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Abstract：
African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that encodes its own host-like RNA polymerase (RNAP) and factors required to produce mature mRNA. The formation of accurate mRNA 3\' ends by ASFV RNAP depends on transcription termination, likely enabled by a combination of sequence motifs and transcription factors, although these are poorly understood. The termination of any RNAP is rarely 100% efficient, and the transcriptional \"readthrough\" at terminators can generate long mRNAs which may interfere with the expression of downstream genes. ASFV transcriptome analyses reveal a landscape of heterogeneous mRNA 3\' termini, likely a combination of bona fide termination sites and the result of mRNA degradation and processing. While short-read sequencing (SRS) like 3\' RNA-seq indicates an accumulation of mRNA 3\' ends at specific sites, it cannot inform about which promoters and transcription start sites (TSSs) directed their synthesis, i.e., information about the complete and unprocessed mRNAs at nucleotide resolution.
Here, we report a rigorous analysis of full-length ASFV transcripts using long-read sequencing (LRS). We systematically compared transcription termination sites predicted from SRS 3\' RNA-seq with 3\' ends mapped by LRS during early and late infection.
Using in-vitro transcription assays, we show that recombinant ASFV RNAP terminates transcription at polyT stretches in the non-template strand, similar to the archaeal RNAP or eukaryotic RNAPIII, unaided by secondary RNA structures or predicted viral termination factors. Our results cement this T-rich motif (U-rich in the RNA) as a universal transcription termination signal in ASFV. Many genes share the usage of the same terminators, while genes can also use a range of terminators to generate transcript isoforms varying enormously in length. A key factor in the latter phenomenon is the highly abundant terminator readthrough we observed, which is more prevalent during late compared with early infection.
This indicates that ASFV mRNAs under the control of late gene promoters utilize different termination mechanisms and factors to early promoters and/or that cellular factors influence the viral transcriptome landscape differently during the late stages of infection.
Here, we report a rigorous analysis of full-length ASFV transcripts using long-read sequencing (LRS). We systematically compared transcription termination sites predicted from SRS 3\' RNA-seq with 3\' ends mapped by LRS during early and late infection.
Using in-vitro transcription assays, we show that recombinant ASFV RNAP terminates transcription at polyT stretches in the non-template strand, similar to the archaeal RNAP or eukaryotic RNAPIII, unaided by secondary RNA structures or predicted viral termination factors. Our results cement this T-rich motif (U-rich in the RNA) as a universal transcription termination signal in ASFV. Many genes share the usage of the same terminators, while genes can also use a range of terminators to generate transcript isoforms varying enormously in length. A key factor in the latter phenomenon is the highly abundant terminator readthrough we observed, which is more prevalent during late compared with early infection.
This indicates that ASFV mRNAs under the control of late gene promoters utilize different termination mechanisms and factors to early promoters and/or that cellular factors influence the viral transcriptome landscape differently during the late stages of infection.
摘要：
■非洲猪瘟病毒（ASFV）是一种核质大DNA病毒（NCLDV），编码其自身的宿主样RNA聚合酶（RNAP）和产生成熟mRNA所需的因子。通过ASFVRNAP形成准确的mRNA3'末端取决于转录终止，可能是由序列基序和转录因子的组合实现的，虽然人们对这些知之甚少。任何RNAP的终止很少是100%有效的，终止子处的转录“readthrough”可以产生长的mRNAs，这可能会干扰下游基因的表达。ASFV转录组分析揭示了异质mRNA3'末端的景观，可能是真正终止位点和mRNA降解和加工的结果的组合。虽然短读测序（SRS）如3'RNA-seq表明mRNA3'末端在特定位点的积累，它不能告知哪些启动子和转录起始位点（TSS）指导它们的合成，即，关于核苷酸分辨率下完整和未加工的mRNA的信息。
■这里，我们报告了使用长读数测序(LRS)对全长ASFV转录本进行的严格分析.我们系统地比较了在早期和晚期感染期间从SRS3'RNA-seq预测的转录终止位点与LRS绘制的3'末端。
■使用体外转录测定，我们显示重组ASFVRNAP在非模板链的polyT延伸处终止转录，类似于古细菌RNAP或真核RNAPIII，没有二级RNA结构或预测的病毒终止因子的帮助。我们的结果巩固了这种富含T的基序（RNA中富含U）作为ASFV中的通用转录终止信号。许多基因使用相同的终止子，而基因也可以使用一系列终止子来产生长度变化很大的转录同种型。后一种现象的一个关键因素是我们观察到的高度丰富的终止子读通，与早期感染相比，晚期感染更为普遍。
■这表明在晚期基因启动子控制下的ASFVmRNAs利用与早期启动子不同的终止机制和因子,和/或细胞因子在感染晚期对病毒转录组景观的影响不同。
■这里，我们报告了使用长读数测序(LRS)对全长ASFV转录本进行的严格分析.我们系统地比较了在早期和晚期感染期间从SRS3'RNA-seq预测的转录终止位点与LRS绘制的3'末端。
■使用体外转录测定，我们显示重组ASFVRNAP在非模板链的polyT延伸处终止转录，类似于古细菌RNAP或真核RNAPIII，没有二级RNA结构或预测的病毒终止因子的帮助。我们的结果巩固了这种富含T的基序（RNA中富含U）作为ASFV中的通用转录终止信号。许多基因使用相同的终止子，而基因也可以使用一系列终止子来产生长度变化很大的转录同种型。后一种现象的一个关键因素是我们观察到的高度丰富的终止子读通，与早期感染相比，晚期感染更为普遍。
■这表明在晚期基因启动子控制下的ASFVmRNAs利用与早期启动子不同的终止机制和因子,和/或细胞因子在感染晚期对病毒转录组景观的影响不同。
