Role of group A p21-activated kinases in the anti-apoptotic activity of the pseudorabies virus US3 protein kinase

Abstract

The alphaherpesvirus US3 kinase is a conserved multifunctional serine/threonine kinase that plays a role in several processes, including modulation of the actin cytoskeleton, egress of virus particles from the nucleus and inhibition of apoptosis. However, the mechanisms used by the US3 protein to exert its functions remain poorly understood. Recently, we identified the group A p21-activated kinases PAK1 and PAK2 as important effectors in the US3-mediated cytoskeletal rearrangements. Here, we investigated if group A PAKs are also involved in the anti-apoptotic properties of US3. Infection experiments using a group A PAK inhibitor pointed at a moderate role for group A PAKs in the anti-apoptotic properties of US3. Furthermore, infection assays using wild type and US3null PRV in wild type MEF, PAK1(-/-) MEF and PAK2(-/-) MEF indicated that PAK2 does not play a role in US3-mediated inhibition of apoptosis during infection, whereas PAK1 plays a significant, yet limited role. Experiments in US3-transfected MEF using staurosporine as apoptosis trigger confirmed these observations. These results show that PAK1 plays a significant, yet limited, role in the anti-apoptotic activity of US3.

Figure 1. Group A PAKs play a modest role in the ability of US3 to protect PRV-infected cells from apoptotic cell death

Percentage of active caspase 3 positive mock-infected MEF or MEF at 24h post inoculation with wt PRV or US3null PRV in the presence or absence of the group A PAK inhibitor IPA3 (30 μM). Three independent replicates of each experiment were performed, data shown are means and standard deviations, and statistical analysis was performed using the SPSS software. Means were compared with an analysis of variance and a least significant difference post hoc test for a multiple comparison of means (α=0.05). Different letters indicate significant differences at the 0.05 level.

Figure 2. PAK1, but not PAK2, is involved to some extent in the ability of US3 to protect PRV-infected cells from apoptotic cell death

A. MEF, PAK1^−/− MEF and PAK2^−/− MEF at 24hpi with wtPRV or US3null PRV, stained for active caspase-3 (green) and nuclei (blue) B. Percentage of active caspase-3 positive MEF, PAK1^−/− MEF and PAK2^−/− MEF that were either mock infected or infected with wt PRV or US3nullPRV (24 hpi). Three independent replicates of each experiment were performed, data shown are means and standard deviations, and statistical analysis was performed using the SPSS software. Means were compared with an analysis of variance and a least significant difference post hoc test for a multiple comparison of means (α=0.05). Different letters indicate significant differences at the 0.05 level.

Figure 3. PAK1, but not PAK2, is involved in the ability of US3 to protect cells from apoptosis induced by staurosporine

Percentage of active caspase-3 positive, staurosporine-treated mock-transfected or US3-transfected MEF, PAK1^−/− MEF and PAK2^−/− MEF (24h post transfection). Three independent replicates of each experiment were performed, data shown are means and standard deviations, and statistical analysis was performed using the SPSS software. Means were compared with an analysis of variance and a least significant difference post hoc test for a multiple comparison of means (α=0.05). Different letters indicate significant differences at the 0.05 level. Figure 1