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It has been found that certain types of primates have the proteins that are able to inhibit the replication of various viruses. It is not clearly understood which proteins are the cause for the inhibition or why the inhibition varies in different species. In an attempt to discover further understanding inner workings of the protein viral interactions, an experiment was conducted using Herpes simplex virus, HSV, and TRIM5α proteins. The herpes viruses are capable of surpassing many different types of host cell defenses that are meant to hinder viral replication which allows them to infect a large variety of host. Although it has the ability to infect several species, it does not always cause disease, such as in the rhesus macaques. In other cases that were studied using HIV and primates, it was found that TRIM5α played an important role in providing Old World monkeys a sort of immunity to the virus. There are other TRIM proteins that play different roles in normal development of cells, like TRIM19, that could also be a factor in the inhibition of the viral infection. So with all of the data taken into consideration, the experiment was carried out to test how the virus infected different species of primates and what it was responsible for the various responses that the virus had within each of the species. Further experiments were conducted in order to see exactly which proteins were causing the inhibition within the cells and what role they played.
The first experiment that was conducted tested the amount of viral replication in human cells, HeLa cells, versus cells from the rhesus macaques, fibroblasts. The reason that this test was conducted was because it was believed that HSV had a lower rate of susceptibility of infection in rhesus macaques,RM, but there was no definitive cause as to why this was true. The cells were infected with HSV-1 and HSV-2 at an MOI of 3PFU/cell and they were collected at several different times after they were infected with the viruses. The test confirmed the initial hypothesis that the virus would grow better in the human cells than the RM fibroblasts. Because the virus did not grow as well in the RM cells, that led to another test to determine whether TRIM5α played any role in the rhesus macaques lower levels of viral replication.
The experiment was conducted to see how TRIM5α responded to different levels of the virus when infecting HeLa cells that had TRIM5α from the rhesus monkeys and a control cell, H-L cells that did not have the RM proteins. The cells were infected with different amounts of the virus; MOI's ranged from 1-30PFU/cell. The cells were harvested and the levels of HSV that were present in each of the cells were determined by doing a titration on Vero cells. In comparison to the control cells, the cells that had been infected with the lower MOI's had lower levels of replication whereas the cells with higher MOI's were about the same. These results showed that at low MOI's TRIM5α can reduce the levels of HSV replication, but it cannot compensate for high MOI's. It also appears that TRIM5α could possibly be the reason for the inhibition.
Because TRIM5α showed some inhibitory effects on the HeLa rhesus macaque cells, it was necessary to see what results from testing other types of cells as well. HeLa cells were with TRIM5α from several species were infected with HSV-2; African green monkey, squirrel monkey, rhesus monkey, and human, and a control H-L cell. The African green monkey and rhesus monkey had a significant decrease in replication. While the squirrel monkey still showed a reduced amount, it was not as great as in the other monkeys. The human cells showed similar results to that of the control cells. The experiment showed that the Old World Monkeys had a higher reduction rate with HSV-2 than with other species showing that its effects are species specific.
A western blot was done in order to assess whether or not TRIM5α causes inhibition during the early stages, which is what was hypothesized. H-L (control) and H-R (rhesus) cells were infected with HSV-1 and HSV-2. The HSV-1 cells showed reduction in replication at the immediate early stages of gene expression. HSV-2 cells showed reduction at both the early and immediate early stages. This showed that the protein blocks at the early stages of gene expression which is why the viruses are unable to replicate.
TRIM5α does have effects on HSV but the effects on the isolates of these viruses were unknown. Therefore the HeLa cells, H-L and H-R, were combined with isolates from HSV-2. They also showed a reduction in the amount of viral replication. When using the clinical isolates of HSV-1 there was not a notable decrease. This experiment showed that TRIM5α is not species specific but it more likely to be strain specific.
Although the effects of TRIM5α are becoming clearer, it was still no understanding of where it was located within the cell when dealing with the HSV infected cell protein (ICP0). It was hypothesized that it was located in the cytoplasm. H-L, H-R, and H-H cells were infected. The results showed that in the control cells and the H-H cells, the ICP0 was located in the nucleus but it was in the cytoplasm in the H-R cells. It seemed as if the rhesus TRIM5α increased the amount of ICP0 that accumulated in the cytoplasm.
The previous experiment led to that assumption that the reason TRIM5α is able to inhibit is because of the withholding in the cytoplasm. An immunoflourescence was done to observe that amounts of TRIM5α that came about after the cells were infected. Another western blot as well as an SDS-PAGE was conducted in order to gain further knowledge. The rhesus monkey TRIM5α actually prevents the ICP0 from entering into the nucleus, thus enabling it to do viral replication. But, there is no considerable affect of the absence of ICP0 on the restriction of HSV-1. It would be assumed that ICP0 is not responsible for HSV inhibition. In the H-R cells, the rate of reduction occurred more quickly in HSV-1 than HSV-2. The virus also seems to inhibit the TRIM5α. When there are higher levels of TRIM5α the virus seems to have found a way to decrease the amount of the protein in order for it to be able to replicate. This could be due to an evolutionary mutation. If the levels of the protein are not threatening to the virus, the virus will not try to get rid of it.
Although there was a lot that was learned about the effects that TRIM5α has on the virus, it could not be concluded that that protein was indeed the cause of the viral inhibition in the cell. It showed that there is a lot to be learned and taken into consideration when trying to find ways to combat viruses. The mechanisms that are necessary to inhibit viral function can be extremely complex and even when one is found the virus could possibly find a way to combat that as it did with TRIM5α. Although the cause of the inhibition is unknown, it did show that the protein played some role in the inhibition, at least in certain species or strains of the virus. But, even in the species that had proven to be inhibited, the virus still found a way to fight back. The viruses have the keen ability to be able to adapt overtime to any new changes that may post a threat to their overall livelihood. The data was very consistent and each experiment had a logical reason as to why they did it. The progression was logical and went along with how the proteins were interacting with the virus. Next, more experiments need to conducted to see exactly what TRIM5α does and exactly what it is in the rhesus macaques that makes it behave that way.