We next assessed whether treatment with proteasome inhibitors prior to PBMC activation could block HIV-1 replication. We therefore set up three different treatment scenarios and measured viral infectivity by QPCR and RT-activity (Fig. 4B). In the first condition, Itk
LY 450139 was induced by PHA prior to HIV-1 infection and treatment with ARVs, resulting in effective inhibition of viral replication by 2.5 nM Bortezomib (Fig. 4C and D, Treatment 1). In the second setting, a short pulse with Bortezomib was provided concomitantly with PHA activation in order to inhibit Itk
expression and Bortezomib was washed away from the culture at the time of HIV-1 infection (Treatment 2). Depletion of Itk prior to HIV-1 infection led to reduced RT-activity using either 1.25 or 2.5 nM of Bortezomib (Fig. 4C, Treatment 2). However, a dose of 2.5 nM was required to reduce viral copy numbers, regardless of which treatment setting used (Fig. 4D). Moreover, continuous treatment with Bortezomib (2.5 nM) throughout the experiment resulted in effective inhibition of RT-activity and suppression of HIV-1 infection (Fig. 4C and D, Treatment 3).
