Analysis of viral parameters in parental U937 and resistant RU cells after H-1 virus infection. (A) The survival of U937 and RU cells was determined by counting viable cells 4 days after infection with H-1 virus (MOI = 100 pfu/cell), using the Trypan blue exclusion method, and expressed as a percentage of the value measured in mock-infected cultures. Results from a typical experiment are shown. (B) Infectious particles recovered 48 hours after inoculation of 10⁶ cells with H-1 virus (5 pfu/cell) were measured by plaque-assay. Data shown are from a typical experiment. Similar relative values were obtained in three independent experiments (SD < 22%). (C) The virus uptake was assayed after infection with [³H]thymidine-labeled H-1 virus (2 × 10³ pfu [10⁵ cpm]/10⁶ cells), followed by a washing step with 1 mmol/L EDTA to remove noninternalized particles (upper part). Internalized [³⁵S]methionine-labeled capsids were revealed by autoradiography after immunoprecipitation and SDS-PAGE analysis of VP1 and VP2 proteins extracted 2 hours after infection with H-1 virus (4 × 10⁶ pfu [10⁶ cpm]/10⁶ cell) and washing with EDTA (lower part). (D) Amplification of viral DNA was determined by Southern blotting. After virus (10 pfu/cell) or mock infection, cultures were incubated for 40 hours in complete medium. DNA samples extracted by the Hirt procedure were analyzed by agarose gel electrophoresis, blotted, and hybridized with a virus-specific ³²P-labeled DNA probe. RFI and RFII refer to viral DNA replicative forms of monomer and dimer length, respectively. Viral DNA species were positioned relative to HindIII-digested phage λ DNA used as size markers and revealed by ethidium bromide staining. (E) The viral transcripts were detected by Northern blotting. At 48 hours postinfection (10 pfu/cell), total RNA was isolated by the guanidinium thiocyanate method, fractionated by agarose/formaldehyde gel electrophoresis, blotted, and hybridized with a virus-specific ³²P-labeled DNA probe. (F ) The accumulation of viral proteins was determined by immunoprecipitation. At 36 hours postinfection (5 pfu/cell) cultures were incubated for 2 hours in the presence of [³⁵S]methionine and processed for immunoprecipitation with antiserums directed against the viral products NS-1 or VP-1 and VP-2, SDS-PAGE, and autoradiography.