Use of specific radioactive probes to study transcription and replication of the influenza virus genome

Abstract

Specific radioactive probes have been obtained for both influenza virion RNA (vRNA) and for its complement (complementary RNA or cRNA): 32P-labeled complementary DNA (cDNA) synthesized with the avian sarcoma virus reverse transcriptase, and [125I]vRNA, respectively. From the kinetics of annealing of these two probes to RNA from canine kidney cells infected with the WSN strain of influenza virus, we have determined the average number of cRNA and vRNA sequences in the nucleus and cytoplasm as a function of time after infection. Immediately after infection, a small amount of vRNA is detected, presumably from the inoculum virus. As expected, the amount of cRNA is insignificant. During the first 1.75 h of infection, the most significant increase observed is in cRNA sequences. Most of these cRNA sequences are found in the cytoplasm, but a significant amount (30%) is found in the nucleus. During this time, a small but significant increase in vRNA is also detected in the nucleus and cytoplasm. From 1.75 to 2.75 h, the absolute amounts of both cRNA and vRNA increase, predominantly in the cytoplasm, with cRNA remaining as the majority species. Subsequently, the amount of vRNA increases with respect to cRNA and becomes the majority species. At 3.75 h, 95% of both cRNA and vRNA are found in the cytoplasm. Addition of actinomycin D at 1.75 h completely suppresses the subsequent ninefold increase in cRNA and does not have a significant effect on the subsequent 14-fold increase in cytoplasmic vRNA. This assay is also able to detect the cRNA produced as a result of primary transcription, operationally defined as the cRNA produced in the presence of 100 mug of cycloheximide per ml added at zero time of infection. Increases in cRNA in the presence of cycloheximide are detectable in both the nucleus and the cytoplasm. Addition of actinomycin D as well as cycloheximide at zero time completely suppresses the appearance of cRNA in the cytoplasm, whereas a large fraction (50%) of the increase in nuclear cRNA still occurs.