RNA synthesis in cells infected with herpes simplex virus. XII. Sequence complexity and properties of RNA differing in extent of adenylation

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Abstract
Fractionation of polyadenylated RNA from [human epidermoid carcinoma HEp-2] cells infected with herpes simplex virus by affinity chromatography on columns of poly(U) immobilized on glass-fiber filters yielded 3 major classes of RNA-containing poly(A) chains with average lengths of 30, 50, and 155 adenylate residues [poly(A)30, poly(A)50, poly(A)155]. Nitrocellulose membranes bound predominantly a fraction of RNA containing poly(A)155. The distribution of cytoplasmic RNA in the 3 classes was independent of the labeling interval, ranging from 10 min-6 h. Cytoplasmic poly(A) RNA consisted mainly (57-68%) of the poly(A)155 class; this was also the major class (68%) of polyadenylated RNA found in polyribosomes. Nuclear poly(A) RNA consisted largely (42-50%) of poly(A)30 class; high MW nuclear RNA sedimenting at > 45S contained almost exclusively the poly(A)30 tracts. Hybridization experiments involving unlabeled RNA and labeled viral DNA demonstrated the presence of viral RNA sequences complementary to .apprx. 40% of viral DNA in all polyadenylated RNA classes. Since unfractionated cytoplasmic RNA arises from 40% of the viral DNA, most, if not all, viral RNA species present in the cytoplasm are probably adenylated. However, only a fraction of poly(A)155 RNA, complementary to 21% of viral DNA, bound to nitrocellulose filters. The selective binding of poly(A)155, sequences to nitrocellulose filters might be related to its secondary structure, since transcripts homologous to 40% of viral DNA bind to nitrocellulose membranes, provided the RNA is denatured prior to filtration. Poly(A) tracts apparently arise by at least 2 separate steps. The 1st involves the appearance of poly(A)30 tracts in the high MW nuclear transcripts. The 2nd involves polyadenylation to poly(A)50 and poly(A)155 RNA classes concomitant with processing and transport to the cytoplasm.