Quantitation and Structural Analysis of SV40 RNAs

Abstract

The 5243-bp genome of SV40 contains two transcriptional units (see Fig. 1); the early one, which is first expressed early in the lytic cycle of infection, and the late unit, which is expressed at a significant level only after the onset of viral DNA replication (1). The early genes encode the viral regulatory proteins, small, and large T-antigens. These proteins play roles in replication of the viral genome and transcriptional transactivation of the late genes (see refs. 2–4 and references therein). The late genes encode the capsid proteins that encapsulate the viral DNA into new virions. The cis-acting transcriptional elements that regulate the synthesis of both the early and late viral RNAs (see refs. 5,6 and references therein), the mechanisms that regulate the temporal expression of the SV40 genome (see ref. 4 and references therein) and the patterns by which the viral transcripts are spliced (see refs. 7,8 and references therein) are well understood. In this chapter, we describe methods for quantifying and mapping the 5 ends of the viral RNAs synthesized in SV40-transfected cells. Minor variations of these methods can be used to map the 3 ends as well (9) and the splice sites (8) of the SV40 RNAs synthesized, not only in SV40-transfected cells, but also in SV40-infected and transformed cells.