Quantitation of herpes simplex virus type 1 RNA in infected HeLa cells

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Abstract
The size and amount of herpes simplex virus (HSV)-specific RNA synthesized in [human cervical carcinoma] HeLa cells were quantitatively analyzed using DNA and RNA excess hybridization. At 2 h after infection (early), transcripts from 20% of the total HSV DNA are present on polyribosomes as poly(A+) RNA. At this time viral poly(A+) RNA comprises 60-75% of the poly(A+) newly synthesized poly(A+) mRNA on polyribosomes. By 6 h after infection (late), poly(A+) HSV RNA transcribed from 35-40% of the viral DNA is found on polyribosomes. These viral poly(A+) transcripts comprised as much as 90% of newly synthesized poly(A+) mRNA and are measurably larger than viral poly(A+) transcripts isolated early. Some but not all of this size difference is due to the poly(A) tails on early transcripts being shorter than those found on transcripts made late. Even late after infection a small but readily measurable amount of cellular poly(A+) RNA is still being made and entering polyribosome complexes. In the nucleus late after infection, poly(A+) HSV RNA is complementary to 50% of the total HSV DNA. Early and late after infection total nuclear viral transcripts are, on the average, larger than viral transcripts found on polyribosomes, but nuclear HSV poly(A+) RNA is not measurably larger than the corresponding cytoplasmic viral poly(A+) sequences at either time. A major portion (30-40%) of the polyribosomal HSV RNA made early or late after infection is not polyadenylated. This HSV poly(A-) RNA is transcribed from the same sequences as HSV poly(A+) RNA, but when labeled and isolated early or late after infection, both nuclear and polyribosomal viral poly(A-) RNA molecules sediment faster in sucrose-formaldehyde gradients than their polyadenylated counterparts.

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