The length but not the sequence of the polyoma virus late leader exon is important for both late RNA splicing and stability.

Polyoma virus late RNA processing provides a convenient model system in which to study the mechanics of splicing in vivo. In order to understand further the role of the untranslated "late leader" unit in late RNA processing we have constructed a group of polyoma viruses with deletions and substitutions in the leader exon. This has allowed us to determine that there is a minimum exon size required for both pre-mRNA splicing and stability in this system. We show here that the non-viability of a mutant (ALM) with a 9 base late leader unit is due to a general defect in late RNA splicing. In addition, ALM-infected cells show at least 40-fold depression in the accumulation of late nuclear RNA (spliced or unspliced). The ALM late promoter, however, functions nearly normally. Substituted leader variants with 51- to 96-base long exons of unrelated sequence are viable (G. Adami and G. Carmichael, J. Virol. 58, 417-425, 1986). We show here that late RNA from one of these substituted leader mutants (containing a 51-base leader exon) is spliced at wild type levels, with virtually no defect in accumulation. Thus, in the polyoma system, splice sites separated by only 9 bases can inhibit each others usage, presumably by steric interference. We suggest that this type of inhibition leads to extreme RNA instability.     

Electron microscopic demonstration of the presence of amplified sequences at the 5''-ends of the polyoma virus late mRNAs.

Electron microscopic techniques were used to examine the structure of the leader sequences at the 5'-ends of the late polyoma virus mRNAs. The three late mRNA's were partially purified and hybridized to an E. coli plasmid containing two polyoma virus genomes inserted in tandem. The hybrids were spread by the cytochrome c-formamide technique and visualized in the electron microscope. These studies revealed that whereas the body of a given mRNA molecule can hybridize with only one of the two corresponding body sequences in the two adjacent viral genomes, the leader of the same mRNA molecule can hybridize with both copies of the leader sequence-specific DNA. The mVP1 and mVP3 RNA species thus generated hybrids containing two loops, while mVP2 molecules formed hybrids containing one loop. Hence, the leaders of the three polyoma virus late mRNA species must contain two or more repeats of a sequence transcribed from a unique DNA segment. Length measurements showed that most leaders in the late mRNA's consist of at least 200 nucleotides and some contain up to 500 nucleotides, whereas the basic repeat sequence contains about 60 nucleotides.     

Polyoma-induced stimulation of cellular RNA synthesis is paralleled by changed expression of the viral genome.

We studied synthesis of viral and cellular RNA in the presence and absence of 5-fluorodeoxyuridine (FdU, an inhibitor of DNA synthesis) during lytic infection with polyoma virus in confluent, primary mouse kidney cell cultures. In the presence of FdU, synthesis of early 19S polyoma mRNA and of polyoma tumor (T)-antigen, i.e. expression of the early viral gene, is rapidly followed by a mitogenic reaction of the host cell; it leads to an increase of 30 +/- 5% in cellular, mainly 28S and 18S rRNA, followed by activation of the cellular DNA-synthesizing apparatus. Polyoma-induced cellular RNA synthesis is paralleled by increased production of early 19S mRNA and begin of expression of the late viral genes, leading to synthesis of small amounts of late 19S and 16S mRNAs. Changed expression of the viral genome occurs in the absence of detectable synthesis of polyoma DNA I. Infection in the absence of FdU induces the same sequence of events; it is followed, however, by duplication of the mouse cell chromatin (S-phase) and production of progeny virus.     

Polyoma virus DNA: Sequence from the late region that specifies the leader sequence for late mRNA and codes for VP2, VP3, and the N-terminus of VP1.

The DNA sequence of part of the late region of the polyoma virus genome is presented. This sequence of 1,348 nucleotide pairs encompasses the leader region for late mRNA and the coding sequence for the two minor capsid proteins VP2 and VP3. The coding sequence for the N-terminus of the major capsid protein overlaps the C-terminus of VP2/VP3 by 32 nucleotide pairs. From the DNA sequence the sizes and sequences of VP2 and VP3 could be predicted. Potential splicing signals for the processing of late mRNA's could be identified. Comparisons are made between the sequence of polyoma virus DNA and corresponding regions of simian virus 40 DNA.     

Polyoma infected cells contain at least three spliced late RNAs.

Poly(A)-containing polyoma cytoplasmic RNA was hybridized with linear double-stranded polyoma DNA and RNA displacement loops (R-loops) were formed. The structures visualized in the electron microscope are consistent with the conclusion that there are at least three late polyoma specific RNAs and that the leader sequences at the 5' ends of these viral RNAs are not coded immediately adjacent to the bodies of the RNAs. Measurements carried out on the R-loop structures have provided the locations on the physical map of polyoma DNA, for the bodies and leaders of the RNAs and the length of the bodies, leaders and the corresponding intervening DNA sequences.     

Vaccinia virus produces late mRNAs by discontinuous synthesis

We describe the unusual structure of a vaccinia virus late mRNA. In these molecules, the protein-coding sequences of a major late structural polypeptide are preceded by long leader RNAs, which in some cases are thousands of nucleotides long. These sequences map to different regions of the viral genome and in one instance are separated from the late gene by more than 100 kb of DNA. Moreover, the leader sequences map either upstream or downstream of the late gene, are transcribed from either DNA strand, and are fused to the late gene coding sequence via a poly(A) stretch. This demonstrates that vaccinia virus produces late mRNAs by tagging the protein-coding sequences onto the 3' end of other RNAs.     

Multiple 5' terminal cap structures in late polyoma virus RNA.

Nuclear and cytoplasmic polyoma virus-specific RNA extracted from 32P-labeled mouse embryo cells late during productive viral infection was analyzed for the presence of 5' terminal capped structures by complete digestion with RNAases T1, T2 and A, followed by two-dimensional electrophoretic fractionation. Seven major cap I structures (m7 GpppNm1pN2p) were observed in both cases. These termini were further characterized by digestion with penicillium nuclease P1, followed by product analysis in a variety of alternative separate systems. Each structure had an individual combination of N1 and N2 nucleotides, where N1 was always a purine nucleotide but N2 was any nucleotide subject to the single exception that m7GpppGmpCp is found only in low yield. Four different cap II derivatives (m7GpppNm1pNm2pN3p) of four of the cap I structures were also detected in cytoplasmic RNA. None of the termini described derived from contaminating host cell RNA. All of these cap structures mapped on the polyoma viral DNA genome between 66 and 71 map units, a region distant from the 5' end of the bodies of two of the three late polyoma mRNAs. All the polyoma virus-specific cap structures, however, were present in each of the purified 16S, 18S and 19s late mRNAs. These data suggested that families of capped leader sequences of varying sizes are attached to the main body of each late polyoma mRNA species by a splicing mechanism.     

Sequence repeats in a polyoma virus DNA region important for gene expression.

The sequenced prototype strains (A2 and A3) of polyoma virus lack sequence duplications characteristic of other papovaviruses. However, we found that five polyoma virus strains (P16, Toronto large plaque, MV, Ts 48, and NG59R) contain tandemly duplicated sequences in a region near the late RNA leader. Although the duplications vary in size (31 to 84 base pairs) and location (between nucleotide [nt] 5068 and nt 5185), the sequence between nt 5114 and nt 5137 is contained within all five duplicated segments. This region is known to be important in polyoma virus early gene expression, and it contains sequences capable of enhancing the expression of nonviral genes. Inspection of the sequences at and around the ends of the repeats indicated that the duplications do not arise by homologous recombination, and there was no indication that a sequence-specific mechanism results in their formation. However, the variation in the structure of the repeats among different polyoma virus strains suggests that these sequence duplications are a recent evolutionary occurrence. The potential biological significance of this variation is discussed.     

Effect of a tsA mutation of simian virus 40 late gene expression: variations between host cell lines

Infection of AGMK or CV-1 cells by the early simian virus 40 mutant tsA58 at the permissive temperature (32 degrees C) followed by a shift to the nonpermissive temperature (41 degrees C) caused a substantial decrease in the levels of late viral RNA in the cytoplasm of AGMK cells but not CV-1 cells. At the translational level, this depression of late viral RNA levels was reflected by a decrease in late viral protein synthesis. Thus, in AGMK cells, an early region gene product (presumably large T-antigen) appeared to be continuously required for efficient expression of the late viral genes. In contrast, late simian virus 40 gene expression, once it is initiated in CV-1 cells, continued efficiently regardless of the tsA mutation. The difference in expression of the late simian virus 40 genes in these tsA mutant-infected monkey kidney cell lines may reflect a difference in host cell proteins which regulate viral gene expression in conjunction with early viral proteins.