Construction and characterization of an SV40 mutant defective in nuclear transport of T antigen

An SV40-adenovirus 7 hybrid virus, PARA(cT), has been described that is defective for the nuclear transport of SV40 large tumor antigen. An SV40(cT) mutant was constructed using SV40 early and late region DNA fragments derived from PARA(cT) and wild-type SV40 respectively. The SV40(cT)-3 construct is defective for viral replication, but can be propagated in COS-1 cells. T antigen induced by SV40(cT)-3 is localized in the cytoplasm of infected cells. The cT mutation also inhibits the transport of wild-type T antigen; COS-1 cells lose their constitutive expression of nuclear T antigen after infection with SV40(cT)-3. Sequence analysis revealed that the cT mutation results in the replacement of a positively charged lysine in wild-type T antigen with a neutral asparagine at amino acid number 128, demonstrating that the alteration of a single amino acid is sufficient to abolish nuclear transport. Implications of the cT mutation on possible mechanisms for the transport of proteins to the nucleus are discussed.     

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.     

Appropriately spaced nuclear localizing signals are necessary for efficient nuclear import of nonnuclear proteins

To deliver nonnuclear proteins into the nucleus, we have examined the locations and number of nuclear localizing signals by use of simian virus 40 large T-antigen (SV40Ta) and yeast enhanced green fluorescent protein (yEGFP) in Saccharomyces cerevisiae as a model system. When only one SV40Ta was added to either the N- or C-terminus of yEGFP, the fluorescence of yEGFP was detected in both the nucleus and the cytoplasm. When two SV40Ta signals were added, one to the N-terminus and one to the C-terminus of yEGFP (SV40Ta-yEGFP-SV40Ta), the fluorescence of yEGFP was localized in only the nucleus. When the presequence of cytochrome oxidase subunit IV (pCOXIV) was inserted between the SV40Ta and the N-terminus of yEGFP (SV40Ta-pCOXIV-yEGFP-SV40Ta) in this construct, the fluorescence was located in both the nucleus and the cytoplasm, suggesting that the increased distance between the two SV40Ta signals decreased the efficiency of transport into the nucleus. When an additional SV40Ta signal was inserted between pCOXIV and yEGFP (SV40Ta-pCOXIV-SV40Ta-yEGFP), the fluorescence was localized only in the nucleus, indicating that two SV40Ta signals spaced by pCOXIV of 28 amino acid residues forming an alpha-helix are potent in transporting yEGFP into the nucleus. These results indicate that two SV40Ta signals spaced appropriately are essential for the efficient transport of the nonnuclear protein into the nucleus.