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Herpes simplex virus has been known to plague the universe for many years. The two main strains that will be discussed are Herpes simplex virus 1, HSV 1, and Herpes simplex virus 2, HSV2. HSV is an enveloped, DNA virus which has the ability to replicate in various cells and species. HSV encompasses many characteristics mainly to inhibit intracellular blocks to its replication. The experimental subject used in this study is Rhesus Macaques an old world monkey that is resistant to the HSV infection. This phenomenon is unknown, but what is known is that TRIM5 alpha is present in this species of monkeys. TRIM5 alpha is a protein known to inhibit the replication of Human Immunodeficiency Virus and other retroviruses at some stage in the cell's life cycle. TRIM5 alpha protein can be retrieved from the cytoplasm or nucleus of a cell. Due to the TRIM5 proteins ability to interfere with replication the question arises: can this same protein influence HSV replication?
A series of tests were performed based on the replication process of different species, levels of restriction, the effects of protein synthesis and so forth. Each test revolved around the presence or absence of the TRIM5 alpha protein. To begin the array of experiments the first test focused on how and if the replication of the virus differed in Macaques fibroblast cells and HeLa cells. Each cell was infected with HSV-1 and HSV-2 and monitored over a period of time. Results stated that a lower amount of viral content was produced in the Macaques fibroblast cells than in the HeLa cells. Due to the outcome further study arose questioning whether TRIM5 alpha was responsible for the lower levels of viral content in the Macaques cells. The experiment that followed was one that examined whether TRIM5 alpha restricts different HSV infections. HeLa cells expressing rhesus monkey TRIM5 alpha protein and a control cell culture where the main culprits of the experiment. What they found was that the Rhesus protein when infected at lower MOI s yielded lower production of viral content for both HSV 1 and 2 in contrast to the control that contained no protein. In addition to the results, they also saw that at a higher MOI (30) the viral content in both the HeLa cells and Macaques where nearly equal. Only to conclude that TRIM5 alpha is most effective and will decrease HSV 1 and 2 at lower MOI levels.
A new test formulated was one that targeted whether TRIM5 protein was species specific. They gathered molecules from two old world monkeys ( African green monkey and Rhesus Macaques Monkey), a Squirrel Monkey, a human and created a control. Each culture was infected with HSV2 and the results are as followed the human cells and control cell displayed the same viral amount, followed by a great reduction in the African green monkey and the rhesus monkey compared to the control and are said to be the better inhibitors of HSV replication. Lastly the Squirrel monkey showed the least decline in the infection of HeLa cells expressing TRIM5 alpha.
A technique known as a western blot was carried out in order to test how the TRIM5 alpha protein affects the protein synthesis of HSV at an early stage. When using such a technique one will look for thick band patterns which indicate a higher presence or accumulation of the sample. The culprits used were Rhesus Macaques and a control. By doing so the authors hoped to show that HSV IE viral protein synthesis would be decreased in rhesus monkey TRIM alpha. The results showed that although both the control cells and the rhesus monkey cells were infected with HSV, the control was able to display thicker bands meaning more ICP4, ICP8, and ICP27 proteins were produced and synthesized than the rhesus monkey over time. The Rhesus Macaques TRIM5 alpha was successful in inhibiting protein synthesis.
Another experiment performed is one that tests how the rhesus Macaques affects each HSV1 and HSV 2 strains. Two variants of HSV1 and HSV2 samples were tested and it was concluded that the TRIM 5 did inhibit both strains, but all in all is strain specific. The conclusion was also drawn upon the visual evidence that the plaque forming units displayed, the TRIM inhibited the HSV 2 more than HSV1.
The experiment that followed tested the effects of TRIM5 alpha on HSV ICP0. Cytoplasmic PML found in the cytoplasm acts on ICP0 (Infected cell protein 0) and is proven to act as a HSV1 replication reducer. The authors believed that TRIM5 could also mimic the PMLs function because it is also found in the cytoplasm and is a replication reducing protein. Trim19/PML which was mentioned early in the article keeps ICP0 in the cytoplasm which lowers the replication. Herpes viruses need to enter the nucleus to replicate because they are DNA viruses. So since TRIM5 can function like trim 19 and has the same structure this would be a similar model to test on HSV 1&2. First they observed the ICP0 activity in both the cytoplasm and nucleus, followed by monitoring the activity of the TRIM5. They used immunoflorescense to see if the TRIM5 and ICP0 where clustered in the cytoplasm. What they saw was ICP0 was localized near the Trim 5 but in humans there was less ICP0 near the TRIM5. This is what the authors looked forward to happening, and then they tested to see if it was the actual TRIM5 acting on the ICP0, which was proven. This in turn showed that TRIM 5 was able to prevent the ICP0 nuclear functions and inhibit replication of HSV. A concluding experiment was based on how trim 5 acts on ICP0 null mutant virus. What they found was ICP0 null mutant virus yielded lower viral content meaning the presence of ICP0 had nothing to do with the inhibition of viral replication.
The last experiment that was performed was to test the levels of TRIM5 alpha in HSV infections within 24 hours. As carried out in a previous experiment the Western Blot technique was used to differentiate the Macaques cells, HeLa cells, and the rest of the cultures after a viral injection. They tested each culture (African green monkey, Squirrel monkey, human, rhesus monkey and control), and saw that each subject's TRIM5 protein went undetected each at the same times. They noticed that early proteins present were different; meaning the inhibition of replication differs for each subject and is not determined by the amount of TRIM5. It led them to question whether the loss of TRIM5 protein is related to the inhibition of the replication of HSV.
In conclusion the TRIM5 alpha protein from Old world Monkeys, which is known to inhibit the replication of many retroviruses definitely influences HSV replication. Each experiment yielded different aspects of how the TRIM5 alpha protein affects different species, different strains, and overall replication process of Herpes Simplex Virus 1 & 2. What was concluded is that the TRIM5 alpha protein is most active in the early stages of viral infection; once an increased onset of viral content is present the TRIM5 becomes null. TRIM5 was also found to be successful in prohibiting synthesis of IE proteins.
The article was a bit drab and uninteresting. The illustrations actually clarified most of the text and made it easier to interpret. I do however believe that through this research scientist have gained a better knowledge on the TRIM5 protein and how it inhibits HSV. I believe that from here scientist can further explore HSV and HSV when it comes to humans.