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Vaccinia-like cytoplasmic replication of the giant Mimivirus

15 Apr

Mutsafi Y, Zauberman N, Sabanay I, and Minsky A. 2010. Vaccinia-like cytoplasmic replication of the giant Mimivirus. Proc Natl Acad Sci USA 107 (13): 5978-5982.

Summary:
A lot of the current research of the Mimivirus is focused on its lifecycle. The large nature of this virus is interesting because it contains a lot of DNA and transcription factors to create large elaborate replication factories. Since the DNA is so large (more than 100kbp), it makes traveling through the nuclear envelope difficult. This current study suggests that the Mimivirus does not cross the nuclear envelope and that DNA replication occurs entirely in the cytoplasmic factory, similar to the poxvirus. Discovery of the mechanism of replication of the Mimivirus can lead us to a better understanding of other similar viruses.

The study used diverse imaging techniques to analyze genomes crossing into the nucleus. They had two groups of samples. One involved infected Acanthamoeba polyphaga with Mimivirus, while the other (control) involved Acanthamoeba polyphaga without Mimivirus infection. Immunolabeling was done with BrdU labeling to visualize the viral replication. After a set time frame of incubation and infection, the antibodies to BrdU were added and the results of the images were created. Another label used to visualize the genomes was Fluorescent in Situ Hybridization of Poly(dT) probes. For this labeling technique, the Acanthamoeba polyphaga grew on the glass coverslips and was treated with formamide. The slides were then washed and inoculated following the fluorescent hybridization guidelines. After all, steps for this process were followed correctly. DAPI was added as a stain to create the picture. All the experiments were visualized for fluorescence and photographed using Deltavision system. Images were obtained using an application.

Results from these images show that at 2 hours post infection, the Mimivirus appeared in the phagosomes of the host cell. The images also show structures that were detected in the cytoplasm of the infected amoeba, but not in the uninfected amoeba.

proposed

Figure 1 (Mutsafi, 2010).:
Acanthamoeba cells were fixed and observed using transmission electron microscope (TEM) (A) TEM image of the nucleus in an infected cell (B) Magnification of the delineated area from the cores (arrows) when DNA is released.

proposed

Figure 2 (Mutsafi, 2010).:
Mimivirus replication occurs in the cytoplasm. Amoeba cells infected with Mimivirus and labeled with BrdU (A) infected cell contains a viral factory where replication occurs. Fluorescent label is represented by BrdU (red) and DAPI (blue). This revealed a dotted DAPI.

The labeling with anti-BrdU antibody and DAPI stain revealed the viral inclusions. The BrdU blue color indicates DNA replication. The images indicated that replication is occurring in the cytoplasm.

To further assess replication occurs in the cytoplasm another image was viewed 5 hours after infection with increasing Mimivirus titers. There was a correlation between viral titers and the replication factories generated.

proposed

Figure 3 (Mutsafi, 2010).:
This experiment was done to find the relationship between viral particles and factories when infected. Amoeba cells were infected with Mimivirus. (A) DAPI stained cells (B) cytoplasmic viral factories (C-E) fusion of replication factories (C) TEM image (D) electron tomography of two replication factories generated from fusion of viral genomes released from the viral cores (E) two viral cores in gold engulfed by viral DNA (blue)

While this study shows what is happening, the mechanism by which it does this remains to be solved. Finding out the mechanism of the Mimivirus could also help with our understanding of other large DNA replication strategies.

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Posted by on April 15, 2011 in Uncategorized

 

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