The Effect of Selective Pressure on Mimivirus Ability to Infect Human Cells
One of the largest viruses on Earth has been found in human cells and this is how it might have happened.
Until recently, the potential size of a virus was assumed much smaller than even the smallest bacterium. Nothing larger had been found, and nothing with a genome size that even came close to comparing had been discovered. Viral diversity, however, was and still is grossly underestimated. In 1992, isolates of the amoeba Acanthamoeba polyphaga revealed an unusual particle that stained gram-positively, and was initially though to be a bacterium. It was called Bradfordcoccus until 2003, when a team of scientists at the Université de la Méditerranée identified the organism as mimivirus. Until the later discovery of even larger viruses, such as Cafeteria roenbergensis, it was thought to be the largest virus in existence and even today blurs the lines between viruses and intracellular parasites (3).
Mimivirus, also known as Acanthamoeba polyphaga mimivirus (APMV), was initially discovered primarily as a pathogen of amoebas. It was discovered in Acanthamoeba polyphaga while searching for Legionella bacteria, the causative agent of legionellosis, in cooling systems of air-conditioning (6). Mimivirus was initially thought to be a small bacterium due to its size, but was later found to be a massive virus due to its icosahedral form. Mimivirus and the family it is a part of, the Mimiviridae, are unique because of their great size and unique features shared with cellular organisms, particularly elements of protein translation machinery not found in other viruses. Analysis of its genome, which is 1.2Mb in length, reveals it is a relative of the Nucleocytoplasmic Large DNA Viruses (NCLDV). This group includes the Poxviruses and Iridoviruses and are known for their well-defined inclusion bodies and cytoplasmic infection of which mimivirus is no exception, as it has many similarities to the poxviruses (2).
The nature of the Mimiviridae might indicate a lineage descended from vastly simplified intracellular parasites, due to the presence of peptidoglycan in the virus structure and genes for translation only previously found in cellular organisms. In nature, APMV particles are rough mimics of the bacteria that amoebae feed upon. The peptidoglycan in the structure of the virus and the size strongly support this. When an amoeba encounters an APMV virion, it engulfs it as it would any other bacterium. This means that APMV is dependent on a large size in order to successfully transmit to the next host, which indicates heavy selection pressure leading to the advanced size of mimivirus (1). It is through heavy selective pressure for size that APMV is able to infect humans.
APMV is a potential emerging pathogen in humans. Pneumonia is the leading cause of death from infections but the causative agent is unknown in up to 50% of cases (7). Mimivirus was a suspected agent in some pneumonia cases, so a study was done by Bernard La Scola in which 376 serum samples were taken from patients who had pneumonia that had tested negative for any other pneumonia agent. When tested, 2.3% of the patients had a significant mimivirus antibody titer. Genomic mimivirus DNA was even found in lavage specimens of pneumonia patients (5).
This is strange because mimivirus known as primarily a pathogen of protozoa (as well as some corals and sponges) and would need a mechanism through which it could enter in replicate in many types of cells. In humans, APMV has been implicated in some pneumonia cases because it is incredibly unspecific in host. It enters cells via phagocytosis, which means that human macrophages can be easily infected with APMV. It is through a mechanism such as this that APMV could potentially infect humans. Macrophages are large immune cells that ingest pathogenic organisms via phagocytosis (8).
Giant viruses have four possible possible methods of entry into cells. The first two methods are clathrin-mediated endocytosis and fusion-entry. Unfortunately it is not possible to determine the method of entry by viral size alone, but evidence points to neither of these methods being used by APMV as a form of entry. This was discovered by treating cells with inhibitory drugs and discovering that they still uptook APMV despite having their clathrin-mediated endocytosis processes inactivated. In addition, degradative endosomes do not appear to play a part, leaving two potential options: macropinocytosis and phagocytosis. In the lab macrophages can be experimentally infected with APMV and thus phagocytosis is an option. Macropinocytosis has also been observed as a pathway for viral infection so it is the other viable option (4). Both the host amoeba and the macrophage can utilize these forms of viral uptake into the cell and it seems likely that the rest of the Mimiviridae use similar forms of uptake. Distinguishing which process is used, however, is a little more difficult. Macropinocytosis and phagocytosis can look fairly similar and proving them chemically requires multiple tests.
Mimivirus is so massive that its uptake into the cell can be viewed directly without labeling. When APMV is viewed as it enter a host it can be seen either right next to the cell wall or being absorbed on cellular extensions (pseudopodia). Subsequent absorption of the viral particle takes place very rapidly in a large, smooth endocytic vesicles that can be found very deep in the cell, even sometimes fusing together. The characteristic protrusions associated with mimivirus entry can be attributed to either macropinocytosis or phagocytosis as each other these involves a protrusion or rippling of the cell (4). This mechanism for viral uptake makes the most sense in both contexts. A macrophage and an amoeba are rather similar in their processes for capturing organic matter be it for defense or consumption. The extension of the cell to encapsulate food items is exactly what the virus does in nature and is also what macrophages do to pathogens. Macropinocytosis, however, has been proven not to be the mechanism behind mimivirus entry.
Macropinocytosis is a process intended to take large amounts of solute non-selectively by ruffling of the cell to intake a large macropinosome, simply a type of vesicle. Because macropinocytosis is a regulated function, however, many viruses can use it as a pathway to infect a cell (11). When an inhibitor is added to macrophages and APMV is added infection still results. This alone indicates that macropinocytosis does not play a role in APMV infection but further study has been done that shows APMV does not colocalize with rabankyrin-5, a marker for macropinocytosis (4). These two results can definitively allow the ruling out of macropinocytosis as a method of APMV entry into a host.
The last remaining option is for APMV to use phagocytosis to its advantage. In order to test this there must be a process or chemical exclusive to phagocytosis that macropinocytosis lacks. In most cells that can use either dynamin-II is a protein that serves in the ‘pinching off’ of vesicles and is not fount in macropinocytosis. In some cells it is involved in clathrin-mediated endocytosis but not in macrophages. In order to test this clathrin-mediated endocytosis must be tested by itself. A compound like transferrin, which only enters cells via endocytosis, can be used to determine the role of dynamin-II in endocytosis. If the dynamin-II gene is blocked from expressing the uptake of transferrin is not inhibited. This implies no role of dynamin-II in the endocytosis pathway of macrophages.
Mycobacterium avium is an intracellular parasite that uses the phagocytosis pathway to invade macrophages. When M. avium is added to macrophages that have had their dynamin-II genes blocked their uptake into the cell is significantly reduced, proving that dynamin-II is required for phagocytosis. Finally, APMV must be shown to invade cells in conjunction with phagocytosis by blocking dynamin-II and adding APMV. This is easily done by blocking dynamin-II as with M. avium, which results in no virus being taken into the cell. This is enough to definitively show that APMV enters macrophages via phagocytosis (4). This strengthens the idea that the jump to human macrophages from amoebae is not that great since both use actin-mediated phagocytosis pathways. This has implications for the treatment of APMV infections in humans because specific molecules on the surface coating of the virus used to attach to cells and trigger phagocytosis could be used against the virus as a method of treatment. It also indicates the value of Acanthamoeba as a model organism in the study of APMV pathogenicity provided the phagocytosis pathways are similar.
Phagocytosis appears to be the definitive method that APMV uses to enter both human macrophages and organisms like Acanthamoeba. In nature APMV appears to mimic the size of bacteria that amoebae consume and simply wait to be ingested. Because it is most advantageous to be taken up quickly, the virus has developed a variety of ways to appear more attractive to the amoeba (2). This is why APMV can be the massive size that it is. Relyng on phagocytosis as a method of uptake requires a more passive approach since viruses lack motility and because the process of uptake is similar in both amoebae and macrophages it is likely that this life history strategy gives APMV the ability to enter human cells. The question then becomes how this relationship came to be.
Amoebae are plastic in terms of their genome, phenotype, and environment. This means that amoeba hosts may serve as a reservoir that allows and even encourages a large, adaptable genome in APMV. Amoebae inhabit a wide variety of environments which leads to amoebae having a lot of extensive genetic variation which causes their prokaryotic parasites to also have some accompanying variation. These parasites are a possible route through which APMV acquired genes for pathogenicity because the genome of APMV is large and complex enough to allow for lateral gene transfer within the host amoeba (9). This could be how APMV derived its ability to infect more complex organisms, including humans. Because of the proximity of some of these pathogens to humans it is not beyond the realm of imagination that some of them managed to come into contact with other organisms with genes capable of causing human infection.
Amoebae are also capable of causing human infection in a variety of circumstances. In the vein of the evolution of some macroscopic parasites such as lice and ticks, another mechanism that could explain the evolution of APMV pathogenic to humans is that pathogenic or commensal amoebae brought APMV inside of the host and they infected macrophage cells from there. This would be supported by the very wide variety of amoeba infections of humans, particularly ones that are not fatal, which might lead to prolonged contact between APMV bearing parasites and human hosts due to a lack of treatment (10).
It is not difficult to envision a situation in which amoebae are present in water in a human-associated structure, like a water tower or air conditioning unit. The amoebae would be distributed by this structure and as a result come into contact with people who utilized it. Infection might occur opportunistically in the lungs when aerosolized, which would mean that the amoeba was living inside the human, perhaps without any symptoms at all. If the amoebae was infected with APMV, however, it would eventually lyse and release virions that might be taken up into macrophages.
Though macrophage infection in the lab does not produce a systemic, productive infection, it is entirely possible that it could have happened in the past with similar cells (1). Because macrophages and amoebae use similar methods of uptake into cell there is a chance that receptors are shared between them that APMV could use to gain access to the cell via phagocytosis as described above. Repeated exposures could potentially allow one or more strains of APMV to develop that are capable of direct pathogenesis in humans.
Despite the evidence pointing to macrophages and amoebae using similar mechanisms, the question remains as to how APMV could bridge such a massive host gap. Humans to amoebae is a massive leap of faith in terms of virology, because viruses with such host range are virtually unknown. An organism with this much host range is more akin to a small bacterium than a virus, because most viruses does not have nearly enough genetic material to allow such a dramatic shift. This is where the unique structure and lifestyle of APMV comes into play. APMV has a genome even larger than that of some small intracellular parasites which would allow for significant genetic variation which would allow the virus to retain genetic information that might increase its host range.
Because the selection pressure acting on APMV is for a larger size, speed of replication and efficiency fall to the wayside. Instead, the result is a very large virion that is of the right size for an amoeba to consume (1). This large size allows for a larger genome, which permits more genes being stored in a larger genome. Combined with lateral gene transfer, pathogenic genes have a real chance of being retained in the virus even if they do not serve an immediate purpose. Macrophages and amoebae are not known for their sensitive palettes and will consume most organic matter over a certain size, which APMV fits quite nicely. Phagocytosis is a very general system in which cells uptake nutrients and thus APMV can infect a very wide range of cells that use phagocytosis (1).
Even more indicative is the presence of positive Gram-staining materials in the wall of the virion. The size of the virion is increased with thick bundles of fibers which at first do not appear to serve any other purpose. Upon closer examination there are a variety of bacteria-like traits found in this structure that serve as a potent form of mimicry. These are genes that code for surface proteins that make APMV seem a lot like a bacteria to any potential amoeba host (1). This has a dual effect of stimulating the amoeba to consume the virus and also potentially affecting the way the macrophage interacts with the virus. The presence of bacterial chemicals on the wall of the virion may coincidentally make the macrophage more likely to engulf the cell, and thus make human infections more likely. This would be direct evidence that selective pressure on the virus is driving potential infection of humans.
Though human APMV infections are, for the moment rare, the chance of it becoming an emerging pathogen of humans deserves serious attention. Because of the selective pressure it faces it is a generalist in terms of the cells it will infect. Because it has such a general method of uptake there are likely multiple ways it could attack the human body which is a cause for concern, because a great many cells in our body use phagocytosis to attack intruders. Fortunately, the control of APMV infection does not seem to be difficult; in fact it is already in effect right now.
The best way that APMV can be controlled appears to be by simply keeping it amoebae hosts and people separate. Since many amoebae are potentially pathogenic to humans this kind of control is already in effect, which means that the low incidence of APMV seroconversion can likely be kept even lower with minimal effort. The selective pressures that shape the evolution of the Mimiviridae are likely present throughout the lineage, however, and care should be taken to remain vigilant against any others that might appear.
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