Exon mutations that affect the choice of splice sites used in processing the SV40 late transcripts

The 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.     

Altered utilization of splice sites and 5'' termini in late RNAs produced by leader region mutants of simian virus 40.

We compared the 5' termini and splices of the late 16S and 19S RNAs synthesized by wild-type simian virus 40 and five mutants containing deletions in their late leader region. All mutants produced more unspliced 19S RNA than did wild-type virus, and in two mutants, unspliced 19S RNA constituted more than 60% of the total 19S species. The other three mutants each utilized predominantly a different one of the three spliced species of 19S mRNA. All mutants also produced decreased quantities of 16S mRNA, indicating that they may be defective for splicing both late RNAs. None of the 5' termini of the 16S and 19S RNAs made by the five mutants predominated as in those made by the wild type. Some of the mutant 5' termini were the same as those used by the wild type, whereas others were different. Although present, the major 5'-end positions used by the wild type were frequently not used as major sites by the mutants. In addition, mutants with very similar deletion endpoints synthesized RNAs with different 5' ends. Thus, downstream mutations have a pronounced effect on the location of 5' ends of the late RNAs, and there is no obvious involvement of a measuring function in the placement of 5' ends. For all mutants and wild-type virus, the 5' termini used for 16S and 19S RNAs showed major differences, with some degree of correlation found between the 5' ends and the internal splices of specific mRNA species. A model for the regulation of simian virus 40 late gene expression is presented to explain these findings.     

Leader-encoded open reading frames modulate both the absolute and relative rates of synthesis of the virion proteins of simian virus 40.

Numerous viral and cellular RNAs are polycistronic, including several of the late mRNA species encoded by simian virus 40 (SV40). The functionally bicistronic major late 16S and functionally tricistronic major late 19S mRNA species of SV40 contain the leader-encoded open reading frames (ORFs) LP1, located upstream of the sequence encoding the virion protein VP1, and LP1*, located upstream of the sequence encoding the virion proteins VP2 and VP3. To determine how these leader ORFs affect synthesis of the virion proteins, monkey cells were transfected with viral mutants in which either the leader-encoded translation initiation signal was mutated or the length and overlap of the leader ORF relative to the ORFs encoding the virion proteins were altered. The levels of initiation at and leaky scanning past each initiation signal were determined directly by quantitative analysis of the viral proteins synthesized in cells transfected with these mutants. Novel findings from these experiments included the following. (i) At least one-third of ribosomes bypass the leader-encoded translation initiation signal, GCCAUGG, on the SV40 major late 16S mRNA. (ii) At least 20% of ribosomes bypass even the consensus translation initiation signal, ACCAUGG, when it is situated 10 bases from the 5' end on the major late 16S mRNA. (iii)O The presence of the leader ORF on the bicistronic 16S mRNA species reduces VP1 synthesis threefold relative to synthesis from a similar RNA that lacks it. (iv) At least half and possibly all VP1 synthesized from the bicistronic 16S mRNA species is made by a leaky scanning mechanism. (v) LP1 and VP1 are synthesized from the bicistronic 16S mRNA species at approximately equal molar ratios. (vi) Approximately half of the VP1 synthesized in SV40-infected cells is synthesized from the minor, monocistronic 16S mRNA even though it accounts for only 20% of the 16S mRNA present. (vii) The presence and site of termination of translation of the leader ORF on the late 19S mRNAs affect the relative as well as absolute rates of synthesis of VP2 and VP3.     

Both VP2 and VP3 are synthesized from each of the alternative spliced late 19S RNA species of simian virus 40.

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.     

Accumulation of bacteriophage T7 head-related particles in an Escherichia coli mutant.

We have analyzed the structure of the late cytoplasmic RNAs made after infection with wild-type simian virus 40 and a set of viable mutants, four of which have deletions and one an insertion within the nucleotide sequence specifying the leader segment of the 16S and 19S mRNA's. The principal findings are: (i) simian virus 40 16S and 19S mRNA's made during infections with wild-type virnds and possibly in the nucleotide sequence comprising the "leader" segments. (II) "Spliced" 16S and 19S mRNA's are made during infections with each of the mutants although, in some cases, the ratio of 19S to 16S mRNA species is reduced. (iii) The deletion or insertion of nucleotides within the DNA segment defined by map position 0.70 to 0.75 causes striking alterations in the types of leader structures in the late mRNAs. (iv) Many of the late RNA leader segments produced after infection with the mutants appear to be multiply spliced, i.e., instead of the major 200- to 205-nucleotide-long leader segment present in wild-type 16S mRNA, the RNAs produced by several of the deletion mutants have leaders with whort discontiguous segments.