2.5. Translation of Viral Proteins During Infection and in Vitro

To radiolabel viral translation products in infected cells, HeLa cell monolayers were infected with PV1 or PV1-3′MS2 at an MOI of 10. Following a 30 min adsorption, DMEM without methionine (MP)-8% NCS was added to cells. Cells were pulsed with 20 μCi ³⁵S-methionine (PerkinElmer, Waltham, MA, USA) 1 h prior to collection. Cells were washed twice with PBS, harvested, and resuspended in 2X Laemmli sample buffer (LSB). Crude lysates (25% of total) were resolved on a 12.5% polyacrylamide, SDS-containing gel. For in vitro translation assays, HeLa cell S10 cytoplasmic extracts were generated as described elsewhere [32]. HeLa S10 (60% of total volume) was incubated with 250 or 500 ng of in vitro transcribed RNA constructs corresponding to aptamer-tagged viral genomes or poliovirus virion RNA (vRNA), ³⁵S-methionine (PerkinElmer), and all-four buffer (1 mM ATP, 0.25 mM GTP, 0.25 mM UTP, 0.25 mM CTP, 60 mM potassium acetate, 30 mM creatine phosphate, 0.4 mg/mL creatine kinase, 15.5 mM HEPES-KOH [pH 7.4]). Translation was allowed to proceed at 30 °C for 5–6 h. 2X LSB was added to an equal volume of translation reaction and boiled for 3 min. Samples were then subjected to electrophoresis on an SDS-containing 12.5% polyacrylamide gel. Proteins were visualized by autoradiography following fluorography. Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA) was used to quantify VP3 band intensity of in vitro translation reactions that contained 250 ng of RNA relative to the band intensity of the poliovirus vRNA translation.