Enhanced replication of mouse adenovirus type 1 following virus-induced degradation of protein kinase R (PKR)

Danielle E. Goodman; Carla D. Pretto; Tomas A. Krepostman; Kelly E. Carnahan; Katherine R. Spindler

There are two main proteolysis pathways in cells, proteasomal degradation and lysosomal 228 degradation (58). To determine whether MAV-1 depletes PKR by either protein degradation 229 pathway, we first assayed whether PKR is lysosomally degraded as follows. CMT93 cells were 230 mock infected or infected with MAV-1 and treated at the time of infection with the lysosome 231 inhibitors ammonium chloride or chloroquine, or water (as a control). At 24 hpi, we collected 232 lysates and analyzed them by immunoblot with antibodies to PKR. In the presence of the 233 lysosomal degradation inhibitors, PKR was depleted by 24 hpi (Fig. 5A) , indicating that 234 lysosomal degradation was not the cause of PKR depletion during MAV-1 infection. We 235 confirmed that the inhibitor treatment did block lysosomal degradation by incubating cells with 236 dye-quenched bovine serum albumin (DQ BSA) in addition to the lysosomal inhibitors. DQ BSA 237 is self-quenched until it is digested in the lysosome (59, 60), and imaging confirmed that cells 238 treated with lysosome inhibitors did not fluoresce, but cells treated with the vehicle control 239 (H 2 O) did, as expected (Fig. 5B) . 240 241 Next, we examined whether proteasomal degradation is responsible for the degradation of 242 PKR by using proteasome inhibitors MG132 and bortezomib. These inhibit proteasome activity 243 by binding to the active sites in the 20S subunit and blocking the proteolytic activity (61-63). We 244 mock infected or infected C57BL/6 MEFs with MAV-1 and treated with MG132 or bortezomib 245 in DMSO at the time of infection. At 24 hpi, we collected lysates and analyzed them by 246 immunoblot for PKR protein levels. While PKR was depleted in the control DMSO-treated 247 MAV-1-infected cells as expected, PKR protein was present in the MG132-and bortezomib-248 treated cells at levels comparable to mock infected cells ( Fig. 6A and B ). To rule out the 249 possibility that PKR was present (not depleted) because the virus infection itself was inhibited by 250 MG132 or bortezomib, we assayed viral replication of MAV-1 with MG132 and bortezomib 251 treatment by qPCR of viral DNA. Viral replication was equivalent in all three treatment groups 252 ( Fig. S4) , indicating that the treatments did not affect the ability of the virus to productively 253 infect the cells. Taken together, these data indicate that MAV-1 infection results in PKR 254 depletion by causing PKR to be degraded by the proteasome during infection. 255