A hydrophobic protein sequence can override a nuclear localization signal independently of protein context.

Simian virus 40 T antigen is specifically targeted to the nucleus by the signal Pro-Lys-Lys-128-Lys-Arg-Lys-Val. We have previously described the isolation of a simian virus 40 T-antigen mutant, 676FS, which retains a wild-type nuclear localization signal but fails to accumulate properly in the nucleus and interferes with the nuclear localization of heterologous proteins. Here we report that the hydrophobic carboxy-terminal sequence novel to 676FS T antigen overrides the nuclear localization signal if fused to other proteins, thereby anchoring the proteins in the cytoplasm. We discuss possible mechanisms by which missorting of such a fusion protein could interfere with the nuclear transport of heterologous proteins.

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Characterization of a Drosophila phosphorylation-dependent nuclear-localization-signal-binding protein

Biochemical Journal ◽

10.1042/bj3280821 ◽

1997 ◽

Vol 328 (3) ◽

pp. 821-826

Author(s):

Imre CSERPÁN ◽

Endre MÁTHÉ ◽

András PATTHY ◽

Andor UDVARDY

Keyword(s):

Nuclear Localization ◽

Protein Interactions ◽

Nuclear Localization Signal ◽

Binding Protein ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Localization Signal ◽

Co Precipitation ◽

Drosophila Embryos

A 94 kDa nuclear-localization-signal (NLS)-binding protein was purified from Drosophila embryos. The NLS of the simian-virus-40 T-antigen is specifically bound by the dephosphorylated form of the protein. After phosphorylation, the affinity of the protein for the NLS is sharply decreased. In the dephosphorylated form, p94 (protein of 94 kDa) is the major NLS-binding protein in Drosophila embryos. Immunoprecipitation confirmed the ATP-dependent phosphorylation of p94, and co-precipitation of two additional phosphorylated proteins, indicated that the NLS-binding protein is part of a larger complex in Drosophila embryos. In agreement with the immunoprecipitation results, cross-linking experiments demonstrated the interaction of p94 with three additional proteins. These protein-protein interactions were also phosphorylation-dependent.

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Simian Virus 40 Vp1 DNA-Binding Domain Is Functionally Separable from the Overlapping Nuclear Localization Signal and Is Required for Effective Virion Formation and Full Viability

Journal of Virology ◽

10.1128/jvi.75.16.7321-7329.2001 ◽

2001 ◽

Vol 75 (16) ◽

pp. 7321-7329 ◽

Cited By ~ 39

Author(s):

Peggy P. Li ◽

Akira Nakanishi ◽

Dorothy Shum ◽

Peter C.-K. Sun ◽

Adler M. Salazar ◽

...

Keyword(s):

Dna Binding ◽

Nuclear Localization ◽

Nuclear Localization Signal ◽

Simian Virus 40 ◽

Simian Virus ◽

Dna Binding Domain ◽

Localization Signal ◽

Carboxy Terminal

ABSTRACT A DNA-binding domain (DBD) was identified on simian virus 40 (SV40) major capsid protein Vp1, and the domain's function in the SV40 life cycle was examined. The DBD was mapped by assaying various recombinant Vp1 proteins for DNA binding in vitro. The carboxy-terminal 58-residue truncated Vp1ΔC58 pentamer bound DNA with aKd of 1.8 × 10−9 M in terms of the protein pentamer, while full-length Vp1 and carboxy-terminal-17-truncated Vp1ΔC17 had comparable apparentKd s of 5.3 × 10−9 to 7.3 × 10−9 M in terms of the protein monomers. Previously identified on Vp1 was a nuclear localization signal (NLS) consisting of two N-terminal basic clusters, NLS1 (4-KRK-6) and NLS2 (15-KKPK-18). Vp1ΔC58 pentamers harboring multiple-point mutations in NLS1 (NLSm1), NLS2 (NLSm2), or both basic clusters (NLSm1 · 2) had progressively decreased DNA-binding activity, down to 0.7% of the Vp1ΔC58 level for NLSm1 · 2 Vp1. These data, along with those of N-terminally truncated proteins, placed the DBD in overlap with the bipartite NLS. The role of the Vp1 DBD during infection was investigated by taking advantage of NLS phenotypic complementation (N. Ishii, A. Nakanishi, M. Yamada, M. H. Macalalad, and H. Kasamatsu, J. Virol. 68:8209–8216, 1994), in which an NLS-defective Vp1 could localize to the nucleus in the presence of wild-type minor capsid proteins Vp2 and Vp3. This approach made it possible to dissect the role of the bifunctional Vp1 NLS-DBD in virion assembly in the nucleus. Mutants of the viable nonoverlaping SV40 (NO-SV40) DNA NLSm1, NLSm2, and NLSm1 · 2 replicated normally following transfection into host cells and produced capsid proteins at normal levels. All mutant Vp1s were able to interact with Vp3 in vitro. The mutants NLSm1 and NLSm1 · 2 were nonviable, and the mutant Vp1s unexpectedly failed to localize to the nucleus though Vp2 and Vp3 did, suggesting that the mutated NLS1 acted as a dominant signal for the cytoplasmic localization of Vp1. Mutant NLSm2, for which the mutant Vp1's nuclear localization defect was complemented by Vp2 and Vp3, displayed a 5,000-fold reduced viability. Analysis of NLSm2 DNA-transfected cell lysate revealed a 10-fold reduction in the level of DNase I-protected viral DNA, and yet virion-like particles were found among the DNase I-resistant material. Collective results support a role for Vp1 NLS2-DBD2 in the assembly of virion particles. The results also suggest that this determinant can function in the infection of new cells.

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Peptides Containing Cyclin/Cdk-Nuclear Localization Signal Motifs Derived from Viral Initiator Proteins Bind to DNA When Unphosphorylated

Journal of Virology ◽

10.1128/jvi.76.23.11785-11792.2002 ◽

2002 ◽

Vol 76 (23) ◽

pp. 11785-11792 ◽

Cited By ~ 6

Author(s):

Ronald J. Kim ◽

Stephanie Moine ◽

Danielle K. Reese ◽

Peter A. Bullock

Keyword(s):

Dna Replication ◽

Viral Replication ◽

Nuclear Localization ◽

Nuclear Localization Signal ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Bovine Papillomavirus ◽

Phosphorylation Event ◽

Localization Signal

ABSTRACT A single phosphorylation event at T-antigen residue Thr124 regulates initiation of simian virus 40 DNA replication. To explore this regulatory process, a series of peptides were synthesized, centered on Thr124. These peptides contain a nuclear localization signal (NLS) and a recognition site for cyclin/Cdk kinases. When unphosphorylated, the “CDK/NLS” peptides inhibit T-antigen assembly and bind non-sequence specifically to DNA. However, these activities are greatly reduced upon phosphorylation of Thr124. Similar results were obtained by using peptides derived from the CDK/NLS region of bovine papillomavirus E1. Related studies indicate that residues in the NLS bind to DNA, whereas those in the CDK motif regulate binding. These findings are discussed in terms of the control of T-antigen double hexamer assembly and initiation of viral replication.

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The use of the simian virus 40 large tumor antigen nuclear localization signal to generate transgenic bovine and murine embryos

Theriogenology ◽

10.1016/s0093-691x(99)91986-6 ◽

1999 ◽

Vol 51 (1) ◽

pp. 427

Author(s):

L.N Viets ◽

W.A Reed ◽

E Swank ◽

K.L White

Keyword(s):

Nuclear Localization ◽

Tumor Antigen ◽

Nuclear Localization Signal ◽

Simian Virus 40 ◽

Simian Virus ◽

Large Tumor ◽

Large Tumor Antigen ◽

Localization Signal ◽

Murine Embryos

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A nuclear localization signal binding protein in the nucleolus.

The Journal of Cell Biology ◽

10.1083/jcb.111.6.2235 ◽

1990 ◽

Vol 111 (6) ◽

pp. 2235-2245 ◽

Cited By ~ 95

Author(s):

U T Meier ◽

G Blobel

Keyword(s):

Rat Liver ◽

Nuclear Localization ◽

Nuclear Localization Signal ◽

Biochemical Characterization ◽

T Antigen ◽

Single Amino Acid ◽

Wild Type ◽

Western Blots ◽

Peptide Conjugates ◽

Localization Signal

We used functional wild-type and mutant synthetic nuclear localization signal peptides of SV-40 T antigen cross-linked to human serum albumin (peptide conjugates) to assay their binding to proteins of rat liver nuclei on Western blots. Proteins of 140 and 55 kD (p140 and p55) were exclusively recognized by wild-type peptide conjugates. Free wild-type peptides competed for the wild-type peptide conjugate binding to p140 and p55 whereas free mutant peptides, which differed by a single amino acid from the wild type, competed less efficiently. The two proteins were extractable from nuclei by either low or high ionic strength buffers. We purified p140 and raised polyclonal antibodies in chicken against the protein excised from polyacrylamide gels. The anti-p140 antibodies were monospecific as judged by their reactivity with a single nuclear protein band of 140 kD on Western blots of subcellular fractions of whole cells. Indirect immunofluorescence microscopy on fixed and permeabilized Buffalo rat liver (BRL) cells with anti-p140 antibodies exhibited a distinct punctate nucleolar staining. Rhodamine-labeled wild-type peptide conjugates also bound to nucleoli in a similar pattern on fixed and permeabilized BRL cells. Based on biochemical characterization, p140 is a novel nucleolar protein. It is possible that p140 shuttles between the nucleolus and the cytoplasm and functions as a nuclear import carrier.

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Cytomegalovirus Assembly Protein Precursor and Proteinase Precursor Contain Two Nuclear Localization Signals That Mediate Their Own Nuclear Translocation and That of the Major Capsid Protein

Journal of Virology ◽

10.1128/jvi.72.10.7722-7732.1998 ◽

1998 ◽

Vol 72 (10) ◽

pp. 7722-7732 ◽

Cited By ~ 23

Author(s):

Scott M. Plafker ◽

Wade Gibson

Keyword(s):

Capsid Protein ◽

Nuclear Localization ◽

Major Capsid Protein ◽

Nuclear Translocation ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Protein Precursor ◽

Nuclear Localization Signals ◽

Carboxy Terminal

ABSTRACT The cytomegalovirus (CMV) assembly protein precursor (pAP) interacts with the major capsid protein (MCP), and this interaction is required for nuclear translocation of the MCP, which otherwise remains in the cytoplasm of transfected cells (L. J. Wood et al., J. Virol. 71:179–190, 1997). We have interpreted this finding to indicate that the CMV MCP lacks its own nuclear localization signal (NLS) and utilizes the pAP as an NLS-bearing escort into the nucleus. The CMV pAP amino acid sequence has two clusters of basic residues (e.g., KRRRER [NLS1] and KARKRLK [NLS2], for simian CMV) that resemble the simian virus 40 large-T-antigen NLS (D. Kalderon et al., Cell 39:499–509, 1984) and one of these (NLS1) has a counterpart in the pAP homologs of other herpesviruses. The work described here establishes that NLS1 and NLS2 are mutually independent NLS that can act (i) in cisto translocate pAP and the related proteinase precursor (pNP1) into the nucleus and (ii) in trans to transport MCP into the nucleus. By using combinations of NLS mutants and carboxy-terminal deletion constructs, we demonstrated a self-interaction of pAP and cytoplasmic interactions of pAP with pNP1 and of pNP1 with itself. The relevance of these findings to early steps in capsid assembly, the mechanism of MCP nuclear transport, and the possible cytoplasmic formation of protocapsomeric substructures is discussed.

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Interaction of the Vp3 Nuclear Localization Signal with the Importin α2/β Heterodimer Directs Nuclear Entry of Infecting Simian Virus 40

Journal of Virology ◽

10.1128/jvi.76.18.9368-9377.2002 ◽

2002 ◽

Vol 76 (18) ◽

pp. 9368-9377 ◽

Cited By ~ 71

Author(s):

Akira Nakanishi ◽

Dorothy Shum ◽

Hiroshi Morioka ◽

Eiko Otsuka ◽

Harumi Kasamatsu

Keyword(s):

Nuclear Localization ◽

Nuclear Localization Signal ◽

Simian Virus 40 ◽

Simian Virus ◽

Selective Exposure ◽

Nuclear Entry ◽

New Host ◽

Localization Signal ◽

Nucleoprotein Complexes

ABSTRACT For nuclear entry of large nucleoprotein complexes, it is thought that one key nuclear localization signal (NLS) of a protein component becomes exposed to mediate importin recognition. We show that the nuclear entry of simian virus 40 involves a dynamic interplay between two distinct interiorly situated capsid NLSs, the Vp1 NLS and the Vp3 NLS, and the selective exposure and importin recognition of the Vp3 NLS. The Vp3 NLS-null mutants assembled normally into virion-like particles (VLP) in mutant DNA-transfected cells. When used to infect a new host, the null VLP entered the cell normally but was impaired in viral DNA nuclear entry due to a lack of recognition by the importin α2/β heterodimer, leading to reduced viability. Both Vp3 and Vp1 NLSs directed importin interaction in vitro, but the Vp1 NLS, which overlaps the Vp1 DNA binding domain, did not bind importins in the presence of DNA. The results suggest that certain canonical NLSs within a nucleoprotein complex, such as the Vp1 NLS, can be masked from functioning by binding to the nucleic acid component and that the availability of an NLS that is not masked and can become exposed for importin binding, such as the Vp3 NLS, is a general feature of the nuclear entry of the nucleoprotein complexes, including those of other animal viruses.

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Nuclear Localization of Avian Polyomavirus Structural Protein VP1 Is a Prerequisite for the Formation of Virus-Like Particles

Journal of Virology ◽

10.1128/jvi.78.2.930-937.2004 ◽

2004 ◽

Vol 78 (2) ◽

pp. 930-937 ◽

Cited By ~ 12

Author(s):

Reimar Johne ◽

Hermann Müller

Keyword(s):

Nuclear Localization ◽

Nuclear Localization Signal ◽

Simian Virus 40 ◽

Simian Virus ◽

Influenza Viruses ◽

Structural Proteins ◽

Structural Protein ◽

Virus Like Particles ◽

Localization Signal ◽

Avian Polyomavirus

ABSTRACT Virions of polyomaviruses consist of the major structural protein VP1, the minor structural proteins VP2 and VP3, and the viral genome associated with histones. An additional structural protein, VP4, is present in avian polyomavirus (APV) particles. As it had been reported that expression of APV VP1 in insect cells did not result in the formation of virus-like particles (VLP), the prerequisites for particle formation were analyzed. To this end, recombinant influenza viruses were created to (co)express the structural proteins of APV in chicken embryo cells, permissive for APV replication. VP1 expressed individually or coexpressed with VP4 did not result in VLP formation; both proteins (co)localized in the cytoplasm. Transport of VP1, or the VP1-VP4 complex, into the nucleus was facilitated by the coexpression of VP3 and resulted in the formation of VLP. Accordingly, a mutant APV VP1 carrying the N-terminal nuclear localization signal of simian virus 40 VP1 was transported to the nucleus and assembled into VLP. These results support a model of APV capsid assembly in which complexes of the structural proteins VP1, VP3 (or VP2), and VP4, formed within the cytoplasm, are transported to the nucleus using the nuclear localization signal of VP3 (or VP2); there, capsid formation is induced by the nuclear environment.

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The Carboxy Terminus of Simian Virus 40 Large T Antigen Is Required To Disrupt the Yeast Cell Cycle

Journal of Virology ◽

10.1128/jvi.76.9.4621-4624.2002 ◽

2002 ◽

Vol 76 (9) ◽

pp. 4621-4624 ◽

Cited By ~ 1

Author(s):

Sheara W. Fewell ◽

Dena M. Markle ◽

Jeffrey L. Brodsky

Keyword(s):

Cell Cycle ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Yeast Cells ◽

Wild Type ◽

T Antigens ◽

Normal Morphology ◽

Cell Cycle Disruption ◽

Carboxy Terminal

ABSTRACT Wild-type and J domain mutant simian virus 40 large T antigens alter the cell cycle and bud morphology of Saccharomyces cerevisiae. In contrast, yeast cells expressing mutant T antigen lacking the carboxy-terminal 150 aa exhibit normal morphology, indicating that this region of T antigen is required for cell cycle disruption.

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