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

Because MAV-1 did not reduce PKR mRNA steady-state levels, we determined whether 185 MAV-1 causes PKR depletion by reducing translation of its mRNA. We first assayed total PKR 186 mRNA bound to ribosomes during infection. C57BL/6 MEFs were mock infected or infected at 187 an MOI of 5, and lysates were collected at 48 hpi in the presence of cycloheximide to keep the 188 mRNA bound to the ribosomes (56). Lysates were centrifuged through 25% sucrose to pellet 189 ribosomes, and RNA was purified from the pellets. The purified RNAs were used to generate 190 cDNA, which we assayed for PKR mRNA by qPCR. As a control for pelleting of ribosomes, we 191 assayed the pellets and sucrose cushion supernatants by immunoblot with antibodies to 192 ribosomal protein RPL7. We confirmed that RPL7 was only present in the pellets and not the 193 supernatants (Fig. S2 ). There was no significant difference between the amount of PKR mRNA 194 in the ribosome pellet of mock infected lysates compared to MAV-1-infected lysates (Fig. 4A) . 195 196 To confirm the results seen in total mRNA bound to ribosomes, we also centrifuged cell 197 extracts on sucrose gradients to generate polysome profiles. This enabled us to analyze levels of 198 PKR mRNA associated with actively-translating ribosomes during infection. C57BL/6 MEFs 199 were mock infected or infected at an MOI of 2, and lysates were collected at 24 hpi in the 200 presence of cycloheximide, as above. RNA content for mock and infected lysates was estimated 201 by NanoDrop spectrophotometry, and equivalent OD amounts of RNA were centrifuged on 10-202 50% sucrose gradients to sediment 40S and 60S ribosomal subunits, 80S ribosomes 203 (monosomes), and polyribosomes (polysomes). A typical polysome profile was obtained (Fig. 204 4B) . RNA was purified from fractions containing monosomes and polysomes and then used to 205 generate cDNA, which we assayed for PKR mRNA by qPCR (Fig. 4C) . As a control, GAPDH 206 mRNA was measured by qPCR, and PKR mRNA levels in each fraction were normalized to the 207 GAPDH mRNA content. When the data for percentage of PKR mRNA bound to ribosomes was 208 pooled into monosome and polysome fractions and analyzed (Fig. 4D) , 90.1% and 91.8% were 209 bound to polysomes (fractions 7-19) for mock and infected samples, respectively, compared to 210 9.9% and 8.2% bound to monosomes (fractions 1-6). We performed two additional polysome 211 gradient analyses. The pooled data from all three (Fig. 4E ) were similar to the data for Fig. 4D , 212