807. NADPH-Dependent Endosomal ROS Is Critical for rAAV Capsid Processing during Infection
Authors: Liang N. Zhang, Jennifer Marden, Ziying Yan, John F. Engelhardt
Journal: Molecular Therapy (2006)

Reactive oxygen species (ROS) generated by phagocytes are well recognized as critical components in the innate immune system. Furthermore, cellular redox stress generated following infection with a number of pathogenic viruses such as HIV has also been reported. We have discovered that adeno-associated virus type 2 (AAV2) has evolved to both stimulate endosomal ROS production during its infection and utilize the resultant hydrogen peroxide to facilitate endosomal processing of the virion. Infection of HeLa cells, IB3 cells, or primary mouse fibroblasts with recombinant AAV2 (rAAV2) stimulated endosomal NADPH-dependent superoxide production 3 to 4-fold. Removal of hydrogen peroxide from within the endosomal compartment by catalase loading significantly decreased transduction by rAAV2 (as measured by a luciferase transgene) 80-fold. This inhibitory effect was not due to blocking viral entry as assessed by quantification of intracellular viral genome. Given that Rac1 has been shown to be critical for rAAV2 transduction and is known to be an activator of two NADPH oxidases (Nox1 and Nox2), we sought to determine if rAAV2-induced endosomal ROS originated from Nox1 or Nox2 using knockout (KO) and littermate wild type primary dermal fibroblasts generate from these two KO mouse lines. Results from these experiments demonstrated that Nox2-/- fibroblasts failed to induce endosomal ROS following rAAV2 infection and had an 18-fold lower level of transduction as compared to wild type littermate fibroblasts. In contrast, no differences in rAAV2-induced endosomal ROS or transduction were observed in Nox1 KO and wild type littermate fibroblasts. These results suggested that AAV2 infection induces Nox2 in the endosomal compartment to produce ROS and that endosomal exposure of virus to H2O2 is important for productive intracellular processing of the virus. With the hypothesis that ROS-mediated endosomal processing of rAAV2 might involve redox-mediated changes to cysteine residues on the capsids, we began to map structural changes to purified virions exposed to H2O2 using MALDI-TOF MS. Results from these experiments suggest that nM quantities of H2O2 can enhance trypsin sensitivity of intact capsids, suggesting that ROS may help to unfold the capsid while in the endosome and aid in activating certain biological function of the virus. This hypothesis is supported by experiment demonstrating that treatment of intact rAAV2 virions with nM quantities of H2O2 also stimulates phospholipase A2 activity resident in the viral capsids. Interestingly, changes in pH did not appear to alter either the trypsin sensitivity of intact virions or PLA2 activity of the capsid and did not synergistically affect H2O2 capsid sensitivity in either of these two assays. These results suggest that AAV2 has evolved to both induce and utilize Nox2- derived ROS productively to process its virion during infection. Further elucidation of the mechanism may aid in improving rAAV2 vectors for gene therapy and also uncover novel redox-dependent mechanisms of infection. Reactive oxygen species (ROS) generated by phagocytes are well recognized as critical components in the innate immune system. Furthermore, cellular redox stress generated following infection with a number of pathogenic viruses such as HIV has also been reported. We have discovered that adeno-associated virus type 2 (AAV2) has evolved to both stimulate endosomal ROS production during its infection and utilize the resultant hydrogen peroxide to facilitate endosomal processing of the virion. Infection of HeLa cells, IB3 cells, or primary mouse fibroblasts with recombinant AAV2 (rAAV2) stimulated endosomal NADPH-dependent superoxide production 3 to 4-fold. Removal of hydrogen peroxide from within the endosomal compartment by catalase loading significantly decreased transduction by rAAV2 (as measured by a luciferase transgene) 80-fold. This inhibitory effect was not due to blocking viral entry as assessed by quantification of intracellular viral genome. Given that Rac1 has been shown to be critical for rAAV2 transduction and is known to be an activator of two NADPH oxidases (Nox1 and Nox2), we sought to determine if rAAV2-induced endosomal ROS originated from Nox1 or Nox2 using knockout (KO) and littermate wild type primary dermal fibroblasts generate from these two KO mouse lines. Results from these experiments demonstrated that Nox2-/- fibroblasts failed to induce endosomal ROS following rAAV2 infection and had an 18-fold lower level of transduction as compared to wild type littermate fibroblasts. In contrast, no differences in rAAV2-induced endosomal ROS or transduction were observed in Nox1 KO and wild type littermate fibroblasts. These results suggested that AAV2 infection induces Nox2 in the endosomal compartment to produce ROS and that endosomal exposure of virus to H2O2 is important for productive intracellular processing of the virus. With the hypothesis that ROS-mediated endosomal processing of rAAV2 might involve redox-mediated changes to cysteine residues on the capsids, we began to map structural changes to purified virions exposed to H2O2 using MALDI-TOF MS. Results from these experiments suggest that nM quantities of H2O2 can enhance trypsin sensitivity of intact capsids, suggesting that ROS may help to unfold the capsid while in the endosome and aid in activating certain biological function of the virus. This hypothesis is supported by experiment demonstrating that treatment of intact rAAV2 virions with nM quantities of H2O2 also stimulates phospholipase A2 activity resident in the viral capsids. Interestingly, changes in pH did not appear to alter either the trypsin sensitivity of intact virions or PLA2 activity of the capsid and did not synergistically affect H2O2 capsid sensitivity in either of these two assays. These results suggest that AAV2 has evolved to both induce and utilize Nox2- derived ROS productively to process its virion during infection. Further elucidation of the mechanism may aid in improving rAAV2 vectors for gene therapy and also uncover novel redox-dependent mechanisms of infection.