National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-3210, USA.
J Virol. 2011 Oct;85(19):9899-908. doi: 10.1128/JVI.05446-11. Epub 2011 Jul 27.
The double-stranded DNA genome of vaccinia virus (VACV), the prototype poxvirus, contains approximately 200 open reading frames (ORFs) that are transcribed at early, intermediate, and late stages of infection. Previous high-throughput deep RNA sequencing allowed us to map 118 VACV early genes that are expressed before viral DNA replication and 93 postreplicative genes. However, the intermediate- and late-stage postreplicative genes could not be differentiated. Here, we synchronized infections with a reversible inhibitor of DNA replication and used a VACV mutant that conditionally transcribes late genes to sequence the two classes of mRNAs. In addition, each postreplicative ORF was individually expressed under conditions that distinguished intermediate and late classes. We identified 38 VACV genes that belong to the late class and 53 that belong to the intermediate class, with some of the latter continuing to be expressed late. These data allowed us to prepare a genome-wide early, intermediate, and late transcription map. Inspection of sequences upstream of these ORFs revealed distinctive characteristics of intermediate and late promoters and suggested that some promoters have intermediate and late elements. The intermediate genes encoded many DNA binding/packaging and core-associated proteins in addition to late transcription factors; the late genes encoded many morphogenesis and mature virion membrane proteins, including those involved in entry, in addition to early transcription factors. The top-ranked antigens for CD4(+) T cells and B cells were mainly intermediate rather than late gene products. The differentiation of intermediate and late genes may enhance understanding of poxvirus replication and lead to improvements in expression vectors and recombinant vaccines.
痘苗病毒（VACV）的双链 DNA 基因组是正痘病毒的原型，它包含大约 200 个开放阅读框（ORFs），这些 ORFs 在感染的早期、中期和晚期进行转录。以前的高通量深度 RNA 测序使我们能够绘制 118 个 VACV 早期基因，这些基因在病毒 DNA 复制之前表达，还有 93 个复制后基因。然而，无法区分复制后中期和晚期的基因。在这里，我们使用可逆的 DNA 复制抑制剂同步感染，并使用条件性转录晚期基因的 VACV 突变体来测序这两类 mRNA。此外，每个复制后 ORF 在区分中期和晚期类别的条件下分别表达。我们鉴定了 38 个属于晚期类别的 VACV 基因和 53 个属于中期类别的基因，其中一些后者晚期继续表达。这些数据使我们能够制备全基因组的早期、中期和晚期转录图谱。对这些 ORF 上游序列的检查揭示了中期和晚期启动子的独特特征，并表明一些启动子具有中期和晚期元件。中期基因编码许多 DNA 结合/包装和核心相关蛋白，除了晚期转录因子；晚期基因编码许多形态发生和成熟病毒包膜蛋白，包括参与进入的蛋白，以及早期转录因子。CD4(+) T 细胞和 B 细胞的顶级抗原主要是中间基因产物，而不是晚期基因产物。中间和晚期基因的分化可能有助于深入了解痘病毒的复制，并导致表达载体和重组疫苗的改进。
