Department of Medical Microbiology & Immunology, Li Ka Shing Institute of Virology, University of Alberta, Edmonton, AB, Canada.
J Virol. 2013 Apr;87(8):4623-41. doi: 10.1128/JVI.02617-12. Epub 2013 Feb 13.
Myxoma virus (MYXV) provides an important model for investigating host-pathogen interactions. Recent studies have also highlighted how mutations in transformed human cells can expand the host range of this rabbit virus. Although virus growth depends upon interactions between virus and host proteins, the nature of these interactions is poorly understood. To address this matter, we performed small interfering RNA (siRNA) screens for genes affecting MYXV growth in human MDA-MB-231 cells. By using siRNAs targeting the whole human genome (21,585 genes), a subset of human phosphatases and kinases (986 genes), and also a custom siRNA library targeting selected statistically significant genes ("hits") and nonsignificant genes ("nonhits") of the whole human genome screens (88 genes), we identified 711 siRNA pools that promoted MYXV growth and 333 that were inhibitory. Another 32 siRNA pools (mostly targeting the proteasome) were toxic. The overall overlap in the results was about 25% for the hits and 75% for the nonhits. These pro- and antiviral genes can be clustered into pathways and related groups, including well-established inflammatory and mitogen-activated protein kinase pathways, as well as clusters relating to β-catenin and the Wnt signaling cascade, the cell cycle, and cellular metabolism. The validity of a subset of these hits was independently confirmed. For example, treating cells with siRNAs that might stabilize cells in G(1), or inhibit passage into S phase, stimulated MYXV growth, and these effects were reproduced by trapping cells at the G(1)/S boundary with an inhibitor of cyclin-dependent kinases 4/6. By using 2-deoxy-D-glucose and plasmids carrying the gene for phosphofructokinase, we also confirmed that infection is favored by aerobic glycolytic metabolism. These studies provide insights into how the growth state and structure of cells affect MYXV growth and how these factors might be manipulated to advantage in oncolytic virus therapy.
粘液瘤病毒（MYXV）为研究宿主-病原体相互作用提供了一个重要模型。最近的研究还强调了人类转化细胞中的突变如何扩大这种兔病毒的宿主范围。尽管病毒的生长依赖于病毒和宿主蛋白之间的相互作用，但这些相互作用的性质还知之甚少。为了解决这个问题，我们在人 MDA-MB-231 细胞中进行了针对影响 MYXV 生长的基因的小干扰 RNA（siRNA）筛选。通过使用靶向整个人类基因组（21585 个基因）、一组人类磷酸酶和激酶（986 个基因）的 siRNA，以及靶向整个人类基因组筛选中选定的具有统计学意义的基因（“命中”）和无意义基因（“非命中”）的定制 siRNA 文库（88 个基因），我们鉴定了 711 个促进 MYXV 生长的 siRNA 池和 333 个抑制生长的 siRNA 池。另外 32 个 siRNA 池（主要针对蛋白酶体）具有毒性。命中基因的结果总体重叠约为 25%，而非命中基因的结果总体重叠约为 75%。这些促进病毒和抗病毒基因可以聚类成途径和相关组，包括成熟的炎症和有丝分裂原激活的蛋白激酶途径，以及与 β-连环蛋白和 Wnt 信号级联、细胞周期和细胞代谢有关的簇。其中一些命中基因的子集的有效性被独立证实。例如，用可能使细胞稳定在 G1 期或抑制进入 S 期的 siRNA 处理细胞，会刺激 MYXV 的生长，并且用细胞周期蛋白依赖性激酶 4/6 的抑制剂将细胞困在 G1/S 边界也会产生同样的效果。通过使用 2-脱氧-D-葡萄糖和携带磷酸果糖激酶基因的质粒，我们还证实感染有利于有氧糖酵解代谢。这些研究深入了解细胞的生长状态和结构如何影响 MYXV 的生长，以及如何利用这些因素在溶瘤病毒治疗中获得优势。
