Targeting of T7 RNA polymerase to tobacco nuclei mediated by an SV40 nuclear location signal

We have expressed two T7 RNA polymerase genes by electroporation into tobacco protoplasts. One of the genes was modified by inserting nucleotides encoding a viral nuclear localization signal (NLS) from the large T antigen of SV40. Both T7 RNA polymerase genes directed synthesis of a ca. 100 kDa protein in the electroporated protoplasts. T7 RNA polymerase activity was detected in extracts of protoplasts electroporated with both genes. Immunofluorescence analysis of these protoplasts indicated that only the polymerase carrying the NLS accumulated in the cell nucleus. These experiments suggest that mechanisms involved in the transport from the cytoplasm to the nucleus are similar in plant and animal cells. This system demonstrates the feasibility of T7 RNA polymerase-based approaches for the high-level expression of introduced genes in plant cells.     

The nuclear migration signal of Xenopus laevis nucleoplasmin.

Nucleoplasmin is the most abundant protein in the nucleus of Xenopus laevis oocytes. Its ability to target to the nucleus when microinjected into the cytoplasm has been the subject of many studies central to our understanding of how proteins segregate into nuclei. Using a cDNA clone we constructed beta-galactosidase-nucleoplasmin hybrids in modified bacterial expression vectors. The fusion proteins were expressed in Escherichia coli, purified and injected into the cytoplasm of X. laevis oocytes. The distribution of the fusion proteins between the cytoplasmic and nuclear compartments were analysed after incubation of various lengths of time. The results show that the signal sequence for nuclear transport is located close to the carboxy terminus of the protein. The signal sequence has been mapped to a small stretch of amino acids, containing a stretch of four lysines analogous to the SV40 large-T antigen signal.     

Simian virus 40 cRNA is processed into functional mRNA in microinjected monkey cells.

Monkey cells, microinjected with simian virus 40 (SV40) in vitro synthesized cRNA produce full-size tumor (T)-antigen. This was verified by analyzing immunoprecipitates of microinjected cells by polyacrylamide gel electrophoresis. Early SV40 DNA contains an intron within the large T-antigen coding sequences. Therefore, cRNA copied in vitro from the early DNA strand requires removal of the intron in order to become a functional mRNA. Polyadenylation of the cRNA in vitro by Escherichia coli poly(A)-polymerase increased the biological activity of the RNA. Detection of T-antigen by gel electrophoresis required as little as 50 poly(A)-cRNA injected cells. Splicing of the microinjected cRNA appears to be a nuclear process. Cells enucleated by cytochalasin B prior to injection do not synthesize large T-antigen. However, small t-antigen, a protein with a continuous sequence, is synthesized in these cells. Finally, it is shown that the process of splicing is not required for the transport of mRNA from the nucleus into the cytoplasm. Authentic T-antigen mRNA, isolated from virus infected cells, induced T-antigen synthesis with similar efficiency after either nuclear or cytoplasmic injection.     

Intracellular localization of the viral polymerase proteins in cells infected with influenza virus and cells expressing PB1 protein from cloned cDNA.

The biosynthesis, nuclear transport, and formation of a complex among the influenza polymerase proteins were studied in influenza virus-infected MDBK cells by using monospecific antisera. To obtain these monospecific antisera, portions of cloned cDNAs encoding the individual polymerase proteins (PB1, PB2, or PA) of A/WSN/33 influenza virus were expressed as fusion proteins in Escherichia coli, and the purified fusion proteins were injected into rabbits. Studies using indirect immunofluorescence showed that early in the infectious cycle (4 h postinfection) of influenza virus, PB1 and PB2 are present mainly in the nucleus, whereas PA is predominantly present in the cytoplasm of the virus-infected cells. Later, at 6 to 8 h postinfection, all three polymerase proteins are apparent both in the cytoplasm as well as the nucleus. Radiolabeling and immunoprecipitation analyses showed that the three polymerase proteins remain physically associated as a complex in either the presence or the absence of ribonucleoproteins. In the cytoplasm, the majority of the polymerase proteins remain unassociated, whereas in the nucleus they are present as a complex of three polymerase proteins. To determine whether a polymerase protein is transported into the nucleus individually, PB1 was expressed from the cloned cDNA by using the simian virus 40 late promoter expression vector. PB1 alone, in the absence of the other polymerase proteins or the nucleoprotein, accumulates in the nucleus. This suggests that the formation of a complex with other viral protein(s) is not required for either nuclear transport or nuclear accumulation of PB1 protein and that the PB1 protein may contain an intrinsic signal(s) for nuclear transport.     

Nuclear location of all three influenza polymerase proteins and a nuclear signal in polymerase PB2.

In order to re-examine the sub-cellular location of the three influenza A/NT/60/68 polymerase proteins PB1, PB2 and PA in infected cells, specific antisera for each polymerase component have been prepared by immunizing rabbits with polymerase-beta-galactosidase fusion proteins synthesized in Escherichia coli. We show that polymerase PB1, PB2, and PA are predominantly associated with the nucleus of influenza-infected MDCK cells by immunocytochemical techniques. In the case of polymerase PB2 we investigate the possibility that nuclear accumulation is an intrinsic property of the PB2 protein. Using a vaccinia-PB2 recombinant virus, we show that PB2 accumulates intra-nuclearly in monkey CV-1 cells in the absence of any other influenza protein, suggesting it contains an intrinsic nuclear signal.