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.     

A splice junction deletion deficient in the transport of RNA does not polyadenylate nuclear RNA.

A late region deletion mutant of simian virus 40 (dl5) was previously shown to be deficient in the transport of nuclear RNA. This is a splice junction deletion that has lost the 3' end of an RNA leader, an intervening sequence, and the 5' end of the splice acceptor site on the body of the mRNA. In this report, we analyzed the steady-state structure of the untransported nuclear RNA. The 5' ends of this RNA are heterogeneous but contain a prominent 5' end at the normal position (nucleotide 325) in addition to several other prominent 5' ends not seen in wild-type RNA. The 3' end of this RNA does not occur at the usual position (nucleotide 2674) of polyadenylation; instead, this RNA is non-polyadenylated, with the 3' end occurring either downstream or upstream of the normal position.     

Mechanisms of synthesis of virion proteins from the functionally bigenic late mRNAs of simian virus 40.

The late 19S RNAs of simian virus 40 (SV40) are functionally polycistronic, i.e., all encode both VP2 and VP3. The VP3-coding sequences are situated in the same reading frame as the VP2-coding sequences, within the carboxy-terminal two-thirds of the VP2-coding sequences. To test whether VP3 is produced by proteolytic processing of VP2, we introduced a variety of deletion and insertion mutations within the amino-terminal end of the VP2-coding sequences. Genetic and biochemical analysis of the proteins synthesized in cells transfected with these mutants indicated that VP2 and VP3 were synthesized independently of each other. A leaky scanning model for the synthesis of VP3 was tested by the insertion of a strong initiation signal (CCAACATGG) upstream of the VP3-coding sequences. When the signal was placed in the same reading frame as VP3, synthesis of VP3 was reduced by a factor of 10 to 20, whereas synthesis of the expected VP3-related fusion protein occurred at a rate similar to that observed for VP3 in cells transfected with wild-type SV40 DNA. Insertion of this strong initiation signal at the same site, but in a different reading frame, resulted in the synthesis of VP3 at one-third of the wild-type rate. Mutation of the VP2 initiator AUG resulted in a small but reproducible (1.6-fold) increase in VP3 accumulation. From these experiments we conclude that (i) VP3 is synthesized predominantly by independent initiation of translation via a leaky scanning mechanism, rather than by proteolytic processing of VP2 or direct internal initiation of translation; (ii) a strong initiation signal 5' of the VP3-coding sequences can significantly inhibit synthesis of VP3, but does not act as an absolute barrier to scanning ribosomes; (iii) approximately 70% of scanning ribosomes bypass the VP2 initiator AUG, which is present in a weak context (GGUCCAUGG), and initiate at the VP3 initiation signal located downstream; and (iv) reinitiation of translation appears to occur on the SV40 late 19S mRNAs at an efficiency of 25 to 50%.     

Electron microscopic evidence for splicing of SV40 late mRNAs.

Poly(A)-containing SV40 cytoplasmic RNA was hybridized with linear double-stranded SV40 DNA and formed RNA displacement loops (R loops). The structures visualized in the electron microscope are consistent with the conclusion that the leader sequences at the 5' ends of the 16S and 19S late mRNAs are not coded immediately adjacent to the main portions of the mRNAs. These data are consistent with either segmentation of the leaders or heterogeneity of their lengths. Measurements carried out on the R loop structures have provided the locations, on the physical map of SV40 DNA, for the bodies and leaders of the 16S and 19S late mRNAs, and the lengths of the bodies, leaders and the corresponding intervening DNA sequences.     

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.     

The genome of human papovavirus BKV.

The complete DNA sequence of human papovavirus BKV(Dun), consisting of 5153 nucleotide pairs, is presented. We describe the segments of the genome which correspond to the replication origin, the tandem repeated sequences, the 5' and 3' ends of the mRNAs, the splice sites, the early and late viral proteins and the putative viral polypeptides. These BKV DNA sequences are compared with analogous regions in the SV40 and Py virus genomes in an attempt to localize viral functions for lytic growth and transformation.