ScienceDaily — 來自 Weizmann 研究所的科學家們提供了一項有關病毒感染過程中的重要發現，他們揭露了一種被稱呼為「擬菌病毒（mimivirus）」的感染機轉，這種病毒在阿米巴原蟲當中被發現，它之所以有這樣的稱呼全是因為它的體積比一般的病毒大上許多，體積幾乎和小型細菌類似，而且用普通的顯微鏡就可以觀察到它的存在。這項研究發表已在 PLoS Biology 期刊線上版本中。
圖1. Mimivirus Star-Shaped Structures.
圖2. (A) TEM image of cryo-fixed sectioned and stained extracellular Mimivirus particles revealing a star-shaped structure at a unique vertex. (B) Cryo-TEM image of a whole vitrified fiber-less Mimivirus. (C) SEM image of the star-shaped structure in a mature extracellular Mimivirus particle. (D) Cryo-SEM of an immature, fiber-less particle. (E) Tomographic slice of a mature intracellular Mimivirus particle captured at a late (12 h post infection) infection stage. As shown in Video S1, at this late stage the host cell is packed with mature viral particles. (F and G) Volume reconstruction of the particle shown in (E), revealing the presence of an outer (red) and inner (orange) capsid shells. The star-shaped structure is present in both shells but adopts partially open (dark, star-like region), and completely sealed configurations in the outer and inner shells, respectively. (H) Superposition of the two shells in (F) and (G).
圖3. Morphological Aspects of Phagosome-Enclosed Mimivirus Particles
(A) TEM projection of a phagosome-enclosed particle sectioned along a plane that contains the whole star-shaped structure. The observed features are similar to those characterizing extracellular Mimivirus particles (Figure 1A) and mature intracellular particles (Figure 1E), thus indicating that the starshape assembly is present in the capsid throughout the life cycle of the Mimivirus. The inset provides the various possibilities for random sectioning of the Mimivirus particle. (B) TEM projection of a phagosome-enclosed particle sectioned along a plane that does not contain the star-shaped assembly, thus revealing only unmodified vertices. (C) TEM projection of a phagosome-enclosed Mimivirus particle, revealing the star-shape structure (blue arrowhead) sliced in its center along the plane depicted by a blue line in the inset in (A).
Scale bars, 100nm. doi:10.1371/journal.pbio.0060114.g002
病毒感染活體細胞的過程分為兩個步驟，第一：病毒以滲透方式進入細胞。接著，第二步驟就相當重要，細胞開始製造新的病毒，然後藉著擴散來感染其他細胞。當這個生產程序開始進行，細胞會製造出病毒的外殼，它包含有排序後的蛋白質也就是所謂的殼體（capsid）。接著細胞會開始複製病毒的 DNA，並將這些 DNA 插入殼體中。其結果便是產生一個嶄新的、具備好感染其他細胞的病毒，最後這些病毒便能夠離開它的宿主細胞而後感染更多的細胞。
在瞭解了病毒是如何感染細胞以及新病毒的感染路徑之後，這便能夠讓科學家們找到能夠終止感染週期的方法，如此便能夠阻止因病毒所產生的相關疾病。然而，要進行這項研究有一個最大的困難就在於不同的病毒其侵入策略都大相逕庭。
擬菌病毒是目前已知體積最龐大的病毒，它的體積比一般常見的病毒還要大上五至十倍左右。這種病毒是在二十世紀末期才被發現，他龐大的尺寸使得它不可能藉著正規的方法來鑑定它。此外，它也攜帶著比一般病毒更多的遺傳性物質，這也意味著這種病毒有能力發展出足夠的方法使它的病毒 DNA 進到宿主細胞中，同時將遺傳性包裹於宿主細胞內製造新病毒的過程中插入其蛋白質中。
來自 Weizmann 研究所的教授 Abraham Minsky 與他的研究生 Nathan Zauberman 和來自有機化學系的 Yael Mutsafi，以及提供相關化學研究協助的 Eugenia Klein 博士與 Eyal Shimoni 博士共同進行。他們揭開了這種病毒所使用的方法的某些細節。在這項新研究當中，科學家們首次取得了這種病毒將病毒性遺傳物質注入被感染的宿主細胞時的三維圖像（three-dimensional pictures，3-D 圖像），以及它將遺傳性物質插入蛋白質載體中的程序。
圖4. 研究擬菌病毒的 Abraham Minsky 教授
先前有關病毒的所有研究中，病毒性遺傳物質會在細胞感染的過程中注入細胞中，接著在細胞內產生新病毒時進入新形成的蛋白質容器中，接著通過相同的通道，這個通道在病毒容器中被製造出來。相較之下，研究所的科學家們發現這種巨大的病毒使用存在於殼體上的另一個不同通道來達到這兩個目標。科學家們同時也發現 DNA 螺旋在這兩個程序中並不會形成長絲狀，但會形成一個濃密的塊狀包裝。研究人員相信就是這些獨特的特色促進了兩者注入到宿主細胞當中，而且可以將大量的擬菌病毒之遺傳物質插入其中。
圖5. Intracellular Viral Factories and DNA Packaging. (A) TEM of an intracellular viral factory, revealing Mimivirus particles at various assembly stages. Empty, fiber-less particles at initial assembly stages, appearing at close vicinity to the periphery of the viral factory (purple arrowheads); partially assembled empty, fiber-less particles (blue); and mature, fiber-covered particles located further away from the viral factory than immature particles (yellow). A stargate that is consistently located at the distal site of the factory can be discerned in several particles (red arrowheads). VF and Cyt stand for viral factory and cytoplasm. (B and C) TEM of Mimivirus particles undergoing DNA packaging through a non-vertex face-centered site (green arrowheads). Two edges of a stargate located at the opposite site of the DNA packaging site are indicated with red arrowheads. Approximately 60% of viral particles undergoing DNA packaging (;120 particles out of ;200 analyzed in this study) reveal a face-centered DNA packaging site, while in other virions the packaging site is unclear. As discussed in the text, the reason for this apparent ambiguity in the packaging site results from intrinsic constraints in the interpretation of data derived from projection TEM studies of thin sections. More inclusive data on this issue can, however, be obtained from electron tomography
analyses of thick sections, as demonstrated in Figure 7 and in Video S3.
Scale bars: 500 nm in (A), 200 nm in (B), 100 nm in (C).
圖6. Electron Tomography of a DNA Packaging Site. (A) Tomographic slice of a procapsid undergoing DNA packaging. The DNA-loaded packaging gateway and the stargate are highlighted (green and red arrowheads, respectively). Scale bar, 100 nm. (B) Volume reconstruction of the particle depicted in (A) showing the orifice through which DNA (green) is packaged, which spans the outer and inner capsid shells, and the internal membrane (red, orange, and blue, respectively). The protein core underlying the membrane is shown in gray (See Video S3 for the whole tomogram).
在 Weizmann 的研究中，他們揭露了入侵阿米巴原蟲後的擬菌病毒電子顯微影像，殼體蛋白質的細胞壁 － 一種由 20 個三角形所組成的多邊型 － 彼此相互分離，並且就像花朵的花瓣般的打開，形成一個大的、星狀結構，可以暱稱它為「星閘（stargate）」。
圖7. Stargate Opening. (A) SEM image depicting the release of membranal structures following exposure of mature, extracellular Mimivirus particles to 83 8C for 30 min. Membrane release consistently occurs at the stargate (arrows pointing to the fiber-less edges of the stargate). (B) Extracellular Mimivirus particle revealing a conspicuous 5-fold opening. Such open stargates were detected in a small population of extracellular particles, and may represent empty viral particles released upon the viral-induced lysis of the host cells. Scale bars, 100nm. doi:10.1371/journal.pbio.
在病毒胞膜的星閘下方會與阿米巴原蟲的細胞膜融合在一起，然後殺出一條寬廣的通道直達阿米巴原蟲內部。當細胞壁突然被打開時所釋放出來的壓力會比我們打開香檳時還要大上二十倍，這樣剛好可以將病毒 DNA 擠壓進入通道中，並使其快速的通過管道而進入阿米巴原蟲的細胞中。
圖8. Mimivirus Uncoating and Membrane Fusion. (A) Tomographic slice of a late phagosome enclosing three Mimivirus particles at early, advanced, and final uncoating stages (particles 1, 2, and 3, respectively). At the early uncoating stage, a partial opening of the inner protein shell at the stargate assembly is initiated. The red arrowheads highlight the star-shaped structure sectioned along the plane depicted by a red line in the inset in Figure 2A. The opening of the stargate allows for the extrusion of the viral membrane towards the phagosome membrane, a stage characterizing particle 2. In the final uncoating stage, fusion between viral and phagosome membranes occurs, as revealed in particle 3. The lysosomes surrounding the phagosome should be noted. The reconstructed volume of the tomographic slice is shown in Video S2. Scale bar, 100nm. (B) Volume reconstruction of particle 1 in (A), showing the outer (red) and inner (orange) capsid shells and the closely apposed inner membrane (light blue). The opening of the star-shaped structure in the inner shell (in contrast to its closed configuration in extracellular particles or in particles enclosed in early phagosomes) should be noted. (C) Surface rendering of particle 3 in (A), showing fusion of the viral and phagosome membranes (light and dark blue, respectively) at the site of the opened star-shaped structure. The boundary between the viral and phagosome membranes is arbitrary.
圖9. Schematic Representation of a Mimivirus Particle at Its Final Uncoating Stage. The capsid (red) is opened at the stargate, allowing for fusion of the viral and phagosome membranes (light and dark blue, respectively), thus forming a star-shaped membrane conduit (See Video S2 for the tomogram from which the model was derived).
圖10. (A) SEM of a viral factory within an amoeba cell lysed 8 h post infection. A high-magnification image of the site indicated by the yellow arrowhead (inset) shows assembling fiber-less particles with stargates (red arrowheads), as well as mature fiber-coated particles. (B) SEM of a viral factory isolated 8 h post infection. The factory is studded with viral particles at various assembly stages. Stargates are indicated with red arrowheads. (C) SEM of a viral factory isolated 10 hours post infection. Only mature, fiber-covered particles can be detected. Scale bars are 2 lm in (A), 300 nm in (B),
and 500 nm in (C).
此外，影像也顯示出當病毒在宿主細胞中被製造出來時，病毒的遺傳物質是怎麼插入新形成的蛋白質容器中。在這個程序裡，病毒的遺傳物質會相反方向的經由星閘之容器的新細胞壁而被交付至目的地。插入的過程必須克服容器裡面的壓力，這也許必須藉著當細胞壁開啟時裡面的引擎來驅動。
科學家們相信研究擬菌病毒的生命週期，從感染細胞那一刻起，一直到新病毒的形成為止，也許可以提供有用的資料，包含其他病毒的數種反應機轉是如何運作等等，因為這都和人類遭受病毒感染所產生的疾病相關。
Journal reference:
Zauberman N, Mutsafi Y, Halevy DB, Shimoni E, Klein E, et al.
Distinct DNA Exit and Packaging Portals in the Virus Acanthamoeba polyphaga mimivirus.
PLoS Biology, Vol. 6, No. 5, e114 DOI: 10.1371/journal.pbio.0060114
Adapted from materials provided by Weizmann Institute of Science.
原始報導：
ScienceDaily：Invasion Strategy Of World's Largest Virus Revealed
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