Herpes simplex virus is a DNA virus that causes cold sores and the disease genital herpes. The virus is spread by direct contact, and is a chronic infection that exhibits latency, meaning that the host cycles through symptomatic and asymptomatic phases. Many species have shown susceptibility to HSV. However, there are several species that are resistant to HSV infection, including rhesus macaques, which serve as non-human models for HIV infection in humans (1). Since vaccines for HIV are often tested on rhesus macaques, any innate mechanisms for fighting viral infections that the monkeys posses will undermine the efforts towards vaccine efficacy. Therefore, these mechanisms need to be understood to determine if rhesus monkeys are good candidates for HIV vaccine research.
Reszka et al. chose to study the tripartite motif 5α (TRIM5α) protein, a protein believed to be a key factor in preventing HIV-1 infection in rhesus monkeys. Members of the TRIM protein family have been found in several species, including humans and other primates, and are believed to be part of the innate immune response to viral infection (1). The researchers sought to determine if TRIM5α did influence HSV replication and attempted to elucidate the mechanisms involved.
First and foremost, the researchers wanted to determine if HSV-1 and HSV-2 replication was diminished in rhesus monkeys. This was a direct measure of whether or not rhesus monkeys were resistant to HSV infection, and was also a fairly straightforward experiment. HeLa cells infected with HSV-1 and HSV-2 were grown along with rhesus fibroblast cells also infected with the viruses. Both cells were grown at a multiplicity of infection (MOI) of 3 PFU/cell, harvested at the same post-infection time, and virus yields were determined on Vero cells via plaque assay. Viral yield peaked for both cell lines and virus types at approximately 40 h.p.i.; the rhesus monkey fibroblasts showed significantly lower virus yield compared to the HeLa cells for both virus types. Therefore, rhesus monkeys showed a resistance to HSV infection (1).
Next, the experimenters wanted to see if cells expressing rhesus monkey TRIM5α could resist viral infection, and if this phenomenon was observed across species. For this experiment, HeLa cells containing several different vectors were utilized. The cells contained empty vector (H-L), TRIM5α from rhesus monkey (H-R), squirrel monkey (H-Sq), African green monkey (H-AGM), or human (H-H). For the TRIM5α resistance, HSV-1 and HSV-2 were used to infect H-L and H-R cells at MOIs between 1 and 30 PFU/cell. A significant drop in viral yield was shown for H-R compared to H-L for MOIs 1 to 10 for HSV-1 and for MOIs 1-3 for HSV-2 (1). These results indicate that TRIM5α conveys viral resistance to HSV-1 and HSV-2. For the TRIM5α of various species, a similar methodology was used, except only HSV-2 at a MOI of 3 PFU/cell was used. The results showed a significant drop in virus yield for H-AGM and H-R compared to H-L, while H-H and H-Sq did not show a significant drop (1). Therefore, Old World Monkey (OWM) TRIM5α conveyed a resistance to HSV.
The investigators wanted to see if other strains of HSV would show the same repression by TRIM5α as the strains that had been used in the other experiments, HSV-1 strain KOS and HSV-2 strain 186 syn+. Several strains of HSV-1 and HSV-2 were used to infect H-L and H-R cells as described previously. All results showed a significant reduction in viral replication except for the laboratory strain 17 syn+ (1). Therefore, the action of TRIM5α may be strain-specific rather than species-specific.
Having determined that TRIM5α did in fact play a part in inhibiting HSV synthesis, the researchers sought to understand the mechanism by which TRIM5α worked. The effect of TRIM5α on HSV protein synthesis was analyzed using Western Blot. Western blot is performed by subjecting samples to an SDS-PAGE to separate proteins by molecular weight, and then transferring the separated proteins to a membrane such as nitrocellulose. The membrane is then treated with a modified antibody that is specific to the protein of interest and reacts in the presence of a detector, resulting in a visible band (2). For this experiment, Western blot was used to determine the presence of several immediate-early (IE) viral proteins in H-L and H-R cells infected with either HSV-1 or HSV-2. IE proteins were chosen due to earlier experiments with TRIM5α indicating that it acted early in viral replication (1). Infected cells were harvested and assayed at 4, 6, and 8 hpi (10 hpi for HSV-2), then subsequently subjected to Western blot. The results of this experiment indicated that IE viral proteins were significantly diminished in H-R cells compared to H-L cells, although some cultures from later hpi still had a substantial amount of IE proteins in H-R cells (1). Also, the reduction in protein was greater for HSV-2 infected cells than for HSV-1 infected cells (1). The researchers concluded that these results indicated that TRIM5α repressed viral synthesis at early stages of infection.
The researchers, using the mechanism of promyelocytic leukemia (PML) as a model, wanted to see if the action of TRIM5α involved sequestering viral proteins. PML was shown to sequester infected cell protein 0 (ICP0) in the cytoplasm, preventing it from making its way to the nucleus and therefore inhibiting viral replication. The effect of TRIM5α on (ICP0) was studied using an immunofluorescence assay. In this case, indirect immunofluorescence was utilized; the cells were treated with an antibody that was specific to either the ICP0 or a polyhistidine tag that was fused to the TRIM5α proteins. The sample was then treated with a second antibody, specific for the first antibody, containing the fluorophore, allowing for the proteins to be observed under a light microscope. HSV-1 was used to infect H-L, H-R and H-H cells, and then the cells were harvested at 4 and 8 hpi and observed. ICP0 was found in the nucleus of the H-L and H-H cells at both hpi, and punctate (dotted) structures were observable in the cytoplasm at 8 hpi. These results indicate normal viral replication. In the H-R cells, increased cytoplasmic ICP0 was observable at 4 hpi relative to the other cells. Also there was virtually no ICP0 in the nucleus as 8 hpi. A second immunofluorescent assay, one using different stains to differentiate ICP0 from TRIM5α, showed that ICP0 associated with TRIM5α outside the nucleus in H-R cells but not H-H cells (1). This indicated that TRIM5α sequestered ICP0 in the cytoplasm at early stages of infection and may have lead to a decrease in viral replication at later stages.
To investigate if the effect of TRIM5α on ICP0 was a major mode of repression for the protein, H-L and H-R cells were infected with HSV-1 that either had the ICP0 gene removed (ICP0-) or restored (ICP0+). While the H-R cells showed a lower virus yield compared to the H-L cells, the presence of ICP0 appeared to not have a significant impact (1). Therefore, TRIM5α most likely operated independently of ICP0 when repressing viral replication.
An interesting phenomenon observed during the immunofluorescence assay was that the concentration of TRIM5α appears to decrease over time, even in the presence of viral proteins (1). To test this, a Western blot was performed on H-L and H-R cells infected with HSV-1 and HSV-2. Cells were harvested at 2, 4, 6, 12 and 24 hpi. In the presence of virus, the level of TRIM5α in H-R cells diminished over time and was undetectable at 24 hpi (1). In cells that had been mock infected, the level of TRIM5α remained relatively constant, though there was a slight decrease. This indicates that TRIM5α is highly upregulated in response to host infection but levels drop off as the infection progresses. A second Western blot was performed to see how HSV protein levels may alter TRIM5α levels. Infected cell protein 8 (ICP8), an IE protein, was detected by Western blot against TRIM5α from the different species utilized earlier. It was found that as ICP8 emerged, levels of TRIM5α diminished by roughly the same rate in all species (1). Levels of ICP8 varied across species however, with the lowest level of ICP8 being in the H-R cells.
Here is a summary of the results. HSV replication is inhibited in rhesus monkeys, and TRIM5α plays a part in this inhibition, though the exact mechanism is not understood. OWM TRIM5α is the only form of the protein that can significantly inhibit viral replication, and this effect appears to be strain-specific. TRIM5α exerts its effect on IE viral proteins, possibly sequestering them in the cytoplasm and preventing them from entering the nucleus. The level of TRIM5α diminishes in cells overtime, and appears to coincide with an increase in viral replication, indicating that the virus may have evolved a form of overcoming the effect of TRIM5α.
The results of this series of experiments indicate that TRIM5α has a definite role in the inhibition of HSV in rhesus monkey cells, though it is apparent that TRIM5α is not the only factor involved. The fact that TRIM5α works on IE proteins and diminishes over time means that it likely serves to slow initial viral replication to allow the host time to rally other defenses. TRIM5α may also play an indirect role in adaptive immunity, as it associates with IE viral proteins in the cytoplasm. Future research should aim to better understand the role of the punctate structures formed by TRIM5α and IE viral proteins, as well as how TRIM5α is degraded in the cytoplasm during viral infection. While it is easy to assume that HSV may have adapted to degrade TRIM5α, there may be other mechanisms at work that are part of the host cells immunity. Finally, the experiment has important implications for the use of rhesus monkeys as non-human HIV models. If there are innate mechanisms at work in the rhesus monkey that are inhibiting viral replication, and these mechanism work on HIV the same way they work on HSV, vaccine research would be significantly hindered. Therefore, factors such as TRIM5α need to be controlled or a new model for HIV vaccines needs to be picked.
