ABSTRACrThe spliced species of late SV40 RNAs present in the cytoplasm of cells infected with various wild-type and mutant strains of SV40 that differ in their leader regions were determined using a novel modification of the primer extension method and the S1 nuclease mapping technique. These data indicated that mutations within the first exon of the late RNAs can affect dramatically the utilization of downstream donor and acceptor splice sites. In one instance, a ten base pair insertion within the predominant first exon increased utilization of an infrequently utilized donor splice site such that the small alteration became part of an intervening sequence, thereby suggesting a novel mechanism for regulation of gene expression. In addition, our method enabled detection of a previously unidentified spliced species, representing less than one percent of the SV40 late 19S RNA present in cells infected with wild-type virus, that may be an intermediate in the synthesis of a known doubly spliced 16S RNA species of SV40.

The late 19S RNAs of simian virus 40 consist of a family of alternatively spliced RNAs, each of which contains open reading frames corresponding to all three of the virion proteins. Two approaches were used to test the hypothesis that each alternatively spliced 19S RNA species is translated to synthesize preferentially only one of the virion proteins. First, we analyzed the synthesis of virion proteins in simian virus 40 mutant-infected monkey cells that accumulate predominantly either only one spliced 19S RNA species or only the 19S RNAs. Second, we determined the virion proteins synthesized in a rabbit reticulocyte lysate programmed with specific, in vitro-transcribed 19S RNA species. These results indicated that VP2 and VP3, but not VP1, are synthesized from all 19S RNA species. Quantitative analysis of these data indicated that individual 19S RNA species containing a translation initiation signal upstream of the VP2 AUG codon were translated in a cell extract three- to fivefold less efficiently than were 19S RNA species lacking this signal and that the precise rate of synthesis of VP2 relative to VP3 varied somewhat with the sequence of the leader region. These data are consistent with the synthesis of VP2 and VP3 occurring by a leaky scanning mechanism in which initiation of translation at a specific AUG codon is affected by both (i) the intrinsic efficiency of ribosomes recognizing the sequences surrounding the AUG codon as an initiation signal and (ii) partial interference from 5'-proximal initiation signals and their corresponding open reading frames.

The 5' ends of the simian virus 40 (SV40) late RNAs are heterogeneous in location, spanning a 300-nucleotide region from residues 28 to 325. To examine whether upstream or downstream measuring functions analogous to the TATA box play roles in positioning the 5' ends of these RNAs, we determined by S1 and primer extension mapping the locations of the 5' ends of the late viral RNAs made in monkey cells infected with: (i) three wild-type strains of SV40 that contain tandem duplications of the enhancer region that are 64, 85, and 91, rather than 72, base pairs in length; (ii) four viable mutants that contain alterations in the 21-base-pair tandem repeats; and (iii) four viable mutants that possess small deletions or insertions at or near the major cap site at residue 325. Most of the 5' ends of the RNAs were identical in location to those seen with wild-type strain 776. The only exceptions were the absence of RNAs whose 5' ends mapped to within three bases upstream or downstream of a sequence alteration. In addition, the sequences within residues 251 to 277 that function as transcriptional initiation sites in wild-type strain 776 also did so in their second locations in the wild-type strains in which these sequences are duplicated. Differences were noted in the relative abundances of the numerous 5' ends of the late RNAs, even among the wild-type strains. These findings indicate that many (and likely all) of the approximately two dozen locations of 5' ends of SV40 late RNAs are each determined largely by sequences within their immediate vicinity. However, sequences somewhat removed from these transcriptional initiation sites may modulate the efficiencies with which they are utilized.

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