Simian virus 40-rabbit beta-globin recombinants lacking late mRNA splice sites express cytoplasmic RNAs with altered structures.

Deletions were introduced at exon-intron boundaries in the late region of a simian virus 40-beta-globin cDNA recombinant to study the role of splicing in the formation of simian virus 40 late cytoplasmic RNAs. The recombinant was used as a wild type because it allowed characterization of mutant RNAs expressed from defective genomes in the presence of comparable RNAs contributed by the coinfecting helper virus. Removal of a 17-base pair segment at map position 0.76, which included a portion of the leader sequence implicated in the splicing of the major 16S mRNA, prevented expression of 16S-type mRNA. The same mutant accumulated cytoplasmic 19S-type mRNA, but the assortment of the 5' ends of these mRNAs differed from the assortment of the wild-type counterparts. Another mutant that lacks nucleotide sequences implicated in the splicing of the major 16S mRNA and one of the principal 19S-type RNAs accumulated a 16S-type mRNA with a previously undetected leader splice, and assortment of 19S mRNAs with new or normally underrepresented splices, and even a species of unspliced cytoplasmic 19S mRNA.     

Nucleotide sequence of the EcoRI D fragment of adenovirus 2 genome

The entire nucleotide sequence of the Ad. 2 EcoRI D fragment has been determined using the Maxam and Gilbert method. This sequence of 2678 bp contains informations relative to late mRNAs ending at position 78 and for which an AATAAA sequence corresponding to their 3' ends is found at residue number 833. Position of the PVIII mRNA is determined thus allowing deduction of the probable amino acid sequence of the PVIII protein. The position and the sequence of the first leader of early 3 mRNAs is determined as well as the sequence and position of the second early leader of region 3 mRNAs, which also correspond to the "y" leader of the fiber mRNA. Following the localization of an open reading frame in which an ATG could initiate protein synthesis it can be predicted that 3a, b, c mRNAs code for the 16K early protein and the probable amino acid sequence of this protein can be deduced. The CAGTTT sequence frequently present at the 5' end of a leader or of a mRNA body as well as the GGTGAG sequence which is found at the 3' end of several leaders were used to postulate the position of various early mRNAs of region 3 and to suggest the existence of an additional splicing event during the processing of mRNAs 3a, b and c. They were also used to predict the position of the additional "x" late leaders. The imbrication of information concerning (i) the family of late mRNAs ending at position 78, (ii) the position of the "x" leader and the "y" leader and (iii) the beginning of early region 3 is also depicted.     

Effect of the tripartite leader on synthesis of a non-viral protein in an adenovirus 5 recombinant.

The EIa region of an Adenovirus 5 recombinant has been substituted by a modular gene encoding dihydrofolate reductase (DHFR). In this recombinant, the mouse DHFR cDNA was positioned behind sequences of the major late promoter and the complete tripartite leader. The leader sequences end in the normal 5' splice site (SS) of the third leader, so that RNA splicing joins the tripartite leader to a 3' splice site immediately upstream of the DHFR cDNA. At late stages of infection, high levels of DHFR mRNAs were synthesized. At early times in the late stage, this mRNA was efficiently translated; however, at later times translation of DHFR decreased probably due to poor competition with other late mRNAs. Synthesis of DHFR protein from an analogous Adenovirus 5 recombinant containing only the first late leader was studied in parallel. Equivalent levels of DHFR mRNA were expressed after infection with this recombinant virus; however, the efficiency of DHFR translation was at least 20 fold lower than that of the DHFR mRNA containing the tripartite leader. This suggests that the tripartite leader sequence is important for translation in the late stage of infection. As reported previously, the Ad5 recombinant containing only the first leader vastly overexpresses polypeptide IX from a novel mRNA, formed by the splicing of the first leader in the modular DHFR gene to the 3' splice site in the EIb region. Cells infected with this recombinant synthesize very little normal mRNA from the EIb region. Here, we demonstrated that coinfection of 293 cells with this recombinant and wild type Adenovirus 5 also results in decreased EIb mRNA synthesis. We propose that the overproduction of polypeptide IX suppresses mRNA expression from the EIb and IX promoter sites, probably by an autoregulation loop active during lytic growth.     

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.