Phage morphological and structural diversity
噬菌体形态和结构多样性
As indicated above, phage genomes are either composed of DNA or RNA and it may be double-stranded (ds) or single-stranded (ss). This genetic material is packaged into a capsid that can be polyhedral (Microviridae, Corticoviridae, Tectiviridae, Leviviridae, Cystoviridae), filamentous (Inoviridae), pleomorphic (Plasmaviridae) or connected to a tail (Caudovirales)12 (Fig. 1). Up to date, most isolated phages are tailed and have dsDNA genomes13. Taxonomic classification of phage taxa is carried out by the International Committee on Taxonomy of Viruses (ICTV)14. While phage classification was historically based on characteristics such as genome type (DNA, RNA, ss, ds), viral morphology, and host range, it is currently undergoing a major overhaul, primarily using mostly genomic-based methods to classify them. For example, the 1999 ICTV report classified tailed phages into 3 families, 16 genera and 30 species, while the 2018 version grouped them into 5 families, 26 subfamilies, 363 genera and 1320 species (https://talk.ictvonline.org/taxonomy/p/taxonomy_releases). Comprehensive guidelines have been proposed for phage classification and it is expected that the list of virus taxa will significantly increase in the upcoming years15.
如上所述，噬菌体基因组由 DNA 或 RNA 组成，可能是双链（ds）或单链（ss）。这些遗传物质被包装在噬菌体的噬菌体囊中，噬菌体囊可以是多面体（细小病毒科、皮层病毒科、Tectivir 科、Levivir 科、Cystovir 科）、丝状（Inovir 科）、多形性（Plasmavir 科）或与尾部相连（Caudovirales）12（图 1）。迄今为止，大多数分离出的噬菌体都是有尾的，并具有 dsDNA 基因组13。噬菌体类群的分类由国际病毒分类委员会（ICTV）14 进行。噬菌体分类历来以基因组类型（DNA、RNA、ss、ds）、病毒形态和宿主范围等特征为基础，但目前正在进行重大改革，主要采用基于基因组的方法对噬菌体进行分类。例如，1999 年的 ICTV 报告将有尾噬菌体分为 3 科、16 属和 30 种，而 2018 年的版本则将其分为 5 科、26 亚科、363 属和 1320 种 (https://talk.ictvonline.org/taxonomy/p/taxonomy_releases)。目前已提出了噬菌体分类的综合指南，预计未来几年病毒类群列表将大幅增加15。
Tailed bacteriophages 尾部噬菌体
The large majority of phages described to date have a tailed morphology with dsDNA genome and belong to the Caudovirales order13. This viral order, while under re-classification16 is currently comprised of five families: Myoviridae, Siphoviridae, Podoviridae, Ackermannviridae and Herelleviridae. The last two families were created only recently as network and meta-analyses indicated that they represented distinct clusters within the Myoviridae family16,17. A large variation in capsid size can be observed among members of Caudovirales, with diameters ranging from 45 nm to 185 nm, which is usually linked to genome size18. Most of the tailed phages (75%) have icosahedral capsid structures and around 15% have an elongated capsid aligned with the axis of the tail13. Interestingly, all members of Caudovirales share the same major capsid protein (MCP) fold (HK97-fold)19. The HK97-fold was identified following X-ray crystallography of the capsid of phage HK97. The capsid is connected to its tail through a connector complex often composed of a portal protein and two head completion/connector proteins. Structural studies have revealed that the portal complex is a dodecameric ring with a similar overall structure shared between most tailed phages despite low sequence similarity20–22. Capsid completion/connector proteins also form dodecameric rings as observed in the Siphoviridae SPP1 and HK97 phages. Homologs of these proteins are found in a variety of phages that have contractile and non-contractile tails23. Moreover, the head-to-tail connector protein gp4 of the Podoviridae P22 has a similar structure to the ones present in siphophages SPP1 and HK97, despite no observable trace of sequence similarity24.
迄今描述的大多数噬菌体都有尾部形态和 dsDNA 基因组，属于 Caudovirales 目13。该病毒目目前由 5 个科组成，但正在重新分类16 ：Myoviridae、Siphoviridae、Podoviridae、Ackermannviridae 和 Herelleviridae。后两个科是最近才建立的，因为网络和元分析表明，它们代表了肌病毒科内不同的群16,17。Caudovirales 成员的噬菌体大小差异很大，直径从 45 纳米到 185 纳米不等，这通常与基因组大小有关18。大多数有尾噬菌体（75%）具有二十面体噬菌体结构，约 15%的噬菌体具有与尾部轴线对齐的拉长噬菌体13。有趣的是，Caudovirales 的所有成员都具有相同的主要噬菌体蛋白（MCP）折叠（HK97-fold）19。HK97 折叠是在对噬菌体 HK97 的囊膜进行 X 射线晶体学研究后发现的。噬菌体通过一个通常由一个入口蛋白和两个头部完成蛋白/连接蛋白组成的连接复合体与其尾部相连。结构研究表明，入口复合体是一个十二分子环，尽管序列相似性较低，但大多数尾部噬菌体都具有类似的整体结构20-22。正如在 Siphoviridae SPP1 和 HK97 噬菌体中观察到的那样，头盖完成/连接蛋白也形成十二分子环。这些蛋白的同源物存在于多种噬菌体中，它们的尾部有收缩和非收缩两种23。此外，Podoviridae P22 的头尾连接蛋白 gp4 与虹吸噬菌体 SPP1 和 HK97 中的头尾连接蛋白具有相似的结构，尽管在序列上看不出相似之处24。
The tails of Siphoviridae are composed of a central tape measure protein surrounded by a tail tube and ends with a terminator protein25. A similar architecture is observed for phages of the Myoviridae family, where an additional layer, the protein sheath, enables contraction for the insertion of the tail tube into the bacterial host during infection26. Interestingly, the capsid-tail joining protein gpFII of the Siphoviridae phage λ has a similar tertiary fold to its tail tube protein gpV and adopts the same quaternary structure when assembled in the phage27. GpV also shares structural homologies with the tail tube of Myoviridae phages as well as some components of the bacterial type VI secretion system like the Hcp1 protein28. Moreover, the folds of proteins gpFII and gpV are similar to those of the baseplate hub of the myophages T4 and Mu, once again without any sequence homology27. These observations suggest that the tail tube-like fold adopted by the capsid-tail connector, the tail tube protein itself and the base plate is an important building block for members of Siphoviridae and Myoviridae. Members of the Podoviridae family, such as E. coli phage T7, have very short and non-contractile tails. A tube-like extension of the tail that penetrates both cell membranes was observed to be essential for genome delivery into the host29.
Siphoviridae噬菌体的尾部由一个中央卷尺蛋白组成，周围是尾管，末端是终结蛋白25。肌病毒科的噬菌体也有类似的结构，其中的附加层–蛋白鞘–可使尾管在感染过程中收缩插入细菌宿主体内26。有趣的是，Siphoviridae λ噬菌体的囊膜-尾管连接蛋白 gpFII 与其尾管蛋白 gpV 具有相似的三级折叠，并且在噬菌体中组装时采用相同的四级结构27。GpV 还与 Myoviridae 噬菌体的尾管以及细菌 VI 型分泌系统的某些成分（如 Hcp1 蛋白）具有相同的结构28。此外，gpFII 和 gpV 蛋白的褶皱与噬菌体 T4 和 Mu 的基板枢纽相似，但同样没有任何序列同源性27。这些观察结果表明，囊尾连接体、尾管蛋白本身和基板所采用的类似尾管的折叠是虹彩病毒科和肌病毒科成员的重要结构单元。Podoviridae 科的成员，如大肠杆菌噬菌体 T7，具有非常短且不收缩的尾部。据观察，可穿透两层细胞膜的管状尾部延伸是将基因组送入宿主体内的关键29。
Finally, receptor binding proteins (RBP) present at the tip of the tail or at the baseplate were characterized at the structural level in siphophages and showed high levels of structural similarity despite low sequence homology30–32. Moreover, RBP domains are interchangeable between different phages and are homologous with mammalian adenovirus33. Members of the Ackermannviridae family, formerly known as Viunalikevirus, have a myovirus-like morphology but they differ by their complex and unique adsorption structures. Short filaments with bulbous tips that resemble an umbrella and prong-like structures are attached to the baseplate17.
最后，在虹吸噬菌体中，存在于尾端或基板上的受体结合蛋白（RBP）在结构水平上表现出特征性，尽管序列同源性较低，但结构相似性很高30-32。此外，不同噬菌体之间的 RBP 结构域可以互换，并且与哺乳动物腺病毒具有同源性33。阿克曼病毒科（Ackermannviridae）的成员以前被称为 Viunalikevirus，其形态与肌病毒相似，但它们的不同之处在于复杂而独特的吸附结构。短丝状物的顶端呈球状，类似伞状和棱状结构，附着在基板上17。
Membrane-containing bacteriophages
含膜噬菌体
Phages belonging to the Tectiviridae (phage PRD1) and Corticoviridae (phage PM2) families comprise icosahedral tailless virions that have an internal lipidic membrane and linear or circular dsDNA genomes, respectively. A hallmark characteristic of these two phage families is their trimeric major capsid protein, which is composed of a double β-barrel structure34,35. Structural
属于 Tectiviridae（噬菌体 PRD1）和 Corticoviridae（噬菌体 PM2）家族的噬菌体由二十面体无尾病毒组成，分别具有内部脂质膜和线性或环状 dsDNA 基因组。这两个噬菌体家族的一个标志性特征是它们的三聚体主要囊膜蛋白，由双 β 管结构组成34,35。结构
analyses of the MCP of phage PRD1 revealed an N-terminal alpha helix, which interacts directly with the phage inner membrane and shares structural homologies with the MCP of adenoviruses34. Furthermore, the RBP present at the icosahedral vertices of phage PRD1 and PM2 capsids also share N-terminal domains with human adenovirus35–37. Phage PRD1 does not have a tail to deliver its DNA to its Gram-negative host, but its membrane was observed to transform into a proteo- lipidic tube, which can pierce host envelopes38. Unlike Corticoviridae and Tectiviridae which have inner lipidic membranes, members of the Cystoviridae family including phage phi6 have lipidic membranes that surround their icosahedral capsids39. Finally, Acholeplasma virus L2 (AVL2 or also referred as MVL2) is currently the only classified member of the Plasmaviridae family. It infects the wall-less Acholeplasma bacterial species and new virions are released by membrane budding without causing cell lysis40. Plasmaviridae phages do not possess any capsid but their genomes are enclosed in a proteinaceous lipid vesicle that has a similar composition to the outer membrane of phi641.
对噬菌体 PRD1 的 MCP 的分析表明，其 N 端 alpha 螺旋直接与噬菌体内膜相互作用，并与腺病毒的 MCP 具有相同的结构34。此外，存在于噬菌体 PRD1 和 PM2 外壳二十面体顶点的 RBP 也与人类腺病毒共享 N 端结构域35-37。噬菌体 PRD1 没有将其 DNA 运送到革兰氏阴性宿主体内的尾部，但观察到其膜转变为蛋白脂质管，可以刺穿宿主包膜38。Corticoviridae 和 Tectiviridae 具有内脂质膜，而囊尾蚴科的成员（包括 phi6 噬菌体）则不同，它们的二十面体囊壳周围有脂质膜39。最后，Acholeplasma 病毒 L2（AVL2 或又称 MVL2）是目前唯一归类为浆液病毒科的成员。它感染无壁的 Acholeplasma 细菌，通过膜出芽释放新病毒，不会导致细胞裂解40。质病毒科噬菌体不具有任何外壳，但其基因组被包裹在一个蛋白脂质囊泡中，该囊泡的成分与 phi641 的外膜相似。
Phages with small icosahedral capsids or filamentous morphology
具有二十面体小菌体或丝状形态的噬菌体
Microviridae and its most studied member, phage phiX174, have small icosahedral capsids (26 nm) and ssDNA genomes (5,386 bp) (Fig. 1) 42. The capsid is built on a protein fold that has a “jelly roll” β-barrel structure and has similarities with ssDNA eukaryotic viruses, including rhinoviruses42. They are currently classified in two subfamilies named Bullavirinae and Gokushovirinae. DNA delivery in the bacterial host relies on a protein, which oligomerizes to form a tube that crosses the host’s periplasmic space by joining the outer and inner membranes43. Structural differences in proteins mediating host attachment have been observed for both subfamilies. Bullavirinae have pentameric major spike protein (MSP) complexes at the end of each capsid vertex44, while Gokushovirinae have “mushroom-like” protrusions that extend along the threefold icosahedral axes of the capsid44. The MSP complexes in the Bullavirinae clade are also divergent, but as their structures are superimposable, they can be exchanged between phages45.
微小病毒科及其研究最多的成员–噬菌体 phiX174–具有小的二十面体病毒盖（26 nm）和 ssDNA 基因组（5,386 bp）（图 1）42。噬菌体建立在具有 “果冻卷 “β-桶状结构的蛋白质折叠上，与 ssDNA 真核病毒（包括鼻病毒）有相似之处42。目前，这些病毒被分为两个亚科，分别名为 “牛病毒科”（Bullavirinae）和 “Gokushovirinae”（Gokushovirinae）。DNA 在细菌宿主中的传递依赖于一种蛋白质，这种蛋白质通过连接外膜和内膜形成一个管子，穿过宿主的周质空间43。在这两个亚家族中都观察到了介导宿主附着的蛋白质的结构差异。公牛病毒亚科在每个噬菌体顶点的末端都有五聚体的主要尖峰蛋白（MSP）复合物44，而鹅膏病毒亚科则有 “蘑菇状 “突起，沿着噬菌体的三倍二十面体轴延伸44。Bullavirinae 支系中的 MSP 复合物也存在差异，但由于它们的结构可以叠加，因此可以在噬菌体之间交换45。
Other small icosahedral viruses include members of the Leviviridae family, such as the phage MS2 that has a ssRNA genome (Fig. 1). The MS2 viral particle has only two proteins: a major capsid protein and a single copy of the maturation protein that interacts with the genomic RNA during packaging and with the host receptor during adsorption46. The MCP of phage MS2 can control replication by interacting with the initiation codon of the replicase-encoding gene,
其他小型二十面体病毒包括 Leviviridae 家族成员，如具有 ssRNA 基因组的噬菌体 MS2（图 1）。MS2 病毒粒子只有两种蛋白：一种是主要的囊膜蛋白，另一种是单拷贝的成熟蛋白，成熟蛋白在包装过程中与基因组 RNA 相互作用，在吸附过程中与宿主受体相互作用46。噬菌体 MS2 的 MCP 可通过与复制酶编码基因的起始密码子相互作用来控制复制、
which switches the replication cycle to viral assembly47. Recent cryo-EM reconstruction of the viral capsid also revealed that the RNA genome is highly involved in virion assembly since it can adopt secondary structures that act as a scaffold48. Of note, this system of genome packing is radically different from the Caudovirales, in which an empty capsid is first assembled and then filled with the phage genome and the packaged capsid is then connected to its tail49.
从而将复制周期切换到病毒组装阶段47。最近对病毒外壳的低温电子显微镜重建也显示，RNA 基因组高度参与病毒组装，因为它可以采用二级结构作为支架48。值得注意的是，这种基因组包装系统与 Caudovirales 完全不同，Caudovirales 首先组装一个空的囊膜，然后填充噬菌体基因组，包装好的囊膜再与其尾部连接49。
Finally, members of the family Inoviridae are dramatically different in terms of morphology and lifestyle (Fig. 1). Phage particles contained a dsDNA genome surrounded by thousands of copies of MCP that are assembled and then extruded from the host in a continuous manner50. The MCPs of filamentous phages are unique in their architecture, which consists of a long alpha-helix with an N-terminal signal peptide for membrane translocation51. The signal peptide is then cleaved before the proteins are assembled in a long cylindrical shell spiral with the C-terminal end interacting directly with the viral DNA52.
最后，噬菌体科的成员在形态和生活方式上存在巨大差异（图 1）。噬菌体颗粒包含一个 dsDNA 基因组，周围有数千个 MCP 复制件，这些复制件组装后以连续的方式从宿主体内挤出50。丝状噬菌体的 MCPs 结构独特，由一个长的α-螺旋线和一个 N 端信号肽组成，用于膜转运51。然后，信号肽被裂解，蛋白再组装成螺旋状长圆柱形外壳，C 端直接与病毒 DNA 相互作用52。
Two sides of the same coin
一枚硬币的两面
From a morphological point of view, several diverse phages still share some commonalities. One example is the MCP fold, which is conserved at the structural level between all tailed phages but also extends to archaeal viruses and adenoviruses53,54. For the majority of these proteins where conservation is observed in their structure, no trace of homology can be detected, both at the amino acid and nucleic acid levels. This paradigm is explored further in Box 1, where convergent evolution or a common ancestor are discussed as possible explanation for structural similarities among viruses infecting the three domains of life.
从形态学的角度来看，几种不同的噬菌体仍有一些共性。其中一个例子是 MCP 折叠，它在所有有尾噬菌体之间的结构水平上都是保守的，而且还延伸到了古细菌病毒和腺病毒53,54。对于大多数结构保持不变的蛋白质，在氨基酸和核酸水平上都检测不到任何同源性。方框 1 进一步探讨了这一模式，其中讨论了聚合进化或共同祖先作为感染三个生命领域的病毒之间结构相似性的可能解释。