Polyoma virus giant RNAs contain tandem repeats of the nucleotide sequence of the entire viral genome.

Abstract

ABSTRACr The bulk of late virusspecific RNA synthesized in polyoma virus-infected mouse cells is larger than a single strand of polyoma DNA. The arrangement of viral nucleotide sequences in these giant polyoma RNAs was studied by electron microscopy of hybrids between purified high molecular weight viral RNA and the Hindu-i fragment of polyoma DNA, which contains 91% of the viral genome. Hybridimolecules containing a short single-stranded gap (corresponding to the 9% of viral sequences not present in Hind]-i), flanked by double-stranded regions, were photographed and measured. The majority of hybrid molecules contained no single-stranded loops or branches, showing that all viral sequences are transcribed contiguously and that no nonviral sequences are present in the RNA. Hybrid molecules, containing RNA up to 3.5 times the genome length, had a repeating structure of single-stranded gaps 8% of genome length interspersed with double-stranded regions 89% of genome length, showing that giant polyoma RNAs contain tandem repeats of the nucleotide sequence of the entire viral DNA. A small proportion of hybrid molecules contained single-stranded branches or deletion loops in characteristic positions, indicating that RNA "splicing" may occur on high molecular weight nuclear polyoma RNA.Transcription of polyoma DNA during the late phase of productive infection (for review, see ref. 1) gives rise to "giant" RNA molecules up to 3-4 times the size of the viral genome (2-5). These giant polyoma-specific RNAs are limited to the nucleus (6) where transcription takes place; viral mRNAs in the cytoplasm are one-quarter to one-half the size of the viral DNA (6-8). Most late nuclear polyoma-specific RNA is transcribed from the L strand of viral DNA (9); all sequences of the L strand are represented in late nuclear transcripts (10,11).To try to understand the mechanism by which these giant RNA molecules are synthesized, I have hybridized this RNA with a fragment (HindII-l) of polyoma DNA consisting of 91% of the genome (12,13) (Amersham,25 Ci/mmol) per culture dish in 1 ml of reinforced Eagle's medium. Cells were scraped into cold 10 mM triethanolamine, pH 8.5/10 mM NaCl/1.5 mM MgCl2 and lysed by addition of Nonidet P40 (Shell) to 0.5% final concentration. Nuclei were pelleted and resuspended in the same buffer. RNA was extracted by addition of 1 vol of 10 mM triethanolamine (pH 8.5)/100 mM NaCl/10 mM EDTA/4% triisopropylnaphthylenesulfonate (Serva) (14, 15) and 2 vol of phenol/chloroform/isoamyl alcohol, 50:50:1 (vol/vol). After three successive extractions at room temperature, nucleic acids in the aqueous phase were precipitated three times in succession with ethanol and then incubated at 00 for 20 min with DNase I (RNase-free; Worthington; 10,Mg/ml) in 10 mM Na acetate, pH 5.1/100 mM NaCI/2 mM MnCI2. RNA was extracted with phenol/chloroform in the presence of 1% sodium dodecyl sulfate (NaDodSO4) and ethanol-precipitated two times.Preparative Hybridization of RNA to Polyoma DNA. Polyoma form I DNA was denatured and bound to nitr...

Transcription from the polyoma late promoter in cells stably transformed by chimeric plasmids.

“…Mutation within these sequences results in an extended message which can be unstable (25). Since giant nuclear late transcripts are observed during polyoma lytic infection, the possibility exists that the polyoma late polyadenylation signal functions inefficiently in generating a proper 3' terminus of a message (1,2). Consequently, it is also possible that the inability to detect significant levels of late messages in polyoma-transformed cells is not due to an inactive promoter but instead to inefficient message cleavage or transcription termination and subsequent message instability.…”

“…Late lytic transcription is characterized by a heterogeneous mixture of 5' initiation sites (13,50). Additional diversity is generated by the splicing of giant multimeric nuclear transcripts that presumably result from repeated transcription of the circular genome (1). This splicing produces a noncoding leader sequence that contains a variable number of repeats of a 57-nucleotide segment and precedes the bodies of the messages for the three late viral proteins (21,28,32).…”

Use of a novel S1 nuclease RNA-mapping technique to measure efficiency of transcription termination on polyomavirus DNA.

“…To simplify the discussion, polyomavirus DNA is shown as an infinitely long linear tandem repeat, each repeat unit consisting of an entire 5.3-kb genome. This is formally equivalent to a circle from the point of view of an RNA polymerase molecule which is traversing the DNA unidirectionally and is in fact the form in which multigenome transcripts are produced (1). The left-hand end of this linear DNA molecule is defined as the site where transcription initiates, which we shall consider unique for the sake of argument.…”

“…RNA polymerase II does not efficiently terminate transcription on the L strand of polyomavirus DNA during the late phase of productive infection (1). As a result, some RNA polymerase molecules traverse the circular, 5.3-kilobase (kb), double-stranded polyomavirus genome several times before dissociating and releasing their RNA chains.…”

Synthesis of Prematurely Terminated Late Transcripts of Polyoma Virus DNA is Resistant to Inhibition by 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole

“…2(a) shows that virus RNA synthesized in untreated cells sedimented as a heterogeneous collection of molecules mainly in the region of 18S to 60S. These RNAs, some of which are already processed within minutes of their synthesis (N. H. Acheson, unpublished results), probably arise by continuous transcription of the L strand of circular virus DNA for several cycles before termination occurs (Acheson et al, 1971;Acheson, 1978). A small amount of labelled virus RNA sedimented near the top of the gradient in the 4S region.…”