Analysis of a nuclear localization signal of simian virus 40 major capsid protein Vp1.

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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JC Virus Minor Capsid Proteins Vp2 and Vp3 Are Essential for Virus Propagation

Journal of Virology ◽

10.1128/jvi.01298-06 ◽

2006 ◽

Vol 80 (21) ◽

pp. 10858-10861 ◽

Cited By ~ 28

Author(s):

M. L. Gasparovic ◽

G. V. Gee ◽

W. J. Atwood

Keyword(s):

Nuclear Localization ◽

Nuclear Localization Signal ◽

Major Capsid Protein ◽

Jc Virus ◽

Life Cycles ◽

Capsid Proteins ◽

Coat Proteins ◽

Virus Propagation ◽

Localization Signal ◽

Capsid Protein Vp1

ABSTRACT Virus-encoded capsid proteins play a major role in the life cycles of all viruses. The JC virus capsid is composed of 72 pentamers of the major capsid protein Vp1, with one of the minor coat proteins Vp2 or Vp3 in the center of each pentamer. Vp3 is identical to two-thirds of Vp2, and these proteins share a DNA binding domain, a nuclear localization signal, and a Vp1-interacting domain. We demonstrate here that both the minor proteins and the myristylation site on Vp2 are essential for the viral life cycle, including the proper packaging of its genome.

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The VP2/VP3 Minor Capsid Protein of Simian Virus 40 Promotes thein VitroAssembly of the Major Capsid Protein VP1 into Particles

Journal of Biological Chemistry ◽

10.1074/jbc.m511261200 ◽

2006 ◽

Vol 281 (15) ◽

pp. 10164-10173 ◽

Cited By ~ 27

Author(s):

Masa-aki Kawano ◽

Takamasa Inoue ◽

Hiroko Tsukamoto ◽

Tatsuya Takaya ◽

Teruya Enomoto ◽

...

Keyword(s):

Capsid Protein ◽

Major Capsid Protein ◽

Simian Virus 40 ◽

Simian Virus ◽

Minor Capsid Protein ◽

Capsid Protein Vp1

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

Molecular and Cellular Biology ◽

10.1128/mcb.11.10.5137-5146.1991 ◽

1991 ◽

Vol 11 (10) ◽

pp. 5137-5146

Author(s):

K van Zee ◽

F Appel ◽

E Fanning

Keyword(s):

Nuclear Localization ◽

Nuclear Localization Signal ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Heterologous Proteins ◽

Wild Type ◽

Terminal Sequence ◽

Localization Signal ◽

Carboxy Terminal

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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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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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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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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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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