进行核糖体分析，分别区分起始和延伸核糖体。宿主细胞基因表达的荟萃分析表明，RiboSeq 数据集特别以高分辨率捕获了翻译核糖体的足迹。对 MuLV 基因组 RNA 中核糖体占有率的直接测量表明，~7%的核糖体通过 gag 终止密码子通读进入 pol 基因。在 gag 和 env 转录物的 5' 先导区，在它们各自的注释起始密码子之前，发现翻译起始发生在几个其他位点。
这些实验揭示了 MuLV 基因组中存在一些以前未被表征的、核糖体占据的开放阅读框，可能具有调节作用。此外，我们提供了细胞感染过程中终止密码子通读效率的首次直接测量。
