Characterization of early simian virus 40 transcriptional complexes: late transcription in the absence of detectable DNA replication.

Abstract

Isolation of early viral transcriptional complexes and incorporation in vitro of radiolabeled precursors into nascent RNA has permitted an analysis of early simian virus 40 (SV40) transcription. Under conditions such that viral DNA replication was undetectable, both early and late SV40 RNA were synthesized. This finding provides evidence that viral DNA replication is not an absolute requirement for late transcription and supports earlier observations that late viral RNA is synthesized in SV40-infected nonpermissive mouse cells. The majority of the early viral transcriptional activity can be solubilized, indicating that a substantial portion of this RNA is transcribed from free rather than integrated templates. Sedimentation analysis of the transcriptional complexes resulted in the detection of two separate peaks of activity, suggesting the possibility of two distinct types of early SV40 templates.It has been known for some time that simian virus 40 (SV40) RNA is synthesized in two phases (1-3). Prior to viral DNA replication, most of the viral mRNA appears to be transcribed from the early DNA strand (4-6). Subsequently, the bulk of viral transcription occurs from the late DNA strand and is processed into late 16S and 19S viral mRNAs (7,8). It was generally assumed that this late phase of viral transcription was inextricably linked to viral DNA replication (see refs. 9 and 10). The absence of the late SV40 transcripts in most SV40-transformed cell lines supported this concept (11). Nevertheless, we consistently found late viral RNA sequences in abortively infected mouse cells, in which SV40 DNA replication could not be detected (5, 12). Furthermore, recent studies with early temperature-sensitive mutants of SV40 grown at 410 (the nonpermissive temperature) indicated the presence of a small but reproducible fraction of late virus-specific cytoplasmic RNA (13). This led us to speculate that viral DNA replication was not an absolute requirement for the transcription of sequences on the late SV40 DNA strand. It was difficult to test this hypothesis directly, because of the low levels of late virus-specific RNA found prior to viral DNA replication (1-6, 14) and the potential rapidity and efficiency of the RNA processing system (15).We recently described the adaptation of an in vitro transcriptional system (16) established by Gariglio and Mousset (17). The supernatant fraction of SV40-infected cell nuclei, lysed in the presence of Sarkosyl, actively incorporates radiolabeled ribonucleotide triphosphates, and the newly synthesized RNA can be easily analyzed. Although it is unclear whether putative termination signals are recognized under such incorporation conditions, the absence of reinitiation allows one to assess accurately the frequency of in vivo transcription of the two viral DNA strands (16). Further advantages of this in vitro transcriptional system include: (i) the enrichment of virus-specific RNA sequences in the nuclear supernatant fraction; (ii) an increase in the specific activity of the viral RNA t...

Methylation of nuclear simian virus 40 RNAs

“…In contrast, these transcripts represent only 1 or 2% of the cytoplasmic SV40 RNA ( Fig. 5B and C), confirming previous observations (12,22,23). Since the proportion of E transcripts to L transcripts in the cytoplasm remains almost constant for various labeling periods (22), it seems likely that the E transcripts are less effi- Fig.…”

“…The molecular biology of simian virus 40 (SV40) has been under intensive investigation for a number of years, and these studies l;ave provided considerable information regarding the regulation of gene expression, in particular the transcriptional and posttranscriptional processing of mRNA (for review see references 1 and 19). At least two control mechanisms regulate SV40 gene expression in productively infected cells; one operates at the transcriptional level, and the second operates at the posttranscriptional level (9,12,20,22,23). Evidence for the first control mechanism is the observation that at late times after infection about 10% of the nuclear viral RNA is transcribed from the early (E) strand and 90% is transcribed from the late (L) strand (12,22,23).…”

“…At least two control mechanisms regulate SV40 gene expression in productively infected cells; one operates at the transcriptional level, and the second operates at the posttranscriptional level (9,12,20,22,23). Evidence for the first control mechanism is the observation that at late times after infection about 10% of the nuclear viral RNA is transcribed from the early (E) strand and 90% is transcribed from the late (L) strand (12,22,23). A posttranscriptional control mechanism is suggested by the different frequencies of E-and L-RNA transcripts found in the cytoplasm.…”

Effect of sodium butyrate on induction of cellular and viral DNA syntheses in polyoma virus-infected mouse kidney cells

“…This has often been interpreted to indicate that the switch from the expression of early viral genes to that of late genes depends on viral DNA replication or at least its initiation (15,46). More recent experiments have shown that in both polyoma virus-infected mouse cells and simian virus 40-infected monkey cells, late transcripts can be detected in the absence of viral DNA synthesis (3,9,18,30,32). As butyrate exerts its effect in the Gl period of the cell cycle, about 4 h before the onset of cellular and viral DNA synthesis in polyoma virus-infected mouse kidney cells, this system is particularly suitable to study the potential role of virus replication in the expression of late viral genes.…”

Stimulation of simian virus 40 late gene expression by simian virus 40 tumor antigen.

“…1). The factors responsible for the early-to-late transition in SV40 gene expression has been the subject of a number of reports (2)(3)(4)(5)(6). This transition results, at least in part, from autoregulation by the SV40 large T antigen, which inhibits early gene transcription by binding to the region of the early promoter (7)(8)(9)(10)(11)(12)(13)(14).…”