RSS

Category Archives: Uncategorized

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

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.

Advertisements
 
Leave a comment

Posted by on April 15, 2011 in Uncategorized

 

New Scientist: Giant Virus Shapes Life (video)

Source of Video

Summary:
The world’s largest known virus known so far. The Mimivirus is a monster compared to the classic viruses such as HIV or the flu virus, which do not have more than 10 genes. Radio Wammo and Janine Young from Kiwi FM will discuss this topic.

 
Leave a comment

Posted by on April 15, 2011 in Uncategorized

 

Screening Pneumonia Patients for Mimivirus

Dare RK, Chittaganpitch M, Erdman DD 2008. Screening Pneumonia Patients for Mimivirus. Emerg Infect Dis. 14:465-467.

Summary:

Since the discovery of the Mimivirus, scientists have been trying to figure out what role the virus plays. So far, a high number of pneumonia cases have unknown causes. New PCR assay tests are developed to detect presence of Mimivirus, particularly on patients with pneumonia. In this study, scientists took a sample of 496 pneumonia specimens from 9 pneumonia-patient populations in Thailand and United States. These patients were screened for genes found in the Mimivirus. The genes were notably from L396, R596, L65, and R656. The real time PCR was used to identify these primers. Total nucleic acid was extracted from all specimens by using either an automated extractor or the automated BioRobot MDx. In the end, genes L396 and R596 from the Q-PCR results detected few 10 copies of plasmid DNA per reaction. Of all the 496 specimens taken, there were no indications of positives for the Mimivirus DNA.

This study suggests that the Mimivirus role with human pneumonia is still unknown. It doesn’t seem to target a human host for replication. This study is not recent (2008), but it does provide information on the Mimivirus and that it is mainly an amoeba parasite and does not have huge impact as a human pathogen.

 
Leave a comment

Posted by on April 15, 2011 in Uncategorized

 

Listen to our podcast

PODCAST #1

Click here to listen or download to podcast #1 on Mimivirus. Extreme virology episode #23.
Speakers Vincent, Dick, and Alan will review the largest virion and DNA genome on the Mimivirus and the satellite virus Sputnik virophage.

Summary:

Since the discovery of the Mimivirus in 2003, scientists have been rethinking the way to define viruses. The Mimivirus may give an evolutionary clue about viruses. Other interesting research that begun, since its discovery is to perform a PCR analysis to test for Mimivirus in brain disease from amoeba and pneumonia cases. More research needs to be done to find out where the proteins/receptors the Mimivirus attaches to in the host cell before the host cell does phagocytosis. This could help to see what the Mimivirus targets.

PODCAST #2

Click here to listen or download to podcast #2 on Mimivirus. Speakers Vincent, Alan, and Raul Rabadan converse about the structure of mimivirus based on this journal.

Summary:

This podcast discusses the large capsid of the Mimivirus as well as other unique structures. This relates to virology because the asymmetry may help deliver the genome to the host. This makes it a little unique from other viruses.

***SOURCE OF PODCAST***

 
Leave a comment

Posted by on April 15, 2011 in Uncategorized

 

The 1.2-Megabase Genome Sequence of Mimivirus

Didier R, Audic S, Robert C, Abergel C, Renesto P, Ogata H, La Scola B, Suzan M, Claverie JM. 2004. The 1.2-Megabase Genome Sequence of Mimivirus. Science 306:1344-1350.

Summary:

This journal article summarizes the unique findings of the Mimivirus. It is one of the few viruses that have both type one and type two topoisomerases, DNA repair enzymes, and polysaccharide synthesis enzymes. The findings associated with this virus have highlighted the existence of a 4th domain of life. It is interesting because the large DNA virus had proofreading enzymes that are unique to this type of virus.

Having proofreading enzymes to cut out mutations in DNA is important for eukaryotes to be functional, but are often missing in viruses. Since the virus have such a large output of functioning proteins it would not be evolutionarily favorable to leave it unchecked from these unique proofreaders. If other virus genomes could be as stable as the Mimivirus it would make treating them simpler due to the lack of antigenic drift.

 
Leave a comment

Posted by on April 15, 2011 in Uncategorized

 

Vaccinia-like cytoplasmic replication of the giant Mimivirus

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.

 
Leave a comment

Posted by on April 15, 2011 in Uncategorized