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Viruses cause a variety of illness ranging from minor common colds to life-threatening acquired immunodeficiency syndrome (AIDS). Having become increasingly harder to treat, viral infections are an important topic of focus in the medical and, especially, research arena. Nonhuman primates such as rhesus macaques (Old World monkeys) are used as models for studying the mechanisms which take place in host viral replication (Reszka et al., 2009). The herpes simplex virus (HSV), which causes herpes, is capable of infecting a broad range of hosts; however, one species where infection of HSV has not lead to disease is in rhesus macaques. There are many factors which allow HSV to infect a wide range of hosts. One such factor is that HSV is able to enter into different types of cells within the host due to having twelve different surface glycoproteins on its envelope. Another factor is that beyond the glycoproteins located on its envelope, HSV also contains heparan glycosaminoglycans and three receptors for attachment and entry into host cells. One of the most important factors for the successful infection of HSV into many different hosts is its ability to continue replication of its nucleic acid sequence into the host's genome despite the presence of the host's intracellular blocks to replication. The rhesus macaques have, however, overcome these infectious methods of HSV. (Reszka et al., 2009).
To try to gain a better understanding of why rhesus macaques have resistance to certain viral infections and to, then, apply the gained knowledge to help better treat herpes and other viral infections in humans, the rhesus macaques' mechanisms to regulate viral infection have been closely studied. As with HSV, rhesus macaques have also gained avirulence to the human immunodeficiency virus type-1 (HIV-1) (Reszka et al., 2009). The reason for resistance of the HIV-1 is due to the tripartite motif 5 alpha (TRIM5α) which allows the host to detect and break apart the viral capsid. The part of the primary transcript of the TRIM5α gene which has the ability to constrain the viral infection is its largest exon containing the B.30.2 domain. Another protein, TRIM19/PML, is also found in rhesus macaques that serves the same function of restricting viral replication, specifically for the HSV type 1. This protein, along with TRIM5α, is stimulated by type 1 interferons; unlike TRIM5α, the TRIM19/PML protein interacts with other proteins to exert its effects. (Reszka et al., 2009).
Since TRIM19/PML hinders HSV-1 replication and TRIM5α hinders HIV-1 replication, both these proteins serve the same type of function (Reszka et al., 2009). From this concept, we hypothesize that TRIM5α will be able to hinder HSV-1 replication and other retroviral replication along with HIV-1 replication. Thus, the aim of this study is to determine whether TRIM5α is able to regulate and restrict viral replication of HSV. (Reszka et al., 2009).
HSV-1 and HSV-2 replication is reduced in rhesus macaque fibroblasts:
Rhesus macaque fibroblasts and HeLa cells were infected with HSV-1 and HSV-2 (Reszka et al., 2009). Cells were examined for the amount of viral progeny present in them at various times after the infection. The rhesus macaque fibroblast cells had fewer amount of viral progeny by a hundred times than the HeLa cells. (Reszka et al., 2009).
Rhesus TRIM5α protein restricts HSV infection:
HeLa cells that expressed the TRIM5α protein found in rhesus macaques, denoted as H-R cells, and HeLa cells not expressing the TRIM5α protein were infected with HSV-1 and HSV-2 (Reszka et al., 2009). Twenty-four hours after infection, the cells were examined for the amount of viral progeny using titration on Vero cells. It was found that H-R cells infected with HSV-1 had two times lower amount of viral progeny than HeLa cells without TRIM5α protein; H-R cells infected with HSV-2 had five times lower amount of viral progeny than HeLa cells without TRIM5α protein. When the cells were extracted at later time than this, it was found that the amount of viral progeny in both cell types were similar. (Reszka et al., 2009).
Effect of other primate TRIM5α molecules on HSV infection:
Cells were infected with HSV-2 (Reszka et al., 2009). All cells except HeLa expressed species-specific TRIM5α protein. Human cells and HeLa cells showed similar amounts of viral progeny. African green monkey cells had 3.6 times lower viral progeny than HeLa cells. Rhesus macaques cells showed 4.5 times less viral progeny than HeLa cells. Squirrel monkey cells showed a thirty-five percent decrease in viral progeny than HeLa cells. (Reszka et al., 2009).
Effects of rhesus TRIM5α protein on HSV-1 and HSV-2 protein synthesis:
Rhesus macaques and HeLa cells were infected with HSV-1 and HSV-2 (Reszka et al., 2009). Synthesized viral protein was examined on a Western blot. For the HSV-1 infection, the ICP4 protein was seen for both rhesus and HeLa cells at an early stage. At a later stage, ICP4 was lower for rhesus. The ICP8 and IEICP27 proteins were lower in rhesus cells at an early stage than late stage. For the HSV-2 infection, a greater amount of ICP8 and ICP27 were reduced than in HSV-1. (Reszka et al., 2009).
Effects of rhesus TRIM5α on different HSV strains:
Various species of the same stains of HSV-1 and HSV-2 and HSV were used to infect rhesus and HeLa cells (Reszka et al., 2009). Despite which species of a particular HSV strain was used, the TRIM5α protein had the same effected on lowering the viral progeny when compared to HeLa control cells. (Reszka et al., 2009).
Effect of TRIM5α on HSV ICP0 distribution:
Various cells were infected with HSV-1 (Reszka et al., 2009). The amount of ICPO protein was determined to increase in the cytoplasm. (Reszka et al., 2009).
Effect of TRIM5α on HSV-1 ICP0-mutant replication:
HeLa and rhesus cells were infected with mutated ICP0 and HSV-1 (Reszka et al., 2009). The viral progeny in rhesus were lower than in HeLa cells. (Reszka et al., 2009).
Levels of TRIM5α decrease in HSV-infected cells:
A western blot was performed on rhesus and HeLa cells infected with HSV-1 and HSV-2 (Reszka et al., 2009). Data showed HSV-2 infected cells show a smaller decrease in TRIM5α than HSV-1 infected cells after 24hpi of infection. Cells producing different types of TRIM5α proteins were infected with HSV-2. The TRIM5α loss of various types of this protein was detected at an even earlier stage that with just TRIM5α. (Reszka et al., 2009).
The amount of viral progeny in a cell directly correlates to the amount of viral replication which occurred (Reszka et al., 2009). The more a virus's DNA or RNA gets incorporated into the host's genome, the greater the expression for the production of viral progeny. Since the amount of viral progeny in the rhesus macaque for HSV-1 and HSV-2 was less than in HeLa cells, this suggest that the TRIM5α protein might have played a role in restricting viral replication. The HeLa cells are the control cells used throughout this experiment; they contain cells which do not express the TRIM5α protein. (Reszka et al., 2009).
In order to further examine whether or not TRIM5α does play a role in restriction, further experiments were performed. It was found that the TRIM5α protein can reduce HSV-1 and HSV-2 viral replication during a certain amount of time after infection (Reszka et al., 2009). When the cells with TRIM5α where extracted and examined, they contained significantly lower amount of viral progeny than HeLa cells without TRIM5α. But if the time of extraction of the cells exceeded thirty hours after infection, then the TRIM5α protein was not able to restrict viral replication and the amounts of viral progeny were the same. Also, the extent of effect a particular TRIM5α protein will have on an organism is dependent on the species it is. In humans, the TRIM5α protein does not have a significant effect on restricting viral replication of HSV-2; however, in rhesus and African green monkeys the effect is quite strong. (Reszka et al., 2009).
The TRIM5α protein can better restrict viral replication at an early stage of infection (Reszka et al., 2009). Rhesus macaques' TRIM5α protein is better able to restrict viral replication in HSV-2 than HSV-1. This is supported by Western blot examination which showed that a greater amount of viral protein is found in HSV-1 infection than HSV-2. Despite which species of an HSV-1 or HSV-2 or HSV strain are used to infect rhesus cells, the TRIM5α protein has the same effect in lowering the amount of viral progeny within the same strain. (Reszka et al., 2009).
The PML protein functions like TRIM5α in restricting HSV-1 replication (Reszka et al., 2009). The only difference is the PML protein acts to restrict viral protein production in the cytoplasm. The ICPO nuclear protein increased in the cytoplasm so that it would not function and, thus, restricted viral replication. Where or not ICP0 in the cytoplasm affects the restriction of viral replication was examined. Since, the data shows that when mutated ICP0 is added to the cytoplasm, the amount of viral progeny in rhesus and HeLa cells are the same as if a non-mutant ICP0 was present, it can be concluded that restriction of viral replication does not depend on ICP0. (Reszka et al., 2009).
Overall, it can be concluded that as infection time increases, TRIM5α protein's ability to restrict viral replication of HSV-1 and HSV-2 decreases and the amount of TRIM5α production also decreases (Reszka et al., 2009). The HSV infection causes TRIM5α protein synthesis to decrease. Also, the effect of the TRIM5α protein is dependent on a particular species. For example restriction of viral replication in humans is less than that in rhesus macaques. (Reszka et al., 2009).
Due to the limited number of effective antiviral drugs, viral infections are becoming harder to treat by physicians. Their rapid evolution allows them to acquire resistance to many drugs and, also, to host immune systems. Discovering new ways in which the host immune system may be able to suppress the effects of a virus, like that has been done in this experimental study, is important in the field of medicine. The TRIM5α protein in Old World monkeys (rhesus macaques) has an effective way of reducing and restricting the herpes simplex virus' viral replication into the host (Reszka et al., 2009). On a broader scope, this experiment is important because if we can study the mechanisms behind the TRIM5α protein in Old World monkeys, we might be able to successfully apply it to help the human race. (Reszka et al., 2009).
In my opinion, I believe where the authors of the journal article could improve upon based on their experimental approach is to clarify specifically how many samples were tested. For example, the author merely states that rhesus macaques cells were infected with HSV-1, but does not state how large of a sample was used and how many cells were examined. I think the authors of this article should have used a large enough population of cells when testing for the amount of viral progeny produced to gather greater data which will lead to less chances of random error. Another suggestion for the authors of the paper is to indicate what the abbreviations in their paper stands for such as when referring to units. The authors should further their investigation on their topic of restriction of viral replication on other strains of retroviruses beyond HSV and HIV. They could even extend their study of viral replication restriction on non-retroviruses.