We have recently demonstrated that the poly(A) moieties of short RNAs obtained from both in vitro transcription and from vaccinia virus (VV)-infected cells exhibit dissimilar effects on the in vitro translation of cellular and VV mRNAs (R. Bablanian, G. Coppola, P. Masters, and A. K. Banerjee, Virology 148:375-380, 1986; M. J. Su and R. Bablanian, Virology 179:679-693, 1990). In the present study, we have investigated the roles of poly(A), m7GTP, and initiation factors in the mechanism of selective translation of VV mRNAs. The effects of unfractionated poly(A) [termed poly(A)un, with various chain lengths up to 3,000 nucleotides] and a 150- to 300-nucleotide fraction of synthetic poly(A) [termed poly(A)150-300] on the translation of HeLa cell mRNAs and early and late VV mRNAs were studied. Both the poly(A)un and the poly(A)150-300 completely inhibited the translation of HeLa cell mRNAs obtained from total cytoplasmic RNA in the nuclease-treated reticulocyte lysates. Viral mRNAs from total cytoplasmic RNA also were slightly inhibited (15 to 38%) by the poly(A)un, whereas the poly(A)150-300 had no significant effect on their translation. The translation of oligo(dT)-cellulose-selected HeLa mRNAs was as sensitive to inhibition by poly(A)150-300 as the mRNAs found in total cytoplasmic RNA. However, the translations of oligo(dT)-cellulose-selected viral mRNAs become more sensitive to the inhibitory effect of poly(A)150-300 than the translations of viral mRNAs found in the total cytoplasmic RNA. Both HeLa and VV mRNAs became more resistant to the poly(A)-mediated inhibition when these mRNAs were deadenylated, but the relative resistance to inhibition by poly(A)150-300 of deadenylated VV mRNAs was much greater than that of HeLa cell mRNAs. The translation of VV mRNAs was significantly less inhibited than the translation of HeLa mRNAs when the cap analog, m7GTP, was added to the cell-free system. The inhibition of HeLa cell mRNA translation by both poly(A)un and poly(A)150-300 was completely restored when poly(A)-binding protein (PAB) was added to the cell-free translational system. The addition of eukaryotic initiation factor 4A (eIF-4A) did not restore translation when poly(A)un was used to inhibit translation; however, inhibition by poly(A)150-300 was significantly reversed by this initiation factor. The reversal of poly (A)-mediated inhibition of HeLa cell mRNA translation was additive when PAB was used together with eIF-4A. Early VV mRNA translation was only slightly inhibited by poly(A)un (15%), and this inhibition was completely reversed by either PAB or eIF-4A.(ABSTRACT TRUNCATED AT 400 WORDS)