Comparative analysis of gene expression in virulent and attenuated strains of infectious bronchitis virus at sub-codon resolution
Comparative analysis of gene expression in virulent and attenuated strains of infectious bronchitis virus at sub-codon resolution
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亚密码子分辨率下传染性支气管炎病毒强毒株和减毒株基因表达的比较分析
DOI：
10.1101/612614
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Dinan A
中科院分区：
文献类型：
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作者：
Dinan A
与所有冠状病毒一样，鸡传染性支气管炎病毒（IBV）具有长的单链正义RNA基因组（约27 kb），并具有复杂的复制策略，包括产生嵌套的亚基因组mRNA（sgmRNAs）。在这里，我们使用全转录组测序（RNASeq）和核糖体分析（RiboSeq）来描述IBV M41-CK和Beau-R株在亚密码子分辨率下的基因表达。RNASeq促进了病毒RNA合成的比较分析，并揭示了减毒Beau-R菌株中的两个新的转录连接位点，其中一个将产生编码位于核衣壳编码区下游的核糖体占据的开放阅读框（dORF）的sgmRNA。RiboSeq允许量化病毒基因表达的翻译效率，并首次确定了基因组上核糖体暂停的位点。在基因组RNA ORF 1a/ORF 1b连接处的程序化核糖体移码（PRF）信号侧翼的读段的定量显示，IBV中的PRF是高效的（33%至40%）。RiboSeq数据的三联体定相允许精确确定阅读框，并揭示了两个ORF（sgmRNA IR上的ORF 4 b和ORF 4c）的翻译，这两个ORF在IBV分离株中广泛保守。在感染的原代鸡肾细胞中差异基因表达的分析表明，宿主细胞对IBV的反应主要发生在转录水平上，感染后免疫相关mRNA转录物的整体上调和宿主基因翻译效率的相对温和的变化。细胞基因和基因网络差异表达的病毒感染过程中也被确定，给洞察宿主细胞对IBV infection.IMPORTANCEIBV的反应是一种主要的禽病原体，并提出了大量的经济负担，家禽业。迫切需要改进的疫苗接种策略来遏制这种病毒的全球传播，并且通过对IBV分子生物学的更好理解来帮助开发合适的候选疫苗。我们的高分辨率数据使我们能够精确研究感染了致病性和减毒形式IBV的细胞中的转录和翻译，并扩大了我们对γ冠状病毒基因表达的理解。我们证明，基因表达显示出相当大的种内变异，单核苷酸多态性与sgmRNA转录物的产生改变相关，我们的RiboSeq数据集使我们能够在两种菌株中发现新的核糖体占据的ORF。发现在病毒感染期间差异表达的许多细胞基因和基因网络提供了对宿主细胞对IBV感染的应答的见解。
Like all coronaviruses, avian infectious bronchitis virus (IBV) possesses a long, single-stranded, positive-sense RNA genome (∼27 kb) and has a complex replication strategy that includes the production of a nested set of subgenomic mRNAs (sgmRNAs). Here, we used whole-transcriptome sequencing (RNASeq) and ribosome profiling (RiboSeq) to delineate gene expression in the IBV M41-CK and Beau-R strains at subcodon resolution. RNASeq facilitated a comparative analysis of viral RNA synthesis and revealed two novel transcription junction sites in the attenuated Beau-R strain, one of which would generate a sgmRNA encoding a ribosomally occupied open reading frame (dORF) located downstream of the nucleocapsid coding region. RiboSeq permitted quantification of the translational efficiency of virus gene expression and identified, for the first time, sites of ribosomal pausing on the genome. Quantification of reads flanking the programmed ribosomal frameshifting (PRF) signal at the genomic RNA ORF1a/ORF1b junction revealed that PRF in IBV is highly efficient (33 to 40%). Triplet phasing of RiboSeq data allowed precise determination of reading frames and revealed the translation of two ORFs (ORF4b and ORF4c on sgmRNA IR), which are widely conserved across IBV isolates. Analysis of differential gene expression in infected primary chick kidney cells indicated that the host cell response to IBV occurs primarily at the level of transcription, with global upregulation of immune-related mRNA transcripts following infection and comparatively modest changes in the translation efficiencies of host genes. Cellular genes and gene networks differentially expressed during virus infection were also identified, giving insights into the host cell response to IBV infection.IMPORTANCEIBV is a major avian pathogen and presents a substantial economic burden to the poultry industry. Improved vaccination strategies are urgently needed to curb the global spread of this virus, and the development of suitable vaccine candidates will be aided by an improved understanding of IBV molecular biology. Our high-resolution data have enabled a precise study of transcription and translation in cells infected with both pathogenic and attenuated forms of IBV and expand our understanding of gammacoronaviral gene expression. We demonstrate that gene expression shows considerable intraspecies variation, with single nucleotide polymorphisms being associated with altered production of sgmRNA transcripts, and our RiboSeq data sets enabled us to uncover novel ribosomally occupied ORFs in both strains. The numerous cellular genes and gene networks found to be differentially expressed during virus infection provide insights into the host cell response to IBV infection.