Bam35 Tectivirus Intraviral Interaction Map Unveils New Function and Localization of Phage ORFan Proteins
Bam35 Tectivirus Intraviral Interaction Map Unveils New Function and Localization of Phage ORFan Proteins
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DOI：
10.1128/jvi.00870-17
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发表时间：
2017-10-01
影响因子：
5.4
通讯作者：
Redrejo-Rodriguez, Modesto
中科院分区：
文献类型：
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作者：
Berjon-Otero, Monica;Lechuga, Ana;Redrejo-Rodriguez, Modesto
Tectiviridae家族包括一组无尾的、二十面体的、含膜的噬菌体，其可根据其宿主（革兰氏阴性或革兰氏阳性细菌）分为两组。虽然第一组由PRD 1和几乎相同的充分表征的裂解病毒组成，但第二组包括更易变的温带病毒，如GIL 16或Bam 35，其宿主是蜡样芽孢杆菌和相关的革兰氏阳性细菌。Bam 35基因组中32个开放阅读框（ORF）中有近一半在数据库中没有同源物（ORFans），是功能未知的推定蛋白，这阻碍了对其生物学的理解。为了增加对病毒蛋白质组的了解，我们对Bam 35基因组编码的所有推定蛋白质进行了全面的酵母双杂交分析。由此产生的蛋白质相互作用组包括24种蛋白质之间的76种独特的相互作用，其中12种具有未知的功能。这些结果表明，P17蛋白是Bam 35的次要衣壳蛋白，P24是五邻体蛋白，后者的发现也得到了迭代线程蛋白建模的支持。此外，内膜转糖基酶蛋白P26可能具有额外的结构作用。我们还检测到涉及非结构蛋白的相互作用，如DNA结合蛋白P1和基因组末端蛋白（P4），这是通过重组蛋白的共免疫沉淀证实的。总之，我们的研究结果提供了一个功能视图的Bam 35病毒蛋白质组，重点是组成和组织的病毒particle.IMPORTANCE无尾病毒的家庭Tectiviridae可以感染细菌和致病性革兰氏阳性和革兰氏阴性细菌。此外，它们被认为是几组大型真核DNA病毒和自我复制质粒的进化起源。然而，由于其古老的起源和复杂的多样性，许多tectivirus蛋白是未知功能的ORFan。对病毒蛋白质进行全面的蛋白质-蛋白质相互作用（PPI）分析最终可以揭示生物学机制，从而为逐个研究蛋白质所无法实现的蛋白质功能提供新的见解。在这里，我们全面描述了tectivirus Bam 35蛋白质中的病毒内PPI使用多载体酵母双杂交筛选确定，这些PPI进一步得到了免疫共沉淀试验和蛋白质结构模型的结果的支持。这种方法使我们能够提出已知蛋白质的新功能，并假设一些病毒ORFan蛋白在病毒颗粒内定位的生物学作用，这将有助于理解感染革兰氏阳性菌的tectiviruses的生物学。
The family Tectiviridae comprises a group of tailless, icosahedral, membranecontaining bacteriophages that can be divided into two groups by their hosts, either Gram-negative or Gram-positive bacteria. While the first group is composed of PRD1 and nearly identical well-characterized lytic viruses, the second one includes more variable temperate phages, like GIL16 or Bam35, whose hosts are Bacillus cereus and related Gram-positive bacteria. In the genome of Bam35, nearly half of the 32 annotated open reading frames (ORFs) have no homologs in databases (ORFans), being putative proteins of unknown function, which hinders the understanding of their biology. With the aim of increasing knowledge about the viral proteome, we carried out a comprehensive yeast two-hybrid analysis of all the putative proteins encoded by the Bam35 genome. The resulting protein interactome comprised 76 unique interactions among 24 proteins, of which 12 have an unknown function. These results suggest that the P17 protein is the minor capsid protein of Bam35 and P24 is the penton protein, with the latter finding also being supported by iterative threading protein modeling. Moreover, the inner membrane transglycosylase protein P26 could have an additional structural role. We also detected interactions involving nonstructural proteins, such as the DNA-binding protein P1 and the genome terminal protein (P4), which was confirmed by coimmunoprecipitation of recombinant proteins. Altogether, our results provide a functional view of the Bam35 viral proteome, with a focus on the composition and organization of the viral particle.IMPORTANCE Tailless viruses of the family Tectiviridae can infect commensal and pathogenic Gram-positive and Gram-negative bacteria. Moreover, they have been proposed to be at the evolutionary origin of several groups of large eukaryotic DNA viruses and self-replicating plasmids. However, due to their ancient origin and complex diversity, many tectiviral proteins are ORFans of unknown function. Comprehensive protein-protein interaction (PPI) analysis of viral proteins can eventually disclose biological mechanisms and thus provide new insights into protein function unattainable by studying proteins one by one. Here we comprehensively describe intraviral PPIs among tectivirus Bam35 proteins determined using multivector yeast twohybrid screening, and these PPIs were further supported by the results of coimmunoprecipitation assays and protein structural models. This approach allowed us to propose new functions for known proteins and hypothesize about the biological role of the localization of some viral ORFan proteins within the viral particle that will be helpful for understanding the biology of tectiviruses infecting Gram-positive bacteria.