Hybridization and sedimentation studies on “early” and “late” vaccinia messenger RNA

Abstract The vaccinia virus multiplication cycle can be divided into two portions: the early portion, which precedes viral DNA replication and lasts for about one-and-a-half hours; and the late portion which follows until maturation of progeny is complete. Vaccinia messenger RNA transcribed during these two periods, that is, early and late messenger RNA, has been studied by means of hybridization and density gradient sedimentation. The following results were obtained: 1. (1) Early vaccinia messenger RNA is distinctly smaller than late messenger RNA (10 to 12 s against 16 to 20 s). Late messenger RNA contains nucleotide sequences not present in early messenger RNA; however, all the sequences transcribed early are also transcribed late. If late messenger RNA is transcribed from the entire viral genome, one-half to two-thirds is transcribed early. 2. (2) Messenger RNA transcribed in the presence of cytosine arabinoside, which completely inhibits viral DNA replication, appears to be identical to early messenger RNA. The cytosine arabinoside RNA transcribed in HeLa and L cells contains the same base sequences. 3. (3) The pattern of transcription of early and late vaccinia messenger RNA in HeLa and L cells is quite different. In HeLa cells the total amount of late messenger RNA synthesized greatly exceeds the amount of early; in L cells the position is reversed. 4. (4) The effect of varying the multiplicity of infection on the pattern of early and late messenger RNA transcription has been studied. The rate of early mRNA synthesis is proportional to the multiplicity over a certain limited range. Once the maximum rate of mRNA transcription has been reached, which occurs the earlier the higher the multiplicity, the rate of transcription of messenger RNA falls to a steady low level equal to no more than 5% of the maximum value; this rate of transcription then persists until at least 19 hours after infection. 5. (5) The large messenger RNA molecules transcribed late contain nucleotide sequences also present in small early messenger RNA molecules. Some small messenger RNA molecules are also transcribed late: these contain at least some sequences characteristic of late messenger RNA. 6. (6) At five hours after infection the messenger RNA molecules present in polyribosomes contain all the sequences characteristic of early messenger RNA molecules. By eight hours after infection messenger RNA in polyribosomes is very significantly depleted with respect to sequences characteristic of early messenger RNA. 7. (7) The stability of early and late vaccinia messenger RNA in the presence of actinomycin D has been studied in HeLa and L cells. Early vaccinia messenger RNA is very stable in HeLa cells. Those late messenger RNA molecules which contain sequences characteristic of late messenger RNA are mostly unstable (half-life less than one hour); but those containing sequences characteristic of early messenger RNA are as stable as early messenger RNA itself. In L cells early and late vaccinia messenger RNA are equally stable (half-life two to three hours). mRNA is considerably more stable in extracts of HeLa cells than of L cells; and early mRNA is somewhat more stable in extracts of HeLa cells than is late mRNA. These results are discussed in relation to the pattern of macromolecular biosynthesis during the vaccinia virus multiplication cycle.