Virology is basically the scientific study of viruses, their characteristics, and their modes of operation. In this scientific field, many virologists examine and study these organisms in order to establish ways to combat several actions, such as fighting diseases, finding cures, acquiring vaccines, etc. In this particular article, scientists are studying the effects of the disease known as herpes simplex virus (HSV), which has been known for decades for replicating uncontrollably in many cells. However, Old World monkeys, which are better models for studying HIV/AIDS research since they are hosts for simian immunodeficiency virus, have been found to possess TRIM5α proteins, which can block the replication of HIV and reduce infection in various forms as well. To ascertain how TRIM5α proteins work well to reduce infection, scientists utilized several parameters: HSV-1 and HSV-2 replication in rhesus macaque fibroblasts, effects of TRIM5α protein upon HSV infection, effect of other primate TRIM5α molecules on HSV infection, effects of rhesus TRIM5α protein on HSV-1 and HSV-2 protein synthesis, effects of rhesus TRIM5α on different HSV strains, effect of TRIM5α on HSV ICP0 distribution, effects of TRIM5α on HSV-1 ICP0-mutant replication, and levels of TRIM5α decrease in HSV-infected cells.
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In order to determine HSV growth in rhesus cells, scientists studied replication of HSV-1 and HSV-2 in rhesus monkey fibroblasts and compared this with the replication of HeLa cells. HeLa cells are primary, human cell lines used in virology that were derived from Henrietta Lacks in 1951 and have been used in many laboratories for research purposes (1). HeLa cells and rhesus cell lines were infected with HSV-1 and HSV-2 at an MOI of 3 PFU/cell. The results obtained from this trial indicated that HSV-1 and HSV-2 yields were reduced in comparison to HeLa cells. This information allowed scientists to investigate whether rhesus TRIM5α protein contributed to this effect.
In order to determine if TRIM5α contributed to the restriction of HSV-1 and HSV-2 in rhesus cells, scientists examined HSV infection of HeLa cells that can express TRIM5α protein, known as H-R cells. H-R cells and the control H-L cells were infected with HSV-1 or HSV-2 at MOI's ranging from 1-30 PFU/cell. By comparing the yields obtained from this trial, it was found that at lower MOI's rhesus monkey TRIM5α can reduce HSV replication, but at higher MOI's TRIM5α couldn't reduce HSV replication. This result allowed scientists to establish whether the source of TRIM5α had any effect on HSV infection.
In order to determine the effect of other primate TRIM5α molecules on HSV infection, four primate molecules were acquired to achieve this goal: African green monkey, squirrel monkey, rhesus monkey, and human. By comparing the yields obtained from this trial, it was shown that the TRIM5α protein of rhesus macaques showed the strongest inhibition of HSV infection.
Scientists wanted to determine the effect of rhesus TRIM5α protein on HSV-1 and HSV-2 synthesis since it was thought that the restrictive activity of TRIM5α on retroviruses occurred at an early stage of infection, and for that reason hypothesized the same effect for rhesus monkey TRIM5α. HSV IE viral protein synthesis would be decreased in rhesus monkey TRIM5α-expressing cell lines to demonstrate such an action. To test this hypothesis, H-L control cells and H-R cells expressing rhesus monkey TRIM5α were infected with HSV-1 and HSV-2 and assayed viral protein synthesis by Western blot analysis. The HSV-1 ICP4 protein was detectable at 4 hpi in both cell lines and peaked at 6 hpi, expression of the IE ICP27 protein showed a higher reduction in H-R cells than in H-L cells at 4 hpi, and expression of the early ICP8 protein in the H-R cell line showed a higher reduction in H-R cells than in H-L cells at 4 hp. This means that the reduction of HSV-1 replication in H-R cells is exhibited in IE gene expression at an earlier stage. However, HSV-2 infected cells showed a greater reduction of viral protein synthesis in H-R cells in comparison to HSV-1 cells. This allowed scientists to investigate reduction of various HSV strains.
In order to ascertain the effects of rhesus TRIM5α on different strains, H-R cells and the control H-L cells were infected with HSV-2 clinical isolate SD90-3P or the low passage stain HSV-2 G. Based upon the yields, H-R cells showed greater reduction of 186 syn+ and G as well as the HSV-2 clinical isolate. In the second experiment H-R cells and the control H-L cells were infected with the HSV-1 low passage strain F or laboratory strain 17 syn+. H-R cells showed a greater reduction in comparison to H-L control cells. This shows that both HSV-1 and HSV-2 strains were inhibited by TRIM5α and inhibition of HSV replication in TRIM5-expressing cells is viral strain-specific than species specific.
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It was revealed that ICP0, an HSV infected protein, can help reduce HSV-1 replication in the cytoplasm, and scientists wanted to determine the effect of TRIM5α on HSV ICP0 distribution. To do this ICP0 distribution was examined in HSV-1 KOS virus-infected H-L, H-R, and H-H cells. The yields suggest that rhesus TRIM5α increased the amounts of ICP0 in the cytoplasm. In addition when the cells were stained to detect ICP0 and TRIM5α, HSV-1 infected H-R cells showed that ICP0 was localized to express the structures near the TRIM5α-containing structures, whereas HSV-infected H-H cells showed little structure localization near TRIM5α-containing structures. This results that not only can TRIM5α prevent the nuclear functions of ICP0 in the cytoplasm, but can inhibit HSV replication. This suggested that a cytoplasmic retention of ICP0 contributes to the inhibition of HSV replication at earlier times of infection.
Scientists suggested that reduction would be dependent on ICP0. In order to determine the effect of TRIM5α on HSV-1 ICP0-mutant replication, H-L and H-R cells were infected with the ICP0 null and rescued viruses and measured at 24 hpi. The ICP0 null mutant virus had a lower yield in H-R cells in comparison to H-L cells, and the rescued virus also had a lower yield in H-R cells in comparison to H-L cells. In other words the lack of ICP0 had no effect of TRIM5α on HSV-1 ICP0-mutant replication.
In the final parameter, to determine if HSV infection affected TRIM5α levels, Western blot analysis was done on HSV-infected H-R and H-L cells. Yields exhibited that HSV-1 and HSV-2 infection resulted in decreased levels of TRIM5α. Next, to determine if the loss of TRIM5α was related to the restriction of HSV replication, cells with H-L, H-R, H-Sq, H-H, and H-AGM cells were infected with HSV-2 virus and grown in culture at various hpis. However, the yields suggest that TRIM5α levels based on restriction of HSV replication by the different TRIM5α proteins could not be ascertained by just the TRIM5α protein levels in the infected cells.
HSV is a disease that has been around for many decades. Just like HIV/AIDS, it can replicate in many cell types and can cause infections as well. This article demonstrated that simian TRIM5α proteins have several effects on HSV replication and infection. To further establish this concept, Old World monkeys were utilized due to the fact that they were better models for study and can possess a similar form of HIV known as simian immunodeficiency virus and established several parameters to establish their hypotheses. These experiments went hand in hand in several ways. The first experiment demonstrated that HSV-1 and HSV-2 yields were reduced in comparison to HeLa cells, and this information allowed scientists to investigate whether rhesus TRIM5α protein had any contribution to this effect; the second experiment demonstrated that at lower MOI's rhesus monkey TRIM5α can reduce HSV replication, but at higher MOI's TRIM5α couldn't reduce HSV replication. This in turn allowed scientists to establish whether the source of TRIM5α had any effect on HSV infection. By comparing the yields obtained, it was shown that the TRIM5α protein of rhesus macaques showed the strongest inhibition of HSV infection, and that TRIM5α from certain monkey species can reduce HSV replication. In the fourth experiment, reduction of HSV-1 replication in H-R cells is exhibited in IE gene expression at an earlier stage. However, HSV-2 infected cells showed a greater reduction of viral protein synthesis in H-R cells in comparison to HSV-1 cells. This allowed scientists to investigate reduction of various HSV strains. The fifth experiment demonstrated that both HSV-1 and HSV-2 strains were inhibited by TRIM5α and inhibition of HSV replication in TRIM5-expressing cells is viral strain-specific than species specific. The sixth experiment was done to determine the effect to TRIM5α on HSV ICP0 distribution. The yields suggested that rhesus TRIM5α increased the amounts of ICP0 in the cytoplasm, prevented the nuclear functions of ICP0 in the cytoplasm, and inhibited HSV replication. The seventh experiment, however, was basically a red herring. It was performed in order to determine the effect of TRIM5α on HSV-1 ICP0-mutant replication, but results showed that the lack of ICP0 had no effect of TRIM5α on HSV-1 ICP0-mutant replication. The last experiment exhibited that HSV-1 and HSV-2 infection resulted in decreased levels of TRIM5α.
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I believe that the whole relevance of this paper was to offer an approach of combating a disease by undergoing numerous methods. This paper has opened my eyes to the hectic world that scientists face in attempting to solve and to create new techniques to fight and to rid humankind of deadly diseases. The experiments were necessary to perceive whether the desired results, such as reduction in viral replication, could be achieved and perhaps offer some hope for fighting or mitigating these diseases; they acted as a link to each other with one result leading to a hypothesis, and that hypothesis leading to a result, and etc. In addition, I believe that this study was done to serve as a model for discovering a way to combat the deadly disease HIV/AIDS. This disease has been around for decades with no cure, but only treatments. Perhaps this study can offer some hope as to mitigate the incidence rates of HIV. Since simian TRIM5α proteins can help reduce replication and infection in Old World monkeys, I feel that this same methodology should be attempted for human cells. Overall, I found the article to be very informative and scintillating. I was very hopeful after reading this article that someday there will finally be a cure for HIV/AIDS.