The anti-interferon activity of conserved viral dUTPase ORF54 is essential for an effective MHV-68 infection.
The anti-interferon activity of conserved viral dUTPase ORF54 is essential for an effective MHV-68 infection.
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DOI：
10.1371/journal.ppat.1002292
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发表时间：
2011-10
期刊：
影响因子：
6.7
通讯作者：
Sun R
中科院分区：
文献类型：
--
作者：
Leang RS;Wu TT;Hwang S;Liang LT;Tong L;Truong JT;Sun R
KSHV 和 EBV 等伽玛疱疹病毒通过淋巴细胞中的潜伏期建立终生持续感染。这些病毒已经进化出多种策略来对抗先天性和适应性免疫系统的各个组成部分。我们使用遗传和生物学相关的病毒 MHV-68 进行了公正的筛选，以寻找参与抑制 I 型干扰素信号传导的病毒 ORF，并鉴定了保守的病毒 dUTP 酶 ORF54。在这里，我们通过异位表达和使用转基因 MHV-68 定义了 ORF54 在 I 型干扰素抑制中的贡献。 ORF54和缺乏dUTPase酶活性的ORF54通过诱导I型干扰素受体蛋白IFNAR1的降解来有效抑制I型干扰素信号传导。随后，我们在体外证明，在存在 I 型干扰素信号传导的情况下，ORF54 的缺乏会导致裂解复制减少。对 MHV-68 体内感染期间 IFNAR1 降解的生理后果和 ORF54 重要性的研究表明，ORF54 对持续感染的影响比对裂解复制的影响更大。缺乏ORF54表达的MHV-68无法有效地在淋巴细胞中建立潜伏感染，尽管它在肺组织中相对正常地复制。然而，IFNAR−/−小鼠的感染减轻了这种表型，强调了ORF54在I型干扰素抑制中的特定作用。用 ORF54 中带有位点特异性突变的重组 MHV-68 病毒感染小鼠和细胞，使 dUTPase 失活，表明 dUTPase 酶活性不是 ORF54 的抗干扰素功能所必需的。此外，我们发现 dUTPase 活性在分析的 MHV-68 感染的所有阶段都是可有可无的。总体而言，我们的数据表明，ORF54 除了其 dUTPase 酶活性外，还进化出了抗干扰素活性，并且实际上正是抗干扰素作用使得 ORF54 对于建立 MHV-68 的有效持续感染至关重要。人类伽马疱疹病毒、卡波西肉瘤相关疱疹病毒和 Epstein-Barr 病毒是多种恶性肿瘤的病因，特别是在因 HIV 感染而免疫功能低下的患者中。对这些人类伽马疱疹病毒的研究很困难，因为它们无法在细胞培养物中复制并且缺乏小动物模型。鼠伽马疱疹病毒 68 是一种遗传和生物学相似的病毒，被用作小鼠模型，因为它具有在细胞培养物中复制的能力、可操纵的基因组和感染小鼠等优点。在这项研究中，我们发现病毒开放阅读框 54 (ORF54) 是先天免疫的抑制剂，特别是 I 型干扰素反应的抑制剂。尽管ORF54是一种保守的病毒dUTP酶，但我们发现其抗干扰素活性并不需要其酶活性。通过感染细胞和小鼠，我们确定了 ORF54 在通过诱导 I 型干扰素受体降解建立 MHV-68 持续潜伏感染中的关键作用。我们的研究为 I 型干扰素信号传导的深远影响和 ORF54 的双重作用提供了新的见解。这项工作可能有助于制定针对伽马疱疹病毒感染的疫苗策略。
Gammaherpesviruses such as KSHV and EBV establish lifelong persistent infections through latency in lymphocytes. These viruses have evolved several strategies to counteract the various components of the innate and adaptive immune systems. We conducted an unbiased screen using the genetically and biologically related virus, MHV-68, to find viral ORFs involved in the inhibition of type I interferon signaling and identified a conserved viral dUTPase, ORF54. Here we define the contribution of ORF54 in type I interferon inhibition by ectopic expression and through the use of genetically modified MHV-68. ORF54 and an ORF54 lacking dUTPase enzymatic activity efficiently inhibit type I interferon signaling by inducing the degradation of the type I interferon receptor protein IFNAR1. Subsequently, we show in vitro that the lack of ORF54 causes a reduction in lytic replication in the presence of type I interferon signaling. Investigation of the physiological consequence of IFNAR1 degradation and importance of ORF54 during MHV-68 in vivo infection demonstrates that ORF54 has an even greater impact on persistent infection than on lytic replication. MHV-68 lacking ORF54 expression is unable to efficiently establish latent infection in lymphocytes, although it replicates relatively normally in lung tissues. However, infection of IFNAR−/− mice alleviates this phenotype, emphasizing the specific role of ORF54 in type I interferon inhibition. Infection of mice and cells by a recombinant MHV-68 virus harboring a site specific mutation in ORF54 rendering the dUTPase inactive demonstrates that dUTPase enzymatic activity is not required for anti-interferon function of ORF54. Moreover, we find that dUTPase activity is dispensable at all stages of MHV-68 infection analyzed. Overall, our data suggest that ORF54 has evolved anti-interferon activity in addition to its dUTPase enzymatic activity, and that it is actually the anti-interferon role that renders ORF54 critical for establishing an effective persistent infection of MHV-68. Human gammaherpesviruses, Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus, are the cause of several malignancies, especially in patients immunocompromised due to HIV infection. The study of these human gammaherpesviruses is difficult due to their inability to replicate in cell culture and the lack of a small-animal model. Murine gammaherpesvirus-68 is a genetically and biologically similar virus that is utilized as a mouse model because it offers such advantages as the ability to replicate in cell culture, a manipulatable genome, and infection of mice. In this study, we have identified viral open reading frame 54 (ORF54) as an inhibitor of innate immunity, specifically of the type I interferon response. Although ORF54 is a conserved viral dUTPase, we found that its anti-interferon activity does not require its enzymatic activity. Through infection of cells and mice, we define the critical role of ORF54 in establishing persistent latent infection of MHV-68 by inducing the degradation of the type I interferon receptor. Our studies provide new insights into the far reaching effects of type I interferon signaling and the dual role of ORF54. This work could aid in the development of vaccine strategies to gammaherpesvirus infection.
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