Identification and characterization of interferon-induced proteins that inhibit alphavirus replication
Identification and characterization of interferon-induced proteins that inhibit alphavirus replication
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DOI：
10.1128/jvi.01282-07
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发表时间：
2007-10-01
影响因子：
5.4
通讯作者：
Klimstra, William B.
中科院分区：
文献类型：
--
作者：
Zhang, Yugen;Burke, Crystal W.;Klimstra, William B.
α/β干扰素(α/β干扰素)通过上调许多干扰素刺激基因(ISGs)产生抗病毒作用，这些基因的蛋白产物是抗病毒状态的效应物。我们实验室以前的数据表明，干扰素-α/β可以通过蛋白激酶R(PKR)依赖和不依赖的机制来限制Sindbis病毒(SB)的复制，并且一种PKR不依赖的机制可以抑制感染病毒基因组的翻译(K.D.Ryman等，J.Virol)。79：1487-1499,2005)。此外，利用Affymetrix微阵列技术，我们鉴定了44个基因作为候选基因，这些基因是L干扰素诱导的非PKR/RNA酶诱导的抗病毒活性的候选基因。在目前的研究中，我们已经开始使用三种技术来分析这些基因产物的抗甲病毒活性：(I)从SB载体中过表达蛋白质并评估小鼠的毒力衰减；(Ii)在四环素诱导的稳定的小鼠成纤维细胞培养系统中过表达蛋白质并评估其对SB复制的影响；以及(Iii)小干扰RNA介导的在成纤维细胞培养中下调基因mRNA，随后进行如上所述的SB复制评估。测试的蛋白质包括那些我们假设可能会影响病毒基因组翻译的蛋白质，包括小鼠ISG20、ISG15、锌指抗病毒蛋白(ZAP)、蛇毒蛋白、p56、p54和p49。有趣的是，一些基因产物在体外和体内的抗病毒活性模式是不同的。Viperin和ZAP对小鼠的毒力减弱程度最深。然而，ISG20和ZAP在体外对SB复制有较强的抑制作用，而Viperin、p56和ISG15在体外对SB的复制有轻微的抑制作用。相反，p54和p49在任何检测中都几乎没有作用。
Alpha/beta interferon (IFN-alpha/beta) produces antiviral effects through upregulation of many interferon-stimulated genes (ISGs) whose protein products are effectors of the antiviral state. Previous data from our laboratory have shown that IFN-alpha/beta can limit Sindbis virus (SB) replication through protein kinase R (PKR)-dependent and PKR-independent mechanisms and that one PKR-independent mechanism inhibits translation of the infecting virus genome (K. D. Ryman et al., J. Virol. 79:1487-1499, 2005). Further, using Affymetrix microarray technology, we identified 44 genes as candidates for PKR/RNase L-independent IFN-induced antiviral activities. In the current studies, we have begun analyzing these gene products for antialphavirus activity using three techniques: (i) overexpression of the protein from SB vectors and assessment of virulence attenuation in mice; (ii) overexpression of the proteins in a stable tetracycline-inducible murine fibroblast culture system and assessment of effects upon SB replication; and (iii) small interfering RNA-mediated knockdown of gene mRNA in fibroblast cultures followed by SB replication assessment as above. Tested proteins included those we hypothesized bad potential to affect virus genome translation and included murine ISG20, ISG15, the zinc finger antiviral protein (ZAP), viperin, p56, p54, and p49. Interestingly, the pattern of antiviral activity for some gene products was different between in vitro and in vivo assays. Viperin and ZAP attenuated virulence most profoundly in mice. However, ISG20 and ZAP potently inhibited SB replication in vitro, whereas and viperin, p56, and ISG15 exhibited modest replication inhibition in vitro. In contrast, p54 and p49 had little to no effect in any assay.