The Journal article entitled "Simian TRIM5Î± proteins reduce replication of herpes simplex virus" investigated further into the viral resistant properties of TRIM5Î±, a protein known to block replication of retroviruses mainly HIV. The researchers that created this paper had several questions they wanted answered some of which were spurred on as a result of the outcomes of other experiments. First researchers thought they must confirm what is already known about HSV-1 and HSV-2 viral replication is in Rhesus macaques, which is that they exhibit a reduce rate of viral replication. Secondly they had to make sure that these phenomena can be recreated using a laboratory cell model.
Once researcher s had insured they could perform their line of investigation they performed experiments that prodded at the root causes of the viral resistance. Since HSV replication is reduced in Rhesus Macaque Fibroblast 100 times lower than that of permissive HeLa cells; does TRIM5Î± contribute to this significant reduction? If resistance provided by TRIM5Î± varies among different retro viruses and is also dependant on the species origin of TRIM5Î±: Do these same phenomena affect the HSV replication in infected cells?
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Many virologists believe the theory that the mechanisms employed by TRIM5Î± act on immediate early genes of retroviruses such as HIV in order to restrict their activity. If this is the case, is it also possible that the reductive power of TRIM5Î± on HSV replication is due to a mechanism that acts on the immediate early genes of HSV as well? Does the TRIM5Î± from the Rhesus macaque effects vary with different strain of HSV-1 and HSV-2?
From previous research efforts it was determined that viral protein ICP0 (Infected cell protein) encoded by HSV was bound by PML in the cytoplasm and kept in abeyance. Is it possible that TRIM5Î±'s effectiveness in reducing viral replication is in part also due to binding and suspending the activity of ICP0? If ICP0 is held in cytoplasm by TRIM5Î± like PML the reduction in viral replication would be a result of this process; if this is the case then removing ICP0 expression an HSV strain should knock out its ability to cause infection?
Analysis of TRIM5Î± through immunoflouresence studies is shown to decrease over time. How does this decline in TRIM5Î± occur and why?
In the first experiment encountered in this Journal article researchers thought to explore the reduction in viral replication of HSV-1 and HSV-2, in Rhesus macaques. To do this Fibroblasts of Rhesus Macaques and HeLa cells were infected with HSV-1 KOS and HSV-2 186 at various time intervals post infection, Vero cell plaque assays were performed to quantify viral load. The Fibroblast and HeLa cells were inoculated at 3PFU/cell and reached a maximal viral load 40hpi however, the Fibroblast of the Rhesus Macaque showed a 100 fold decrease in the level of viral replication in comparison to the HeLa cells.
HeLa cells were induced using a pLPCX vector to constitutively express TRIM5Î± of Rhesus Macaques and run against a control that contained an empty pLPCX vector (that is not induced to express Rhesus TRIM5Î±). The H-R cells induced to express TRIM5Î± of Rhesus macaques and the H-L cells containing the empty pLPCX vector were then infected at MOIs that ranged from 1-30PFU/cell and harvested after 24hpi. The yield of HSV-1 and 2 from both cell lines were determined through titration on Vero cell plaque assay which produced significant reduction in H-R cells for MOIs that ranged from 1-3PFU/cell when compared with H-L control; as MOIs increased beyond 3PFUs/Cell the resistance of TRIM5Î± was overcome. In addition to the ability of H-R cells to reduce HSV-1 and HSV-2 replication it was also observed that TRIM5Î± showed varying levels of resistance dependant on the strain with which it was inoculated. HSV-1 KOS produced a lower reduction than HSV-2 186 which produced a HSV reduction 5 times more than that of the control, where as HSV-1 KOS replication only expressed a 2-fold reduction. From these results researchers concluded that Rhesus TRIM5Î± is only able to reduce HSV replications at MOIs lower than 3PFU/cell and furthermore TRIM5Î± resistance varies with the viral strain in which it is inoculated with.
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Researchers then sought to identify how TRIM5Î± from other species affects HSV replication 24hpi with an MOI of 3PFU/cell. In order to perform this task pLPCX vectors were used to induce HeLa cells to express TRIM5Î± of the african green monkey, rhesus macaques, squirrel monkey and Humans, the induced cells where then run against H-L control (empty pLPCX vector). The cells that stably expressed the different forms of TRIM 5Î± were inoculated with HSV-2 186 and plaque assayed using Vero cells. The plaque assay showed that TRIM5Î± from old world monkeys reduced viral replication most significantly for H-AGM cells (HeLa cells expressing african green monkey TRIM5Î±) and H-R cells which exhibited a 3.6 and 4.5 fold reduction in viral replication 24hpi. The H-Sq cells expressing TRIM5Î± of squirrel monkey only produced a 35% reduction in the level viral replication when compared to HeLa cells expressing Human TRIM5Î± (H-H) or H-L cells. H-H and H-L cells exhibited no statistically significant difference in the level at which they reduced viral replication. From this experiment researcher found that Old world monkeys are the most competent at reducing viral replication.
Once researchers established that TRIM5Î± produced HSV resistance, they sought to determine if this resistance acted on IE genes as is currently theorized of TRIM5Î±'s role in HIV resistance. To test this hypothesis H-R and H-L cells were inoculated with HSV-1 KOS and HSV-2 186, and analyzed at 4hpi, 6hpi, 8hpi and an additional 10hpi for HSV-2 186. In order to determine TRIM5Î±'s effect on IE genes a western blot with antibodies specific for HSV viral proteins were used to detect relative abundance of proteins encoded by immediate early genes. The Western blot for HSV-1 tested for three immediate early genes HSV IE ICP4, HSV IE ICP27 and HSV IE ICP8, all of which exhibited a significant decrease between 2 to 4 fold that of H-L cells within 4-6hpi. The Western blot assay for HSV-2 analyzed only the HSV IE ICP8 and HSV IE ICP27 which exhibited a stronger decrease which ranged from 5 to 7 times that of H-L cells within 4-10hpi. Researchers concluded from this data that TRIM 5Î± resistance mechanism acts on immediate early genes expression.
The effects of TRIM5Î± of Rhesus macaques on Immediate Early genes and HSV replication is evident but are these effects an occurrence isolated to the HSV strains tested in this experiment. In order to determine if the effects of TRIM5Î± can be applied to more strains affecting a species H-L & H-R cells were inoculated with HSV-2 SD90-3P a clinical isolate and laboratory strains HSV-2 G both of which are weakly transmitted. The other strains were run along with the existing laboratory strain HSV-2 186 and H-L strains as a control. The HSV-2 SD90-3P clinical isolate and laboratory strains HSV-2 G showed a 2.5 and 4 fold reduction respectively however, the HSV-186 syn+ strain examined in previous experiments expressed a 12 fold reduction in viral replication. The same was examined using HSV-1 laboratory strains F and 17syn+, which when compared against control expressed a 3.6 fold reduction for F and no reduction for 17syn+. From this data the researchers concluded that the effects of Rhesus TRIM5Î± are a strain specific resistance and not a species specific resistance.
In experiments documented by previous researchers cytoplasmic PLM held the HSV-1 protein ICP0 in cytoplasm by binding to it. To determine if TRIM5Î± also bound to ICP0 and held it in abeyance H-R, H-H and H-L cells were stained with immunoflouresence so that ICP0 in HSV-1 KOS infected cells could be observed. The infected cell lines were examined at 4 and 8hpi and showed that some ICP0 was held in abeyance within the cytoplasm in H-R cell lines and some within the nucleus, by 8hpi cytoplasmic ICP0 increased and nuclear ICP0 decreased. In the H-L and H-H cell lines ICP0 was found in the nucleus at 4 and 8hpi with cytoplasmic spotting of ICP0. To further investigate these phenomena the researcher dual stained cells so that the Interaction of TRIM5Î± with ICP0 could also be detected: In H-R cells TRIM5Î± associated with ICP0; however no association with ICP0 was seen in the H-H cells that expressed Human TRIM5Î±. The researcher saw similar effects with other HSV-1 strains and hypothesized that by prevent ICP0 from getting to the nucleus by holding it in abeyance in the cytoplasm, it interferes with a necessary process for HSV replication.
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In the aforementioned experiment PML was responsible for reduction in viral replication of HSV-1, if TRIM5Î± binding to ICP0 is a part of this viral resistance a HSV-1 knockout for ICP0 should not cause infection. To test this hypothesis H-R and H-L cells were infected with an ICP0 null mutant and a rescued virus; the H-R cells when compared to the H-L cell showed approximately a 2 fold reduction of viral replication indicating no significant reduction in either. Since the absence of ICP0 showed no significant difference the antiviral affects of TRIM5Î± are independent of ICP0.
Researchers in the process of trying to determine more of the TRIM5Î± role in viral replication cycle analyzed immunoflouresence studies which showed declines of TRIM5Î± post infection. In order to determine why TRIM5Î± declines, a western blot analysis was performed on lysates of H-R and H-L cell lines infected with and without HSV-1 and HSV-2. For cells infected with HSV there was an apparent decline in expression of TRIM5Î± with after only 4hpi and by 24hpi the expression of TRIM5Î± was undetectable. Researchers wanted to determine if the TRIM5Î± decline was a result of restricting HSV replication, so they infected H-Sq, H-AGM and H-H cell lines also which expressed a decline in TRIM5Î± 6hpi and an undetectable load 16hpi. Researchers also noted that the initial decline coincided with ICP8 expression. For this experiment it was deduced that TRIM5Î± is not the sole responsible party for reducing viral replication.
The journal article provided new insight into the deductive reasoning of viral research and how results of one experiment can create a whole new line of exploration. The Journal article on the other hand did not however provide any insight as to why TRIM5Î± of different species have varying levels of resistance; why didn't the research probe the difference in TRIM5Î± expressed in other species? In short the results for this experiment bringing to light that TRIM5Î± is not the "lone gunman" in the reduction of HSV replication, it opens up different avenues of exploration for other researchers.