Ribonucleic acid synthesis in vaccinia virus: I. The mechanism of synthesis and release of RNA in vaccinia cores

Abstract

Messenger RNA synthesis and release by vaccinia subviral particles (cores) was studied in vitro using in part a nitrocellulose filter binding assay for the detection of core-bound, nascent messenger RNA. During steady-state synthesis of RNA approximately one-half of the particle-bound RNA is sensitive to ribonuclease, and is, presumably, external to the core protein coat. The other half of the nascent RNA is ribonuclease resistant and is probably internal to the viral protein coat. Both ribonuclease-sensitive and resistant core-bound messenger RNA is continuously released as synthesis progresses. Synthesis of RNA by cores is linear for extended periods of time following a lag of approximately 1.5 minutes. This lag can be eliminated by pre-incubation of cores with a combination of ATP and CTP but not with UTP or GTP. Incorporation of γ-32P-labeled nucleoside triphosphates indicated that only ATP and CTP were capable of initiating RNA chains in vaccinia cores in vitro. The maximum rate of RNA chain elongation was estimated at 17 nucleotides per second per chain. A minimum of 40 RNA-chain growing-points occur per subviral particle. Part of a nascent RNA chain is extruded to the exterior of the core protein coat shortly after initiation of chain elongation.DNA-RNA hybridization experiments indicate that a restricted portion, only 7%, of the viral DNA is transcribed by cores in vitro.