Panoply™ Human ITK Knockdown Stable Cell Line

Successful replication of human immunodeficiency virus (HIV)-1 depends on the expression of various cellular host factors, such as interleukin-2 inducible T cell kinase (ITK), a member of the TEC tyrosine kinase protein family. ITK is selectively expressed in T cells and coordinates signaling pathways downstream of T cell receptors and chemokine receptors, including PLC-1 activation, Ca2+-release, transcription factor mobilization, and actin rearrangement. The exact role of ITK in HIV-1 infection remains unclear. Here, researchers analyzed the function of ITK in HIV-1 replication and showed that when ITK was knocked down, the attachment, fusion, and entry of viral particles into Jurkat T cells were inhibited. In contrast, reverse transcription and proviral expression were unaffected by ITK deficiency. Inhibiting ITK expression did not affect cell surface CXCR4 receptors, while levels of CD4 and LFA-1 integrin were slightly increased in ITK knockdown cells, and heparan sulfate (HS) expression was completely absent in ITK-deficient T cells. However, neither HS expression nor other attachment factors could explain the impaired binding of HIV-1 to ITK-deficient cells, suggesting that ITK affects more complex cellular processes or that there are yet undiscovered molecules involved in limiting HIV-1 binding and entry.

To assess HIV-1 replication, researchers infected cells with replication-competent HIV-1 (NL4-3 clone) and monitored viral replication for 12 days. Viral titers were determined by infecting TZM-bl reporter cells with cell culture supernatants. The results showed that wild-type and non-targeting (n.t.) shRNA Jurkat cells supported HIV-1 replication, while ITK knockdown cell lines were resistant to viral infection (Figure 1A). Furthermore, the researchers independently monitored viral replication in these cells by quantitatively measuring reverse transcriptase (RT) activity in the infected cell supernatants. This experiment confirmed that HIV-1 replication was inhibited in ITK knockdown cells. Subsequently, they used a single-round HIV-1 luciferase reporter virus to determine which stage of the viral life cycle was affected by ITK. Compared to Jurkat cells expressing ITK, luciferase reporter gene activity was significantly reduced in ITK knockdown cells. However, when Jurkat cells were transduced with VSV-G-pseudotyped HIV reporter virus, luciferase activity was independent of ITK expression (Figure 1B). The researchers also tested the effect of the ITK inhibitor BIX0252428 on HIV-1 infection using a luciferase reporter virus (Figure 1C). Jurkat cells treated with the ITK inhibitor showed a concentration-dependent decrease in sensitivity to HIV envelope protein-mediated HIV-1 transduction, but infection was not inhibited when tested with VSV-G pseudotyped virus. These findings suggest that ITK deficiency leads to a specific restriction of HIV-1, involving viral fusion and entry mechanisms, but not downstream early steps such as reverse transcription, integration, and protein expression.

Figure 1. Loss of ITK expression blocks HIV-1 replication in Jurkat cells. (Hain A, et al., 2018)