Herpes Simplex Virus 1 Expressing GFP-Tagged Virion Host Shutoff (vhs) Protein Uncouples the Activities of RNA Degradation and Differential Nuclear Retention of the Virus Transcriptome.
Herpes Simplex Virus 1 Expressing GFP-Tagged Virion Host Shutoff (vhs) Protein Uncouples the Activities of RNA Degradation and Differential Nuclear Retention of the Virus Transcriptome.
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DOI：
10.1128/jvi.01926-21
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发表时间：
2022-07-27
影响因子：
5.4
通讯作者：
Elliott, Gillian
中科院分区：
文献类型：
--
作者：
Wise, Emma L.;Samolej, Jerzy;Elliott, Gillian
病毒粒子宿主关闭蛋白(VHS)是由单纯疱疹病毒1型(HSV1)编码的一种内切核酸酶。VHS引起感染细胞环境的几个变化，这些变化有利于晚期(L)病毒蛋白的翻译：细胞mRNAs降解，即刻早期(IE)和早期(E)病毒转录产物与Polya结合蛋白1(PABPC1)隔离在细胞核中，双链RNA被降解以帮助抑制依赖PKR的应激反应。为了进一步了解VHS的细胞生物学，我们构建了一种表达VHS的病毒，该病毒的C端带有GFP标记。首次表达时，VHS-GFP定位于核旁簇，随后与其结合伙伴VP16共定位并相互作用，并被包装成病毒粒子。尽管VHS-GFP在分离表达时保持活性，但它在感染期间未能降解mRNA或重新定位PABPC1，而病毒转录水平与VHS敲除病毒相似。在VHS-GFP感染的细胞中，PKR的磷酸化也被增强，这与未能降解dsRNA是一致的。但FISH结果显示，在VHS-GFP感染的细胞中，IE和E，而不是L的转录本，在晚期仍保留在细胞核内，而在Wt而不是Dvhs感染的细胞核中。这些结果表明，VHS诱导的IE和E转录本的核滞留依赖于VHS的表达，而不依赖于其内切核酸酶的活性，从而解偶联了VHS的这两个功能。与许多病毒一样，单纯疱疹病毒1型(HSV1)表达一种内切核酸酶，即病毒粒子宿主关闭(VHS)蛋白，它调节感染细胞的RNA环境，促进病毒蛋白翻译的经典级联反应。它通过导致一些信使核糖核酸分子的降解和其他分子的核滞留来做到这一点。在这里，我们描述了一种表达VHS的病毒，其C末端标记有绿色荧光蛋白(GFP)，表明VHS-GFP融合蛋白保留了天然VHS的物理性质，但不会导致mRNA的降解。尽管如此，VHS-GFP保持了将早期病毒转录组捕获在细胞核中以利于晚期蛋白质翻译的能力，首次证明了mRNA降解不是VHS对核转录组影响的先决条件。因此，这种病毒解偶联了VHS的核滞留和降解活动，为VHS在感染过程中提供了新的理解。
Virion host shutoff (vhs) protein is an endoribonuclease encoded by herpes simplex virus 1 (HSV1). vhs causes several changes to the infected cell environment that favor the translation of late (L) virus proteins: cellular mRNAs are degraded, immediate early (IE) and early (E) viral transcripts are sequestered in the nucleus with polyA binding protein (PABPC1), and dsRNA is degraded to help dampen the PKR-dependent stress response. To further our understanding of the cell biology of vhs, we constructed a virus expressing vhs tagged at its C terminus with GFP. When first expressed, vhs-GFP localized to juxtanuclear clusters, and later it colocalized and interacted with its binding partner VP16, and was packaged into virions. Despite vhs-GFP maintaining activity when expressed in isolation, it failed to degrade mRNA or relocalise PABPC1 during infection, while viral transcript levels were similar to those seen for a vhs knockout virus. PKR phosphorylation was also enhanced in vhs-GFP infected cells, which is in line with a failure to degrade dsRNA. Nonetheless, mRNA FISH revealed that as in Wt but not Dvhs infection, IE and E, but not L transcripts were retained in the nucleus of vhs-GFP infected cells at late times. These results revealed that the vhs-induced nuclear retention of IE and E transcripts was dependent on vhs expression but not on its endoribonuclease activity, uncoupling these two functions of vhs. IMPORTANCE Like many viruses, herpes simplex virus 1 (HSV1) expresses an endoribonuclease, the virion host shutoff (vhs) protein, which regulates the RNA environment of the infected cell and facilitates the classical cascade of virus protein translation. It does this by causing the degradation of some mRNA molecules and the nuclear retention of others. Here, we describe a virus expressing vhs tagged at its C terminus with a green fluorescent protein (GFP) and show that the vhs-GFP fusion protein retains the physical properties of native vhs but does not induce the degradation of mRNA. Nonetheless, vhs-GFP maintains the ability to trap the early virus transcriptome in the nucleus to favor late protein translation, proving for the first time that mRNA degradation is not a prerequisite for vhs effects on the nuclear transcriptome. This virus, therefore, has uncoupled the nuclear retention and degradation activities of vhs, providing a new understanding of vhs during infection.
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