Poly(A) sequences of vaccinia virus messenger RNA: Nature, mode of addition and function during translation in vitro and in vivo

Abstract

Studies were undertaken of the nature and mode of addition of the poly(A) sequences at the 3′-termini of vaccinia virus messenger RNA, as well as of their function during translation in vivo and in vitro. The median length of poly(A) sequences on messenger RNA synthesized in vivo, both “early” and “late” in infection, and on RNA transcribed by vaccinia virus cores in vitro was the same, namely about 100 residues. Poly(A) on messenger RNA synthesized in vivo was slightly more homogeneous in size than that on RNA transcribed in vitro. No nucleic acid bases other than adenine were present. Base composition analysis of poly(A) liberated by pancreatic and T1 ribonuclease indicated that the average 3′-terminal sequences of vaccinia virus RNA were G(C2 U4 A?)A100 Aoh.When vaccinia virus messenger RNA was hybridized to DNA, the poly(A) sequences remained free, and at least 95% of them could be removed by treatment with pancreatic and/or T1 ribonuclease, with no detectable diminution in size. This was interpreted as evidence that they were added posttranscriptionally, rather than copied from sequences of poly(dT) in the viral genome. In fact, the vaccina virus strain WR genome was found to contain essentially no poly(dT) sequences (less than 0.015% of the virus genome) as judged by poly(A) binding.In the presence of cordycepin, the formation of both early and late vaccinia messenger RNA in infected cells was markedly inhibited (90% inhibition by 50 μg/ml). The residual 10% of messenger RNA that was still formed under these conditions contained poly(A) sequences that were only about one-half their normal length, but gave rise to about one-half of the amount of virus-specified protein that was synthesized in the absence of cordycepin. Halving the length of poly(A) sequences on vaccinia virus messenger RNA therefore appears to have no adverse effects on its ability to be translated in vivo.Not all vaccinia virus messenger RNA molecules possessed poly(A); both early and late messenger RNA synthesized in vivo, as well as RNA transcribed in vitro, contained about 10% of molecules that did not bind to poly(U)-cellulose and contained no poly(A). The poly(A) (−) RNA molecules that were transcribed in vitro had the same size as poly(A) (+) RNA, and possessed the same base sequences as judged by hybridization analysis. Further, they were translated as efficiently as poly(A) (+) RNA molecules in an in vitro Krebs II ascites cell-free protein synthesizing system, yielding the same polypeptides. The presence of poly(A) was therefore not essential for translation in vitro. The pattern of polypeptides that was obtained was complex; this was no doubt due to the fact that, as determined by hybridization analysis, the messenger RNA synthesized by vaccinia virus cores in vitro was transcribed from 50% of the viral genome. This degree of complexity was quantitatively comparable to that of early messenger RNA synthesized in vivo.