Regulation of simian virus 40 gene expression in Xenopus laevis oocytes.

Expression of the simian virus 40 (SV40) early and late regions was examined in Xenopus laevis oocytes microinjected with viral DNA. In contrast to the situation in monkey cells, both late-strand-specific (L-strand) RNA and early-strand-specific (E-strand) RNA could be detected as early as 2 h after injection. At all time points tested thereafter, L-strand RNA was synthesized in excess over E-strand RNA. Significantly greater quantities of L-strand, relative to E-strand, RNA were detected over a 100-fold range of DNA concentrations injected. Analysis of the subcellular distribution of [35S]methionine-labeled viral proteins revealed that while the majority of the VP-1 and all detectable small t antigen were found in the oocyte cytoplasm, most of the large T antigen was located in the oocyte nucleus. The presence of the large T antigen in the nucleus led us to investigate whether this viral product influences the relative synthesis of late or early RNA in the oocyte as it does in infected monkey cells. Microinjection of either mutant C6 SV40 DNA, which encodes a large T antigen unable to bind specifically to viral regulatory sequences, or deleted viral DNA lacking part of the large T antigen coding sequences yielded ratios of L-strand to E-strand RNA that were similar to those observed with wild-type SV40 DNA. Taken together, these observations suggest that the regulation of SV40 RNA synthesis in X. laevis oocytes occurs by a fundamentally different mechanism than that observed in infected monkey cells. This notion was further supported by the observation that the major 5' ends of L-strand RNA synthesized in oocytes were different from those detected in infected cells. Furthermore, only a subset of those L-strand RNAs were polyadenylated.     

Oligonucleotide-targeted degradation of U1 and U2 snRNAs reveals differential interactions of simian virus 40 pre-mRNAs with snRNPs.

We have investigated the roles of U1 and U2 snRNP particles in SV40 pre-mRNA splicing by oligonucleotide-targeted degradation of U1 or U2 snRNAs in Xenopus laevis oocytes. Microinjection of oligonucleotides complementary to regions of U1 or U2 RNAs either in the presence or absence of SV40 DNA resulted in specific cleavage of the corresponding snRNA. Unexpectedly, degradation of U1 or U2 snRNA was far more extensive when the oligonucleotide was injected without, or prior to, introduction of viral DNA. In either co-injected or pre-injected oocytes, these oligonucleotides caused a dramatic reduction in the accumulation of spliced SV40 mRNA expressed from the viral late region, and a commensurate increase in unspliced late RNA. When pre-injected, two different U2 specific oligonucleotides also inhibited the formation of both large and small tumor antigen spliced early mRNAs. However, even when, by pre-injection of a U1 5' end-specific oligonucleotide, greater than 95% degradation of the U1 snRNA 5' ends occurred in oocytes, no reduction in early pre-mRNA splicing was observed. In contrast, the same U1 5' end oligonucleotide, when added to HeLa splicing extracts, substantially inhibited the splicing of SV40 early pre-mRNA, indicating that U1 mRNP is not totally dispensable for early splicing. These findings confirm and extend our earlier observations which suggested that different pre-mRNAs vary in their requirements for snRNPs.     

Efficient expression of small RNA polymerase III genes from a novel simian virus 40 vector and their effect on viral gene expression.

In the past, simian virus 40 (SV40) has been used as a cloning vehicle to clone foreign genes by substituting portions of the viral genome vital for viral replication. Propagation of these defective viruses required a helper virus and the recombinant viruses obtained could be grown only as a mixture. In this study, we describe a novel nondefective SV40 vector to clone small RNA polymerase III genes. Two small RNA polymerase III genes, an amber suppressor human serine tRNA gene and the adenovirus (Ad) VAI RNA gene, were cloned in the intron region of the large-T antigen gene of SV40 after deleting DNA sequences coding for the small-t polypeptide. The recombinant viruses grew to wild type levels and showed no growth defects. When CV-1p cells were infected with these viruses, the cloned RNA polymerase III genes were expressed at high levels at late times. Interestingly, large amounts VAI RNA in CV-1p cells infected with SV40-VA recombinant virus, did not enhance translation of viral mRNAs significantly but did lead to a 3 to 4 fold increase in the steady state levels of large-T mRNA suggesting a novel function for VAI RNA in SV40 infected monkey cells. Furthermore, VAI mutants which fail to function in Ad infected human cells also failed to enhance the levels of large-T mRNAs in monkey cells infected with SV40. The simple SV40 vector described here may be useful to study the structure and function of small RNA polymerase III genes in the context of a eucaryotic chromosome. In addition, the nondefective recombinant SV40 which expresses the suppressor tRNA gene at high levels may provide a useful helper system to propagate animal viruses with amber mutations in essential genes.     

Initiation of simian virus 40 DNA replication in vitro.

Exogenously added simian virus 40 (SV40) DNA can be replicated semiconservatively in vitro by a mixture of a soluble extract of HeLa cell nuclei and the cytoplasm from SV40-infected CosI cells. When cloned DNA was used as a template, the clone containing the SV40 origin of DNA replication was active, but a clone lacking the SV40 origin was inactive. The major products of the in vitro reaction were form I and form II SV40 DNAs and a small amount of form III. DNA synthesis in extracts began at or near the in vivo origin of SV40 DNA synthesis and proceeded bidirectionally. The reaction was inhibited by the addition of anti-large T hamster serum, aphidicolin, or RNase but not by ddNTP. Furthermore, this system was partially reconstituted between HeLa nuclear extract and the semipurified SV40 T antigen instead of the CosI cytoplasm. It is clear from these two systems that the proteins containing SV40 T antigen change the nonspecific repair reaction performed by HeLa nuclear extract alone to the specific semiconservative DNA replication reaction. These results show that these in vitro systems closely resemble SV40 DNA replication in vivo and provide an assay that should be useful for the purification and subsequent characterization of viral and cellular proteins involved in DNA replication.     

Hybrid genomes of the polyomaviruses JC virus, BK virus, and simian virus 40: identification of sequences important for efficient transformation.

Hybrid viral genomes were used to investigate the influence of specific polyomavirus sequences on the transforming behavior of JC virus (JCV). One set of chimeric DNAs was made by exchanging the regulatory regions between JCV and simian virus 40 (SV40) or JCV and BK virus (BKV). A second set of constructs was produced that expressed hybrid JCV-BKV T proteins under the control of either JCV or BKV regulatory signals. Transformation of Rat 2 cells with the parental and chimeric DNAs indicated that both the JCV regulatory signals and the sequence encoding the amino terminus of T protein contributed to the restricted transforming behavior of this virus. Analysis of the viral proteins in the transformed rat cells indicated that the large T antigens of JCV and BKV were less stable than their SV40 counterpart, that small t protein was produced in JCV transformants, and that the subpopulation of T antigen that forms a stable complex with cellular p53 protein was smaller in JCV-transformed cells than in SV40- or BKV-transformed cells.     

Identification and mapping of N6-methyladenosine containing sequences in simian virus 40 RNA.

Late SV40 16S and 19S mRNAs were found to contain an average of three m6A residues per mRNA molecule. The methylated residues of both the viral and cellular mRNAs occur in two sequences; Gpm6ApC and (Ap)nm6ApC, where n = 1-4. More than 60% of the m6A residues in SV40 16S and 19S mRNAs occur in Gpm6ApC even though there are twice as many (A)nAC than GAC sequences in these messengers. The m6A containing oligonucleotides of late SV40 MRNAs were localized in the viral messengers. In the 16S mRNA two m6A oligonucleotides were located at the 5' coding region between 0.95--0.0 map units. The third m6A residue was mapped between 0.0--0.14 map units in the translated portion of this mRNA. The overall pattern of internal methylation in the 19S mRNA is similar. However, some differences between 16S and 19S mRNAs were observed in both the content and location of the longer (Ap)n m6AC nucleotides. These results provide the first example of precise localization of internal methylation sequences in mRNA species with defined coding specificity. It implies that a) location of m6A residues is not random but specific to a particular region of the RNA, b) apart from sequence specificity other structural features of the mRNA may influence internal methylation and c) m6A residues are present in coding regions of SV40 mRNAs.     

SV40 DNA injected into Xenopus oocyte nuclei is transcribed by RNA polymerase B.

SV40 DNA I. injected into Xenopus oocyte nuclei is transcribed. The SV40-specific RNA molecules migrate on sucrose gradients as do viral RNAs formed in infected green monkey cells but a variable proportion of RNA sequences complementary to SV40 DNA is also found in the light region of the gradients. All SV40-specific RNA species seem to be synthesized by RNA polymerase B as their synthesis is completely sensitive to low concentrations (0.1 microgram/ml) of alpha-amanitin. Concomittantly, the formation of SV40-specific proteins (tumor antigens) is inhibited by injecting alpha-amanitin together with the SV40 DNA.     

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.