Simian TRIM5-alpha. Cell line used is HELA cells that siminan and human orthologs of TRIM5alpha. Paper describes how TRIM5alpha seems to restrict HSV viral replication and that it is strain-specific. Acts at early stages of infection and acts on the IE genes. At later stages of infection, the levels of TRIM5 alpha are lower. Therefore, that inhibition is not occurring at a higher rate because the levels of TRIM5 are lower at this point. (?) However, HSV does "fight back" and will reduce TRIM5 levels at later parts of replication.
HSV: family Herpesviradiae? Linear, dsDNA and envelope. Able to replicate in numerous cell types.
HSV vectors in hopes of HIV vaccines. Live attenuated viruses used from HSV and with HIV, in hopes to express HIV.
Experiment: infected 7 rhesus with the most virulent HSV 239 (HSV-1). In the future, want to optimize that
TRIM5alpha responsible for halting HSV replication. When taken into context for aims of a HIV vaccine vectors done in monkeys, all this work done in monkey cells is being halted by TRIM5alpha.
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Concept of specifity: one of the most important concepts?!
old world monkeys such as the rhesus macaques are immune to HIV because of the TRIM5-alpha ability to halt the infection. In humans, it doesn't happen as well.
the researchers used HeLa cells that are an immortal cell line. They infected the cells to express the TRIM5alpha from four different simian species and used a control. The monkeys were: ______________. The virus used the wildtype: HSV-1 and HSV-2
In the initial experiment of the paper, the researcher aimed to look for what was the basis for the lowest susceptibility of the rhesus monkey during HSV infection and is it due to the replication ability of the virus?!?! By infecting two cells lines of rhesus fibroblasts and HeLa cells with HSV-1 and HSV-2 separately, the researchers observed that the yield of viruses produced was significantly lower in the rhesus macaque fibroblasts than the HeLa cells. How well is the virus replicating in human cells vs. monkey cells- Sees that the virus does worse in the rhesus monkey cells. Therefore, the researchers were able to progress and determine if this reduction in HSV replication was due to TRIM5alpha.
Using two HeLa cell lines: one that expresses the TRIM5alpha and one that does not with the two varying HSV viruses. Infected at different multiplicities of infection (MOI). At lower MOIs, HSV replication reduced. However at higher MOIs, the HSV viral replication was not reduced. This could be due to the cell being overwhelmed with the virus; TRIM5 is overwhelmed by the presence of more HSV viruses.
Comparing different monkey TRIM5alpha strains. Possibly think that all TRIM5alphas are the same and act the same way, when in fact, it acts differently. Some monkeys inhibit HSV replication better than others. Their results show that African Green monkeys and rhesus macaque TRIM5alpha inhibit the best and human inhibit the worst. Squirrel monkey kindof intermediate. Surprisingly, African green monkeys and rhesus macaques are both old world monkeys from Africa or Asia. Squirrel world monkeys are new world monkeys from South America. With the monkey species ranging from different parts of the world, the monkeys are likely to have developed evolutionarily different. Therefore, evolutionary distance could have changed TRIM5alpha effect of inhibiting HSV replication.
Through analyzing the TRIM5alpha protein on western blot analysis, the amount of HSV viral protein was observed in HeLa cells expressing the rhesus macaque TRIM5alpha and HeLa control cells. Since TRIM5alpha acts on immediate early genes, the expression of an immediate early gene was observed and showed that there was less viral IE genes in a cell infected with HSV. TRIM5alpha is doing it at the level of IE expression. However, how the protein was doing this is unknown.
The researchers also looked at the effect of rhesus macaque TRIM5 alpha on different HSV strains by using even a clinical isolate virus from a human. From these results, the researchers observed that even different strains of HSV respond differently to TRIM5alpha.
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Therefore, there was a need to understand the link between the virus and the protein. TRIM5alpha was observed to be decreasing as replication continued, which could signify that the virus begins to fight back as replication progresses. The amount of TRIM5alpha protein levels was observed on Western Blot analysis in the differing HeLa cells expressing the different monkey species and human TRIM5alpha that were infected with HSV. They observed that as the hours post infection progressed, the strength in the band representing the amount of protein on the agarose gel would diminish slightly each time. In addition, in all the monkey species and the human sample, all the TRIM proteins are disappearing at the same rate. However, the rate at which this virus is decreasing doesn't give a direct correlation with how well the virus is doing. If TRIM5alpha went away faster, then the virus would do better; however, the virus isn't demonstrating this effect. Therefore, they concluded that there must be something else going on affecting.
However, they still do not understand the interaction between the protein and the virus. Further experimentation would be needed because although there was a loss of the TRIM5alpha protein and there was a reduction in the HSV replication, it does not prove that these two related to one another. There could be some other metabolic process that is using up the TRIM5alpha protein, rather than the virus directly affecting the protein. These experiments do show that the protein and the virus are related to one another. However,
The researchers also found negative results by exploring a known protein, HSV infected cell protein 0 (ICP0) normally found within the nucleus. ICP0 was discovered in the cytoplasm when TRIM5alpha was present in the cell, which was thought to explain why the virus was not doing so well when interacting with TRIM5alpha. Also, the researchers observed the human TRIM5alpha interacting with ICP0 and found that human TRIM5alpha cannot seem to keep ICP0 in the cytoplasm, which could explain why human TRIM5alpha does not do well in inhibiting HSV infection. They discovered that rhesus macaque TRIM5alpha almost kidnaps HSV ICPO in the cytoplasm, but they found that this is not the mechanism by which makes TRIM5alpha successful in inhibiting HSV replication. As the researchers continued to investigate ICPO, they take two viruses: HSV-1 and HSV-1 that is mutated not to have the ICP0 gene. Thus, if TRIM5alpha is known to act on ICP0, then it should not function if the gene is removed. However, they discovered that TRIM5alpha still functions with or without the gene on HSV viral replication. Thus, ICP0 did not lead the researchers to finding the direct link between HSV and TRIM5alpha.
With this in mind, the researchers wanted
The monkeys innate immune system is attacking the HSV. So, if you are using HSV for a vaccine, advise to use a different virus model vector if monkeys are being used for HIV vaccine research.