Biochemical Studies on Adenovirus Multiplication, XIX. Resolution of Late Viral RNA Species in the Nucleus and Cytoplasm

Abstract

Late after infection of cultured human cells (KB) with adenovirus type 2, the nucleus contains heterogeneous viral RNA species ranging in size from 10 to 43 S. Four viral RNA species found in the nucleus (36, 38, 40, and 43 S) Cultured human cells infected with adenovirus type 2 (Ad 2) transcribe specific adenovirus gene sequences during productive infection (1-3). Before viral DNA synthesis (6 hr after infection) virus-specific RNA is transcribed from 15-20% of the Ad 2 genome (2); most of this RNA is detected as two major viral species, with sedimentation coefficients of 23S and 17S (4, 5). Late after infection (16-18 hr), virus-specific RNA is transcribed from 85 to 95% of the adenovirus genome (3). The virus-specific RNA species synthesized late after infection code for the synthesis of the 8-10 known capsid polypeptides, which range in molecular weight from 15,000 to 130,000 (6-9). We report here an analysis of the late-viral RNA species, present at 16-18 hr after infection, which reveals that high molecular weight viral RNA species sedimenting at 36-43 S are found in the nucleus and appear to be precursors of the 10-29S viral-specific RNA species found in the cytoplasm.MATERIALS AND METHODS Infection, cell fractionation, and RNA isolation Suspension cultures of KB cells, at 2-3 X 105 cells/ml, were infected with Ad 2 (strain 38-2) at an input multiplicity of 50 plaque-forming units (PFU)/cell, as described previously (3).[3H]uridine (4 yCi/ml, 20 Ci/mmol) was added to infected suspension cultures, cells were harvested at various times, and These measurements were performed with viral DNA immobilized on nitrocellulose filters as described (10, 11). Ad 2 DNA was prepared from highly purified virus (12).Polyacrylamide gel electrophoresis and zone centrifugation in sucrose density gradients Labeled RNA from infected cells was resolved on either 2.8 or 3.1% polyacrylamide gels, the gels were sliced, the RNA was eluted from each slice, and the viral RNA was quantitated by hybridization with viral DNA (4). RESULTSAd 2 late RNA species in the cell nucleus Nuclear RNA species from KB cells labeled with [3H]uridine for 15 min and 60 min, at 18 hr after infection with Ad 2, were resolved by electrophoresis on 2.8% polyacrylamide gels. Radioactive RNA was eluted from the gel slices and annealed with Ad 2 DNA. As many as 10 virus-specific RNA species larger than 26 S can be detected after 15-and 60-min labeling periods (Fig. 1). Nr-NIv appear to be the major viral RNA species labeled during a 15-min pulse with [8H]uridine (Fig. 1A). Increasing proportions of Nvi-Nx, as well as at least three smaller RNA species, were evident after 60 min of labeling (Fig. 1B). Treatment of nuclear RNA, labeled for 60 min, with 8 M urea (Fig. 2) or95% dimethylsulfoxide (notshown) did not alter the number of RNA species nor their rate of migration. Since it has been demonstrated that such treatment is sufficient to disrupt much of the secondary structure of the 70S RNA of murine leukemia virus (13, 14), we conclude that the...

Species Identification and Genome Mapping of Cytoplasmic Adenovirus Type 2 RNAs Synthesized Late in Infection

“…Late in adenovirus 2 infection, more than 80% of the polyadenylated cytoplasmic RNA is virus specified (22,42); therefore, the total profile of poly(A)+ cytoplasmic RNA is essentially identical to the profile of viral RNA. Although the size distribution obtained with formamide gels was in general agreement with previous studies using aqueous gel systems (6,22,29,36,42), one consistent difference was revealed. Although several earlier studies reported that some cytoplasmic viral RNA migrated significantly slower than 28S rRNA (22,29,42), such size classes have not been detected when formamide gels are used.…”

“…Although these RNAs are probably transcribed by RNA polymerase II (50), other details of their origin are not clear at present. Several observations suggest that the cytoplasmic molecules are not primary transcripts: nuclear RNAs synthesized at late times include sequences that are not in the cytoplasm (33,38,48), and nuclei contain RNAs that are apparently larger than the cytoplasmic species (5,29,48). One possibility is that there is only one major promoter for late transcription from the r strand and that most mRNA's are derived by processing a single large precursor (5).…”

Adenovirus DNA template for late transcription is not a replicative intermediate

“…Thus, although several early genes continue to be expressed (8,16,21), the transition from early to late phase results in a major change in the transcriptional program of adenovirus, in which the viral DNA replication seems to play a central role. This observation was further substantiated by experiments in which DNA replication was prevented by treating the infected cells with inhibitors of DNA (1,9,16,22) or protein synthesis (1,7,11,17), or by incubating at nonpermissive temperature cells that are infected with adenovirus mutants thermosensitive for viral DNA replication (2,5,6). In all cases early transcription occurred normally but there was no shift to late gene expression.…”

Transcription of the Human Cytomegalovirus Genome in Productively Infected Cells

“…Temporal regulation of early and late mRNA species has been demonstrated in a number of animal virus systems, e.g. simian virus 40 (Aloni et al, 1968;Parsons & Green, 1971), adenovirus (Thomas & Green, 1969), poxvirus (Kates & McAuslan, 1967), vaccinia virus (Salzman & Sebring, 1967), HSV (Frenkel & Roizman, 1972) and murine cytomegalovirus (MCMV) (Misra et al, 1978). In these systems, DNA synthesis inhibitors block late mRNA synthesis, indicating that early and late phases of virus gene expression are divided by virus DNA replication.…”