Simian virus 40 early mRNA's contain multiple 5' termini upstream and downstream from a Hogness-Goldberg sequence; a shift in 5' termini during the lytic cycle is mediated by large T antigen

Abstract

We have used primer-directed synthesis, separation, and sequencing of cDNA's to identify and localize the 5' termini of simian virus 40 early mRNA's. We have examined polyadenylated RNAs obtained from whole cytoplasm and polysomes of two transformed lines and from the cytoplasm of infected cells early and late in the lytic cycle, and we have attempted to correlate the results of our cDNA analyses with recent analyses of early cap structures. We have found that early mRNA's from transformed cells have three principal 5' termini, at residues 5,150, 5,154, and 5,155, with terminal transcribed sequences of CU, GC, and GG, respectively. These termini lie 21 to 26 nucleotides downstream from the early Hogness-Goldberg sequence. Transformed cell early mRNA's also contain a series of less abundant 5' termini that are copied from DNA sequences as far as 80 nucleotides downstream and a minimum of 70 to 75 nucleotides upstream from the Hogness-Goldberg sequence. The templates for the upstream 5' termini and the late simian virus 40 mRNA's overlap by a minimum of 60 to 65 nucleotides. Early mRNA's isolated from cells early in infection contain the same three principal 5' termini and downstream minor 5' termini as transformed cell mRNA's, but they lack 5' termini upstream from the Hogness-Goldberg sequence. With the onset of the late lytic phase, there is a progressive decreases in the utilization of the three principal 5' termini and additional downstream 5' termini and a progressive increase in the utilization of four major termini at residues 5,190 to 5,194, which are 10 to 15 nucleotides upstream from the Hogness-Goldberg sequence. With the onset of the late lytic phase, there is a progressive decrease in the utilization of the three principal 5' termini and additional downstream 5' termini and a progressive increase in the utilization of four major termini at residues 5,190 to 5,194, which are 10 to 15 nucleotides upstream from the Hogness-Goldberg sequence. This shift is evident in cells infected with a tsA mutant at the permissive temperature, but is aborted by growth at or shift-up to a restrictive temperature. Thus, this shift is mediated by the gene A product, large T antigen. We present two models, which are mutually exclusive, to account for the role of T antigen in the early-late shift. One involves transcription late in infection on a new DNA template synthesized during DNA replication. The second involves inhibition of initiation of early transcription at residues 5,150 to 5,155 and other downstream sites and a shift of transcription initiation principally to the upstream sites as a result of the binding of T antigen to two sites on simian virus 40 DNA downstream from the Hogness-Goldberg sequence.