The purpose of these series of experiments is to determine how big of a role the TRIM5a protein plays in the reduction of replication in herpes simplex viruses, HSV. The researchers are investigating if the effect of TRIM5a varies between species and strains of HSV-1 and 2 viruses. The investigation will be done on Old World Monkeys, the rhesus macaque and African Green Monkey and the New World Monkey, the squirrel monkey. Human cell lines will also be used during the investigation. This experiment is important in determining how the replication cycle of HSV viruses can be inhibited, and if they can be, how researchers can use this information towards gaining more insight into similar viruses, such as HIV.
The first of many experiments performed during this investigation was to compare the difference in susceptibility of the HSV-1 and HSV-2 viruses to rhesus macaque monkeys and other primates. The rhesus macaque fibroblasts and HeLa cells were infected with the HSV-1 and HSV-2 viruses with a multiplicity of infection (MOI) of 3PFU/cell (plaque forming units). It was found that the rhesus macaque fibroblasts exhibited 100 times less infected cells than the HeLa cells. The researchers were able to conclude that rhesus macaque cells were far less susceptible to HSV viruses than other primate cells, enabling them to further investigate if TRIM5a was responsible for the reduction in infected cell counts.
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In order to determine if TRIM5a was the main cause for inhibiting the replication of HSV-1 and 2 in rhesus macaque cells, H-R cells were infected instead. H-R cells are HeLa cells that have been made to express TRIM5a protein from rhesus monkeys. H-R cells and the control H-L cells, which contain an empty vector, were infected with varying MOIs of both viruses and were all harvested at the same times. The results showed that at low MOIs, of both viruses, the H-R cells had lower infected cell counts than H-L cells. However, at higher MOIs around 30PFU/cell, there was not a big difference in infected cells between the H-R and H-L cells. Thus, TRIM5a does affect the replication of HSV-1 and 2 but it is not as effective at higher MOIs.
The next step in the investigation was to determine how other primate TRIM5a proteins effect replication of HSV-1 and 2. TRIM5a proteins from the African green monkey (H-AGM), squirrel monkey (H-Sq), rhesus monkey (H-R), and human (H-H) were tested. Each cell type was infected with an MOI of 3PFU/cell and harvested 24hpi (hours post infection). H-AGM and H-R cells showed similar results, having the lowest infected cells compared to H-sq and H-H cells. The H-sq cells showed little reduction in viral replication, and the H-H cells had results similar to the control H-L cells. This experiment showed that there was a big difference in the Old World Monkeys, H-AGM and H-R and the New World Monkey, H-Sq tested and the affects of each TRIM5a protein.
From previous experiments, it was thought that TRIM5a is most effective during the early stages of infection. In order to test that in the rhesus TRIM5a protein, HSV-1 and 2 infected H-L and H-R cells were analyzed using the Western blot technique. The genes being investigated were HSV IE (immediate early) proteins that are expressed during early stages of infection, which are involved in early transcription and translation (1). In the HSV-1 infected cells, all of the IE genes were at lower counts in H-R cells than H-L cells. The reduced counts in H-R cells were also seen in HSV-2 infected cells. Thus, during the early stages rhesus TRIM5a reduces synthesis of IE genes, reducing viral replication.
Up until this point, only the HSV-1 KOS strain and HSV-2 186 syn strain had been tested with the rhesus TRIM5a protein. HSV-2 SD90-3P, a low passage clinical isolate strain and HSV-2 G low passage laboratory strain was used for this experiment. A low passage strain is a cell line that may have little original viral content left, and a clinical isolate is a sample that had been taken out of a body recently. These strains were used to infect H-R and H-L cell lines. The results were in conjunction with the previous experiments, the H-R cells showed significantly lower viral replication compared to H-L cells. The strains of HSV-1 used were low passage laboratory strain F and HSV-1 strain 17 syn+. The process of infecting the H-L and H-R cells was the same as the HSV-2 strains. In HSV-1 strain 17 syn+, there was not as significant of an impact on viral replication as in HSV-1 strain F. There also wasn t as much of a difference in comparison to the HSV-2 infected cell numbers. These results showed that the effect of TRIM5a varied depending on the strain of the virus, as well.
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It was found that ICP0, infected cell protein 0, could play a role in the reduction of HSV replication in conjunction with TRIM5a. H-L, H-R, and H-H cell lines were infected with HSV-1 KOS strain and were stained for ICP0 and detected using immunofluorescence assay. . In the H-L and H-H cells, the number of cells containing ICP0 in the nucleus progressively got higher as time passed but not as much in the cytoplasm. In H-R cells, although there were high numbers of nuclear ICP0 present, as time went by there was more cytoplasmic ICP0 present than nuclear. When the all cells were stained to detect both ICP0 and TRIM5a, the H-R infected cells showed the TRIM5a and ICP0 particles close in proximity of one another and the opposite was observed in H-H cells. This could show that the TRIM5a reduces replication by containing the ICP0 proteins to the cytoplasm.
Now, investigators had to determine if ICP0 played a bigger role than that of TRIM5a. This was done by creating a mutant ICP0 virus and infecting the cell lines, to determine if it would cause equal reduction in replication. H-L and H-R cells were infected with ICP0 absent viruses and rescued mutant viruses. The results of the deleted protein were not statistically significant compared to the wild-type of ICP0 viruses in both H-R and H-L cell lines. It was concluded that ICP0 does not play a vital role in replication reduction of HSV.
In earlier experiments it was found that TRIM5a protein levels were high at early stages of infection, yet as time progressed, TRIM5a levels appeared to decrease. The levels of TRIM5a present at various times post infection were analyzed using Western blot of H-R and H-L cells infected with HSV-1 and 2. It was found that although the reduction in TRIM5a occurred at different rates for HSV-1 and 2, at 24 hpi, TRIM5a was not present in either infected cells. All of the cell lines used in this experiment, H-H, H-R, H-L, H-Sq, and H-AGM, were infected with HSV-2 to determine if the loss of TRIM5a coincided with the increased replication of the virus. It was found that as the IE genes turned on for viral protein synthesis, TRIM5a protein levels decreased. Therefore, as the hours post infection increase, the HSV virus replication cycle directly affects TRIM5a levels.
The purpose of these experiments was in order to determine how much of an impact TRIM5a protein makes on the replication of HSV viruses. This was important because of HSV virus similarity to the HIV virus and the possibilities for finding a way around the HIV virus and creating an AIDS vaccine. TRIM5a can inhibit HIV-1 in Old World Monkeys, which is why rhesus macaque monkeys are used in these studies because it is known that this mechanism works on these species. Research using HSV viruses are used because HIV vaccines are often built in HSV viruses.
I think that this data is important in the grand scheme of finding a way to create an HIV or AIDS vaccine. Although this specific set of data did not conclude anything groundbreaking in the search for a vaccine, I think that it further supported the information that is known. This investigation was also able to rule out factors that could have been thought to be relevant, such as the ICP0 protein. I think that more investigating could be done towards finding a way to possibly incorporate rhesus TRIM5a into other species gene expression or finding a way to delete the expression of some IE genes of the HSV virus. I think that another possible idea could be to find an antagonist for the twelve glycoproteins of HSV to prevent entry. However, I think this paper was very important in giving a more detailed description of how TRIM5a works in reducing the replication of the HSV virus, even if it mostly occurs in the early stages. I think any kind of information is better than not knowing where to start on this investigation.