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Vaccine researchers for the retrovirus human immunodeficiency virus (HIV) construct hybrid viruses such as the herpes simplex virus (HSV) in combination with human immunodeficiency virus (HIV) genes. This HSV-HIV vector is injected into a monkey species such as rhesus macaques since all vaccines are tested on animals before they are tested on people. The goal of the vector is that HSV will replicate in the rhesus macaque and cause a number of the HIV proteins to be expressed. This will in turn activate the monkey's immune system to make antibodies to the HIV proteins. Thus, the monkey will be hopefully protected when it is infected with an HIV strain. The researchers from the Department of Microbiology and Molecular Genetics and the Department of Cancer Immunology and AIDS at Harvard Medical School explain in Simian TRIM5α proteins reduce replication of herpes simplex virus that rhesus macaques possess a protein known as TRIM5α that naturally counters HSV infection. TRIM5α is commonly found in human and simian genomes and has been identified to restrict retroviral replication by disabling earlier viral genes. Given that monkeys have an innate ability against HSV vectors, the researchers suggest that the prospect of using HSV-HIV vectors in monkeys is not useful for HIV vaccination studies. The Harvard School of Medicine researchers thus explore the relationship and mechanism between TRIM5α and HSV viral replication to explain why HIV vaccines are not working and the use of HSV as a vector, specifically in monkey models, can be reconsidered for further investigation.
TRIM5α restricts HSV replication in rhesus macaque fibroblasts and at varying MOIs
Herpes simplex virus is an enveloped and double stranded DNA virus that is able to replicate in numerous cell types. Considering TRIM5α being responsible for inhibiting HSV replication, the researchers explored what the basis for the rhesus macaques' low susceptibility during an HSV infection was and if it is due to the replication ability of the virus. They compared HSV replication with two HSV strains in HeLa cells and rhesus macaque fibroblast cells expressing TRIM5α. As a result, the yield of viruses produced was significantly lower in the rhesus macaque fibroblasts than in the HeLa cells [Fig. 1]. Therefore, the researchers were able to progress and investigate if this reduction in HSV replication was directly due to TRIM5α. Interestingly, the researchers found that the functionality of TRIM5α interacting with HSV differed. Two HeLa cell lines- one that expresses TRIM5α and one that does not infected with two varying HSV viruses were infected at different multiplicities of infection (MOI). At lower MOIs, HSV replication was decreased; therefore, TRIM5α halted HSV replication. However at higher MOIs, the HSV viral replication was not reduced [Fig. 2]. This could be due to the cell and TRIM5α being overwhelmed by the presence of more HSV viruses.
Assorted primate TRIM5α influence HSV replication
Hence the rhesus macaque TRIM5α disturbing HSV replication by an unknown mechanism, the researchers compared TRIM5α proteins from different monkey strains consisting of African green monkey, squirrel monkey, and even a human TRIM5α sample. The notion that potentially all TRIM5α proteins performed the same way was contradicted when some monkeys inhibited HSV replication better than others. Their results show that African Green monkeys and rhesus macaque TRIM5α inhibit the best, squirrel monkeys were intermediate, and human TRIM5α was the worst [Fig. 3]. Surprisingly, African green monkeys and rhesus macaques are both old world monkeys from Africa or Asia and squirrel monkeys are new world monkeys from South America. With the monkey species ranging from different areas of the world, the monkeys are likely to have developed evolutionarily different. Therefore, evolutionary distance could have altered TRIM5α's effect of hindering HSV replication.
The TRIM5alpha protein was then examined by western blot analysis. The researchers analyzed the amount of HSV viral protein that was observed in HeLa cells expressing the rhesus macaque TRIM5alpha and HeLa control cells. Since TRIM5alpha acts on immediate early genes, the expression of a HSV immediate early gene proteins was observed. These proteins showed that there was less viral replication in a cell infected expressing TRIM5α and was doing it primarily at the level of IE gene expression. However, how the protein was doing this was still 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. Therefore, there was a need to further understand the link between the virus and the protein.
TRIM5α 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 HSV infected HeLa cells expressing the different monkey species and human TRIM5α. 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 does not 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 was not expressing this effect. Therefore, they concluded that there must be something else going on affecting.
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 TRIM5α was present in the cell, which was thought to explain why the virus was not doing so well when interacting with TRIM5α. The researchers observed the human TRIM5α interacting with ICP0 using immunofluorescence and found that human TRIM5α cannot seem to keep ICP0 in the cytoplasm, which could explain why human TRIM5α does not do well in inhibiting HSV infection. They discovered that rhesus macaque TRIM5α almost kidnaps HSV ICPO in the cytoplasm, but they found that this is not the mechanism by which makes TRIM5α successful in inhibiting HSV replication. As the researchers continued to investigate ICPO, they took 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 TRIM5α.
Although the researchers discovered that rhesus TRIM5α was species and strain specific, the mechanism on how the protein and the virus interact was not fully understood. Further experimentation would be needed because while there was a loss of the TRIM5alpha protein and a reduction in the HSV replication, it does not prove that these two are 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. When taken into context for aims of a HIV vaccine vectors done in monkeys, all this work done in monkey cells that are being halted by TRIM5alpha should be reconsidered. If HSV vectors are used, the mechanism of TRIM5α and HSV needs to be further investigated. Otherwise since the monkeys' innate immune system is attacking the Herpes Simplex virus, a different virus model vector should be used if monkeys are the model used for HIV vaccine research. However, the results did give insight on the potential to revise HIV vaccine models. Research always gives the impression to move forward on positive results and thus fails to reevaluate the negative results. However, this article shows the importance of understanding why negative results can give significant implications to why a result may not have worked. If the researchers would have never explored why simian HIV vaccines were not working because of an innate ability of their experimental model, then the potential of HSV vectors could have been lost.
Bad because lead us to the conclusion that the vaccine does not work, when in fact, the vaccine may work in humans and not monkeys. They are trying to show that monkeys have this natural ability. Important because: 1) if monkeys can do that, then maybe using HSV vectors in monkeys is a bad idea, 2) if monkeys have a natural ability to shut down HSV, then maybe they can figure out how and figure out for humans for HSV infection. The article makes the argument to try different vectors other than HSV, which could explain why HIV vaccines are not working.
Vero cellsà plaques assays- figure out how many
IMPORTANT: TRIM5 works against HSV in the first place.
Strain-specific vs. species-specific- interesting phenomena
Fig. 3- shows species specific. All TRIMs acting different.
Fig. 5- show strain specific. Acting on different strains of virus.
Therefore, TRIM is species and strain specific.
Explain negative results- ICP0. 3 figures on it.
Fig. 8- see that the virus is trying to fight back. (interesting aspect!)
Fig. 9- see TRIM disappearing over time. Shows that there isn't a direct relationship with the protein and the virus, but there could be something else affecting the relationship. This is where further research is needed.
Acts on IE genes.