The family Tectiviridae comprises a group of tailless, icosahedral, membrane-containing 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 two-hybrid 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.

Tectiviridae科包括一组无尾，二十面体，含膜的噬菌体，根据其宿主，革兰氏阴性菌或革兰氏阳性菌可分为两组。第一组由PRD1和几乎相同的特征明确的裂解病毒组成，而第二组则包含更多可变温带噬菌体，例如GIL16或Bam35，其宿主为蜡状芽孢杆菌和相关的革兰氏阳性细菌。在Bam35的基因组中，32个带注释的开放阅读框（ORF）中有近一半在数据库（ORFans）中没有同源物，是功能未知的推定蛋白质，这妨碍了对其生物学的理解。为了增加对病毒蛋白质组学的了解，我们对Bam35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。所得的蛋白质相互作用组包含24种蛋白质之间的76种独特相互作用，其中12种具有未知功能。这些结果表明，P17蛋白是Bam35的次要衣壳蛋白，P24是Penton蛋白，后者的发现也得到迭代穿线蛋白建模的支持。此外，内膜转糖基转移酶蛋白P26可能具有其他结构作用。我们还检测到涉及非结构蛋白的相互作用，例如DNA结合蛋白P1和基因组末端蛋白（P4），这通过重组蛋白的共免疫沉淀得以证实。总之，我们的结果提供了Bam35病毒蛋白质组的功能性观点，重点是病毒颗粒的组成和组织。

重要性无尾病毒家族的Tectiviridae可以感染普通和致病性革兰氏阳性和革兰氏阴性细菌。而且，已经提出它们起源于几组大的真核DNA病毒和自我复制质粒的进化起源。但是，由于其古老的起源和复杂的多样性，许多技术病毒蛋白是功能未知的ORFans。病毒蛋白的全面蛋白-蛋白相互作用（PPI）分析最终可以揭示生物学机制，从而为通过逐一研究蛋白无法获得的蛋白功能提供新的见解。在这里，我们全面描述了使用多载体酵母双杂交筛选技术检测的泰克病毒Bam35蛋白中的病毒内PPI，并且这些PPI进一步得到了共免疫沉淀试验和蛋白质结构模型的支持。