An article by Readinger et al entitled “Selective targeting of ITK blocks multiple steps of HIV replication” and published (free access) in the May 6th edition of the Proceedings of the National Academy of Sciences has been receiving a certain amount of press recently and for good reason.
Most therapeutic approaches to the treatment of HIV infection have been—and continue to be—built on the idea of attacking mechanisms of the virus itself. Highly Active Antiretroviral Therapy (HAART) is an example of this. However, antiretroviral resistant strains of HIV are appearing, thus reducing the efficacy of these treatments.
One potential path for increasing the arsenal of HIV treatments is to target cellular mechanisms that are essential to HIV infection. Indeed HIV, as is the case with all viruses, must hijack cellular mechanisms in order to reproduce itself. In their article, Readinger’s team reports on their research into a cellular protein called “Inducible T cell kinase”, or ITK for short. This cellular protein is essential, at least in vitro, for T-cell activation and appears to play a key role in several steps of HIV replication. Its inactivation, which they experimented using ITK-specific siRNA or a chemical inhibitor with the charming name of BMS509744, may thus be of therapeutic importance.
Their experiments indicate that ITK does indeed play an important role in HIV infection and replication and that its inactivation in CD4+ T-cells may hinder HIV infection.
They found that ITK was important at several steps in the HIV replication cycle:
- Viral entry
ITK inactivation did not affect the production of CD4 receptors or the CXCR4 (co)receptor, and thus viral binding was unaffected. However, they found that viral entry was affected. This was because ITK is a necessary component of cytoskeleton rearrangement, something the HIV particle must induce via its gp120 glycoprotein to be able to enter the cell.
- Transcription from the HIV long terminal repeats (LTR)
Transcription factors, such as NF-κB, need to bind to the LTRs of the viral genome to induce their transcription in activated T-cells. Using a HIV-specific luciferase reporter (a sort of biochemical signaler of transcription factor binding activity) the researchers found that ITK is an upregulator of HIV transcription and blocking it in activated T-cells reduced binding activity by 60-80% (as indicated by the luciferase).
- Viral assembly and escape
The team also looked at ITK’s role later in the viral cycle and found that in a state of overexpressed Gag proteins with active ITK, there was an increase in the creation of virus-like particles. They further explained that this potentiation did not seem to be dependant on kinase activity, but “in a manner dependent on protein interactions and membrane recruitment.”
The also found that the use of ITK-specific siRNA after the establishment of infection reduced virion release by as much as 68% 2 days after treatment and that there were fewer infected cells (6 vs 18% (for controls) after 72 hours). However this effect was limited to 72 hours, which is a result of the transient nature of siRNA.
It is also worth noting that the researchers found that ITK did not seem to have a role in HIV reverse transcription or proviral integration.
A few final points
One of the questions that you would have the right to ask is, “hold on a minute, wouldn’t turning off a protein essential to T-cell activation be a bad idea?” Indeed, you generally want your T-cells to activate when stimulated by antigens. To respond to this question, the researchers underlined previous research, indicating that ITK deficient mice are still capable of mounting a defense against and clearing all viral challenges tested to date. So, although the T-cell response is partial, it is still sufficiently strong to fight off infections. It is this quality that makes ITK so interesting, in that its inactivation may hamper the progression of HIV, without having a major impact on the global T-cell mediated immune response.
Another question that you could ask is, “hold on another minute, although interfering with ITK function hampers HIV, it doesn’t stop it at 100%. Isn’t HIV going to simply evolve to get around the absence of active ITK? In the discussion the researchers are attentive to say that a cellular protein approach may be “less prone” to creating viral resistance. Indeed in the last sentence of their report they are careful to say that their results suggest, “that ITK inhibition provides a model for the study of cellular protein targets that affect HIV infection, which may be useful as part of a multidrug regimen directed against HIV.”
In other words, prudence prudence prucence. Indeed this is strictly an in vitro study in a relatively new field of research. The road ahead is still very long and HIV has proven itself as one real bastard of a virus, and I’m using a euphemism there. This isn’t the first “promising piece of research” and it certainly won’t be the last. It is however an important step forward and a very interesting insight into HIV viral function and its dependence on this cellular ‘tool’ to assure its own reproduction.