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Ultrastructural Characterization of the Giant Volcano-like Virus Factory of Acanthamoeba polyphaga mimivirus

15 Apr

Monti M, La Scola B, Barrassi L, Espinosa L, Raoult D. 2007. Ultrastructural Characterization of the Giant Volcano-like Virus Factory of Acanthamoeba polyphaga mimivirus. PLoS ONE 2:e328.

Summary:

The Mimivirus is a giant icosahedral DNA virus that grows in amoebae. There is a lot of debate about the evolution of the Mimivirus and it has caused scientists to rethink how DNA viruses are categorized. Mimivirus contains ~ 911 protein encoding genes, in which, many of these protein sequences were believed to be found in cellular organisms. Unique gene homologs that encode DNA repair enzymes were also discovered in the Mimivirus, including four that didn’t seem to be found in other dsDNA viruses. The virus also encodes three types of topsomerases, and has two ORFs composed of amino acid that was identified as chaperones.

Studies were performed to figure out the mechanisms of the Mimivirus life cycle. One study used A. polyphaga and seeded them. These cells were then infected with Mimivirus. In order to get a better picture of what was happening in the lifecycle, the fluorescence labeling method was used. This was done both directly and indirectly. In the direct method the cells were ProLong Gold Antifade Reagent. The indirect method used mAb P4C8G2. After that, the stained cells were observed using microscopes with 40x, 63 x, or 100x lenses. The study also generated DIC images and used electron microscopy to look at the entry of the amoebae. The study suggested appearance of Mimivirus entering the amoebae by phagocytic vacuole. The virus fuses with the phagocytic vacuole and delivers itto the cell cytoplasm. The Mimivirus then releases its DNA into the nucleus starting at first round of DNA replication. The experiment then seemed to show the Mimivirus DNA leaving the host nucleus to form the virus factory replication centre. The virus factory (VF) is a structure that is clearly distinct from the nucleus and is surrounded by the mitochondria. The size of this structure increased very rapidly and released virions into the cytoplasmic space by budding.

Before entry into the cell viruses must attach to the host cell in order to get phagocytized. A study in 2009 looked at how the Mimivirus attaches to its target. The Mimivirus attaches through what is believed to be a Star-fish shaped feature. CyroEM studies in the experiment showed that the Mimivirus had special particles on its vertex. With TEM, the scientists sectioned an infected amoeba virus and observed a star-fish structure extending from one of its vertices. This is further seen with AFM, that Mimivirus defibered particles have this star-fish like shape. Other interesting finds in the biosynthesis phase of the Mimivirus was found in a study of the DNA structure. This virus has an unusually large genome. It is made up of approximately 1.2 million bp. The size and linear structure of genome were confirmed by restriction digests and pulse-field gel electrophoresis. There have also been inverted repeats of about 900 nucleotides on both ends suggesting circular topology.

Further results from Monti’s experiment revealed that in the biosynthesis phase, Mimivirus was surrounded by two membrane layers on the interior and a capsid protein coat on the exterior with fibrils attached. During entry, empty particles were observed with an open vertex. The internal Mimivirus membrane also extrudes from the particle in order to merge with the vacuole membrane which the virus will then release itself into the cytoplasm. A condensed genetic material appears to enter the cell nucleus beginning genome replication. In the grand scheme of replication, the Mimivirus involves a rapid takeover of host cell machinery that took place in a unique and autonomous assembly centre called the virus factory (VF). The virus factory is not a part of the nucleus. This just means that Mimivirus replicates and assembles in the cytoplasm. There are three VF zones. They are the inner replication center, intermediate assembly zone, and the peripheral zone.

Other aspects of note in Monti’s 2007 experiment are what the DIC images revealed. The DIC images showed a volcano-like giant Mimivirus factory during the late stage of infection. The experiment also seemed to indicate that the Mimivirus released from the cell through cell lysis once the viral capsids acquire their fibrils.

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Posted by on April 15, 2011 in Uncategorized

 

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