Baculovirus Transregulator IE1 Requires a Dimeric Nuclear Localization Element for Nuclear Import and Promoter Activation

ABSTRACT Immediate-early protein IE1 is a principal regulator of viral transcription and a contributor to origin-specific DNA replication of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Since these viral functions involve interaction of dimeric IE1 with palindromic homologous region (hr) enhancer-origin elements of the AcMNPV genome within the nucleus, it is presumed that proper nuclear transport of IE1 is essential for productive infection. To investigate the mechanisms of IE1 nuclear import, we analyzed the effect of site-directed mutations on IE1 subcellular distribution. As demonstrated by fluorescence microscopy and biochemical fractionation of plasmid-transfected cells, wild-type IE1 localized predominantly to the nucleus. Substitution or deletion of amino acid residues within a positively charged domain (residues 534 to 538) adjacent to IE1's oligomerization motif impaired nuclear import and caused loss of transactivation. Moreover, upon coexpression, these import-defective mutations prevented nuclear entry of wild-type IE1. In contrast, double-mutated IE1 defective for both nuclear import and dimerization failed to block nuclear entry or transactivation by wild-type IE1. Thus, import-defective IE1 dominantly interfered with wild-type IE1 by direct interaction and cytosolic trapping. Collectively, our data indicate that the small basic domain encompassing residues R537 and R538 constitutes a novel nuclear localization element that functions only upon IE1 dimerization. These findings support a model wherein IE1 oligomerizes within the cytosol as a prerequisite for nuclear entry and subsequent high-affinity interaction with the symmetrical binding sites comprising AcMNPV hr enhancer-origin elements.

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Protease-Dependent Uncoating of a Complex Retrovirus

Journal of Virology ◽

10.1128/jvi.79.14.9244-9253.2005 ◽

2005 ◽

Vol 79 (14) ◽

pp. 9244-9253 ◽

Cited By ~ 26

Author(s):

Jacqueline Lehmann-Che ◽

Marie-Lou Giron ◽

Olivier Delelis ◽

Martin Löchelt ◽

Patricia Bittoun ◽

...

Keyword(s):

Nuclear Import ◽

Viral Genome ◽

Productive Infection ◽

Wild Type Virus ◽

Target Cells ◽

Wild Type ◽

Microtubule Organizing Center ◽

Viral Protease ◽

Virus Life Cycle ◽

Organizing Center

ABSTRACT Although retrovirus egress and budding have been partly unraveled, little is known about early stages of the replication cycle. In particular, retroviral uncoating, a process during which incoming retroviral cores are altered to allow the integration of the viral genome into host chromosomes, is poorly understood. To get insights into these early events of the retroviral cycle, we have used foamy complex retroviruses as a model. In this report, we show that a protease-defective foamy retrovirus is noninfectious, although it is still able to bud and enter target cells efficiently. Similarly, a retrovirus mutated in an essential viral protease-dependent cleavage site in the central part of Gag is noninfectious. Following entry, wild-type and mutant retroviruses are able to traffic along microtubules towards the microtubule-organizing center (MTOC). However, whereas nuclear import of Gag and of the viral genome was observed for the wild-type virus as early as 8 hours postinfection, incoming capsids and genome from mutant viruses remained at the MTOC. Interestingly, a specific viral protease-dependent Gag cleavage product was detected only for the wild-type retrovirus early after infection, demonstrating that cleavage of Gag by the viral protease at this stage of the virus life cycle is absolutely required for productive infection, an unprecedented observation among retroviruses.

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TNPO3-Mediated Nuclear Entry of the Rous Sarcoma Virus Gag Protein Is Independent of the Cargo-Binding Domain

Journal of Virology ◽

10.1128/jvi.00640-20 ◽

2020 ◽

Vol 94 (17) ◽

Author(s):

Breanna L. Rice ◽

Matthew S. Stake ◽

Leslie J. Parent

Keyword(s):

Nuclear Localization ◽

Nuclear Import ◽

Rous Sarcoma Virus ◽

Binding Domain ◽

Nuclear Entry ◽

Gag Protein ◽

Nuclear Localization Signals ◽

Rous Sarcoma ◽

Importin Α ◽

Sarcoma Virus

ABSTRACT Retroviral Gag polyproteins orchestrate the assembly and release of nascent virus particles from the plasma membranes of infected cells. Although it was traditionally thought that Gag proteins trafficked directly from the cytosol to the plasma membrane, we discovered that the oncogenic avian alpharetrovirus Rous sarcoma virus (RSV) Gag protein undergoes transient nucleocytoplasmic transport as an intrinsic step in virus assembly. Using a genetic approach in yeast, we identified three karyopherins that engage the two independent nuclear localization signals (NLSs) in Gag. The primary NLS is in the nucleocapsid (NC) domain of Gag and binds directly to importin-α, which recruits importin-β to mediate nuclear entry. The second NLS (TNPO3), which resides in the matrix (MA) domain, is dependent on importin-11 and transportin-3 (TNPO3), which are known as MTR10p and Kap120p in yeast, although it is not clear whether these import factors are independent or additive. The functions of importin-α/importin-β and importin-11 have been verified in avian cells, whereas the role of TNPO3 has not been studied. In this report, we demonstrate that TNPO3 directly binds to Gag and mediates its nuclear entry. To our surprise, this interaction did not require the cargo-binding domain (CBD) of TNPO3, which typically mediates nuclear entry for other binding partners of TNPO3, including SR domain-containing splicing factors and tRNAs that reenter the nucleus. These results suggest that RSV hijacks this host nuclear import pathway using a unique mechanism, potentially allowing other cargo to simultaneously bind TNPO3. IMPORTANCE RSV Gag nuclear entry is facilitated using three distinct host import factors that interact with nuclear localization signals in the Gag MA and NC domains. Here, we show that the MA region is required for nuclear import of Gag through the TNPO3 pathway. Gag nuclear entry does not require the CBD of TNPO3. Understanding the molecular basis for TNPO3-mediated nuclear trafficking of the RSV Gag protein may lead to a deeper appreciation for whether different import factors play distinct roles in retrovirus replication.

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Insertion of a Classical Nuclear Import Signal into the Matrix Domain of the Rous Sarcoma Virus Gag Protein Interferes with Virus Replication

Journal of Virology ◽

10.1128/jvi.78.24.13534-13542.2004 ◽

2004 ◽

Vol 78 (24) ◽

pp. 13534-13542 ◽

Cited By ~ 8

Author(s):

Rachel A. Garbitt ◽

Karen R. Bone ◽

Leslie J. Parent

Keyword(s):

Nuclear Localization ◽

Nuclear Import ◽

Rous Sarcoma Virus ◽

Virus Assembly ◽

Wild Type ◽

Nuclear Targeting ◽

Gag Protein ◽

Rous Sarcoma ◽

Junction Molecules ◽

Sarcoma Virus

ABSTRACT The Rous sarcoma virus Gag protein undergoes transient nuclear trafficking during virus assembly. Nuclear import is mediated by a nuclear targeting sequence within the MA domain. To gain insight into the role of nuclear transport in replication, we investigated whether addition of a “classical ” nuclear localization signal (NLS) in Gag would affect virus assembly or infectivity. A bipartite NLS derived from nucleoplasmin was inserted into a region of the MA domain of Gag that is dispensable for budding and infectivity. Gag proteins bearing the nucleoplasmin NLS insertion displayed an assembly defect. Mutant virus particles (RC.V8.NLS) were not infectious, although they were indistinguishable from wild-type virions in Gag, Gag-Pol, Env, and genomic RNA incorporation and Gag protein processing. Unexpectedly, postinfection viral DNA synthesis was also normal, as similar amounts of two-long-terminal-repeat junction molecules were detected for RC.V8.NLS and wild type, suggesting that the replication block occurred after nuclear entry of proviral DNA. Phenotypically revertant viruses arose after continued passage in culture, and sequence analysis revealed that the nucleoplasmin NLS coding sequence was deleted from the gag gene. To determine whether the nuclear targeting activity of the nucleoplasmin sequence was responsible for the infectivity defect, two critical basic amino acids in the NLS were altered. This virus (RC.V8.KR/AA) had restored infectivity, and the MA.KR/AA protein showed reduced nuclear localization, comparable to the wild-type MA protein. These data demonstrate that addition of a second NLS, which might direct MA and/or Gag into the nucleus by an alternate import pathway, is not compatible with productive virus infection.

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A Small Conserved Domain of Drosophila PERIOD Is Important for Circadian Phosphorylation, Nuclear Localization, and Transcriptional Repressor Activity

Molecular and Cellular Biology ◽

10.1128/mcb.02338-06 ◽

2007 ◽

Vol 27 (13) ◽

pp. 5002-5013 ◽

Cited By ~ 41

Author(s):

Pipat Nawathean ◽

Dan Stoleru ◽

Michael Rosbash

Keyword(s):

Nuclear Localization ◽

Nuclear Import ◽

Transcriptional Repression ◽

Transcriptional Repressor ◽

Nuclear Entry ◽

Conserved Domain ◽

Cell Assays ◽

Import And Export ◽

Repressor Activity

ABSTRACT We identify in this study a 27-amino-acid motif which is conserved between the Drosophila melanogaster period protein (PER) and the three mammalian PERs. Characterization of PER lacking this motif (PERΔ) shows that it is important for phosphorylation of Drosophila PER by casein kinase Iε (CKIε; doubletime protein or DBT) and CKII. S2 cell assays indicate that the domain also contributes significantly to PER nuclear localization as well as to PER transcriptional repressor activity. These two phenomena appear linked, since PERΔ transcriptional repressor activity in S2 cells was restored when nuclear localization was facilitated. Two less direct assays of PERΔ activity in flies can be interpreted similarly. The separate assay of nuclear import and export suggests that the domain functions in part to facilitate PER phosphorylation within the cytoplasm, which in turn promotes nuclear entry. As there is evidence that the kinases also function within the nucleus to promote transcriptional repression, we suggest that there is a subsequent collaboration between phosphorylated PER and the kinases to repress CLK-CYC activity, probably through the phosphorylation of CLK. This is then followed by additional PER phosphorylation, which occurs within the nucleus and leads to PER degradation.

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Wild-Type Levels of Nuclear Localization and Human Immunodeficiency Virus Type 1 Replication in the Absence of the Central DNA Flap

Journal of Virology ◽

10.1128/jvi.76.23.12078-12086.2002 ◽

2002 ◽

Vol 76 (23) ◽

pp. 12078-12086 ◽

Cited By ~ 71

Author(s):

Ana Limón ◽

Noriko Nakajima ◽

Richard Lu ◽

Hina Z. Ghory ◽

Alan Engelman

Keyword(s):

Human Immunodeficiency Virus ◽

T Cells ◽

T Cell ◽

Human Immunodeficiency Virus Type ◽

Nuclear Localization ◽

Nuclear Import ◽

Wild Type ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Numerous factors have been implicated in the nuclear localization of retroviral preintegration complexes. Whereas sequences in human immunodeficiency virus type 1 (HIV-1) matrix, Vpr, and integrase proteins were initially reported to function specifically in nondividing cells, other recently identified sequences apparently function in dividing cells as well. One of these, the central DNA flap formed during reverse transcription, is specific to lentiviruses. It was previously reported that flap-negative (F−) HIV-1LAI was completely defective for viral spread in the MT-4 T-cell line, yet F− HIV-1 vectors were only 2- to 10-fold defective in various single-round transduction assays. To address these different findings, we analyzed the infectivity and nuclear localization phenotypes of two highly related T-cell-tropic strains, HIV-1NL4-3 and a derivative of HIV-1HXBc2 deficient for both Vpr and Nef. In stark contrast to the previous report, F− derivatives of both strains replicated efficiently in MT-4 cells. F− HIV-1NL4-3 also spread like wild-type HIV-1NL4-3 in infected Jurkat and primary T-cell cultures. In contrast, F− HIV-1HXBc2 was replication defective in primary T cells. Results of real-time quantitative PCR assays, however, indicated that F− HIV-1HXBc2 entered primary T-cell nuclei as efficiently as its wild-type counterpart. Thus, the F− HIV-1HXBc2 growth defect did not appear to correlate with defective nuclear import. Consistent with this observation, wild-type nef restored replication to F− HIV-1HXBc2 in primary T cells. Our results indicate that the central DNA flap does not play a major role in either preintegration complex nuclear import or HIV-1 replication in a variety of cell types.

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Nuclear import of the human androgen receptor

Biochemical Journal ◽

10.1042/bj2930761 ◽

1993 ◽

Vol 293 (3) ◽

pp. 761-768 ◽

Cited By ~ 176

Author(s):

G Jenster ◽

J Trapman ◽

A O Brinkmann

Keyword(s):

Androgen Receptor ◽

Cell Line ◽

Nuclear Localization ◽

Nuclear Import ◽

Nuclear Localization Signal ◽

Subcellular Distribution ◽

Wild Type ◽

Cytoplasmic Staining ◽

Localization Signal ◽

Human Androgen Receptor

Nuclear import of the human androgen receptor was investigated by immunocytochemical analysis of androgen receptor deletion and substitution mutants, which were transiently expressed in COS-1 cells. The signal responsible for nuclear import is encoded by amino-acid residues 608-625 and is functionally similar to the bipartite nucleoplasmin nuclear-localization signal. Although the subcellular distribution of androgen receptors mutated in the DNA-binding domain was unchanged compared with the wild-type androgen receptor, in the presence of ligand these mutations resulted in part of the receptor population forming clusters. Depending on the presence or absence of the bipartite nuclear localization signal, clusters were formed in the nucleus or in the cytoplasm, respectively. Expression of the wild-type androgen receptor in different cell lines revealed a cell-line-specific subcellular distribution of the unliganded receptor. The androgen receptor was predominantly nuclear when expressed in HeLa cells, whereas mainly cytoplasmic staining was observed when it was expressed in COS-1 cells. In the presence of hormone, the androgen receptor was located in the nucleus, independent of the cell line that was expressing the receptor. Anti-androgens and various steroid hormones induced the nuclear localization of the wild-type androgen receptor in a dose-dependent way, without activating transcription of an androgen-regulated reporter gene. This indicates that the inability of the tested compounds to activate transcription is not due to inhibited nuclear import.

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Dimerization and nuclear entry of mPER proteins in mammalian cells

Genes & Development ◽

10.1101/gad.14.11.1353 ◽

2000 ◽

Vol 14 (11) ◽

pp. 1353-1363 ◽

Cited By ~ 3

Author(s):

Kazuhiro Yagita ◽

Shun Yamaguchi ◽

Filippo Tamanini ◽

Gijsbertus T.J. van der Horst ◽

Jan H.J. Hoeijmakers ◽

...

Keyword(s):

Nuclear Localization ◽

Nuclear Import ◽

Mammalian Cells ◽

Nuclear Translocation ◽

Cultured Cells ◽

Biological Clock ◽

Nuclear Entry ◽

Cry Protein ◽

Intact Mouse ◽

Clock Proteins

Nuclear entry of circadian oscillatory gene products is a key step for the generation of a 24-hr cycle of the biological clock. We have examined nuclear import of clock proteins of the mammalianperiod gene family and the effect of serum shock, which induces a synchronous clock in cultured cells. Previously, mCRY1 and mCRY2 have been found to complex with PER proteins leading to nuclear import. Here we report that nuclear translocation of mPER1 and mPER2 (1) involves physical interactions with mPER3, (2) is accelerated by serum treatment, and (3) still occurs in mCry1/mCry2double-deficient cells lacking a functional biological clock. Moreover, nuclear localization of endogenous mPER1 was observed in culturedmCry1/mCry2 double-deficient cells as well as in the liver and the suprachiasmatic nuclei (SCN) ofmCry1/mCry2 double-mutant mice. This indicates that nuclear translocation of at least mPER1 also can occur under physiological conditions (i.e., in the intact mouse) in the absence of any CRY protein. The mPER3 amino acid sequence predicts the presence of a cytoplasmic localization domain (CLD) and a nuclear localization signal (NLS). Deletion analysis suggests that the interplay of the CLD and NLS proposed to regulate nuclear entry of PER in Drosophilais conserved in mammals, but with the novel twist that mPER3 can act as the dimerizing partner.

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Nup124p Is a Nuclear Pore Factor ofSchizosaccharomyces pombe That Is Important for Nuclear Import and Activity of Retrotransposon Tf1

Molecular and Cellular Biology ◽

10.1128/mcb.19.8.5768 ◽

1999 ◽

Vol 19 (8) ◽

pp. 5768-5784 ◽

Cited By ~ 32

Author(s):

David Balasundaram ◽

Michael J. Benedik ◽

Mary Morphew ◽

Van-Dinh Dang ◽

Henry L. Levin

Keyword(s):

Nuclear Localization ◽

Nuclear Import ◽

Direct Interaction ◽

Host Factors ◽

Nuclear Pore ◽

Cytoplasmic Localization ◽

Hybrid Analysis ◽

Localization Signal ◽

Two Hybrid

ABSTRACT The long terminal repeat (LTR)-containing retrotransposon Tf1 propagates within the fission yeast Schizosaccharomyces pombe as the result of several mechanisms that are typical of both retrotransposons and retroviruses. To identify host factors that contribute to the transposition process, we mutagenized cultures ofS. pombe and screened them for strains that were unable to support Tf1 transposition. One such strain contained a mutation in a gene we named nup124. The product of this gene contains 11 FXFG repeats and is a component of the nuclear pore complex. In addition to the reduced levels of Tf1 transposition, thenup124-1 allele caused a significant reduction in the nuclear localization of Tf1 Gag. Surprisingly, the mutation innup124-1 did not cause any reduction in the growth rate, the nuclear localization of specific nuclear localization signal-containing proteins, or the cytoplasmic localization of poly(A) mRNA. A two-hybrid analysis and an in vitro precipitation assay both identified an interaction between Tf1 Gag and the N terminus of Nup124p. These results provide evidence for an unusual mechanism of nuclear import that relies on a direct interaction between a nuclear pore factor and Tf1 Gag.

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Nuclear import of activated D-ERK by DIM-7, an importin family member encoded by the gene moleskin

Development ◽

10.1242/dev.128.8.1403 ◽

2001 ◽

Vol 128 (8) ◽

pp. 1403-1414 ◽

Cited By ~ 1

Author(s):

J.A. Lorenzen ◽

S.E. Baker ◽

F. Denhez ◽

M.B. Melnick ◽

D.L. Brower ◽

...

Keyword(s):

Nuclear Localization ◽

Receptor Tyrosine Kinase ◽

Nuclear Import ◽

S2 Cells ◽

Wild Type ◽

Tyrosine Kinase Signaling ◽

Its Gene ◽

Drosophila Homolog ◽

Drosophila S2 Cells ◽

Mutant Phenotypes

The initiation of gene expression in response to Drosophila receptor tyrosine kinase signaling requires the nuclear import of the MAP kinase, D-ERK. However, the molecular details of D-ERK translocation are largely unknown. In this regard, we have identified D-Importin-7 (DIM-7), the Drosophila homolog of vertebrate importin 7, and its gene moleskin. DIM-7 exhibits a dynamic nuclear localization pattern that overlaps the spatial and temporal profile of nuclear, activated D-ERK. Co-immunoprecipitation experiments show that DIM-7 associates with phosphorylated D-ERK in Drosophila S2 cells. Furthermore, moleskin mutations enhance hypomorphic and suppress hypermorphic D-ERK mutant phenotypes. Deletion or mutation of moleskin dramatically reduces the nuclear localization of activated D-ERK. Directly linking DIM-7 to its nuclear import, this defect can be rescued by the expression of wild-type DIM-7. Mutations in the Drosophila Importin (β) homolog Ketel, also reduce the nuclear localization of activated D-ERK. Together, these data indicate that DIM-7 and Ketel are components of the nuclear import machinery for activated D-ERK.

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Yeast Chromatin Assembly Complex 1 Protein Excludes Nonacetylatable Forms of Histone H4 from Chromatin and the Nucleus

Molecular and Cellular Biology ◽

10.1128/mcb.24.23.10180-10192.2004 ◽

2004 ◽

Vol 24 (23) ◽

pp. 10180-10192 ◽

Cited By ~ 35

Author(s):

Lynn Glowczewski ◽

Jakob H. Waterborg ◽

Judith G. Berman

Keyword(s):

Nuclear Localization ◽

Nuclear Import ◽

Fluorescent Protein ◽

Chromatin Assembly ◽

Histone H4 ◽

Wild Type ◽

Acetylation State ◽

Assembly Factor ◽

Heterochromatin Silencing ◽

Green Fluorescent

ABSTRACT In yeast, the establishment and maintenance of a transcriptionally silent chromatin state are dependent upon the acetylation state of the N terminus of histone proteins. Histone H4 proteins that contain mutations in N-terminal lysines disrupt heterochromatin and result in yeast that cannot mate. Introduction of a wild-type copy of histone H4 restores mating, despite the presence of the mutant protein, suggesting that mutant H4 protein is either excluded from, or tolerated in, chromatin. To understand how the cell differentiates wild-type histone and mutant histone in which the four N-terminal lysines were replaced with alanine (H4-4A), we analyzed silencing, growth phenotypes, and the histone composition of chromatin in yeast strains coexpressing equal amounts of wild-type and mutant H4 proteins (histone H4 heterozygote). We found that histone H4 heterozygotes have defects in heterochromatin silencing and growth, implying that mutations in H4 are not completely recessive. Nuclear preparations from histone H4 heterozygotes contained less mutant H4 than wild-type H4, consistent with the idea that cells exclude some of the mutant histone. Surprisingly, the N-terminal nuclear localization signal of H4-4A fused to green fluorescent protein was defective in nuclear localization, while a mutant in which the four lysines were replaced with arginine (H4-4R) appeared to have normal nuclear import, implying a role for the charged state of the acetylatable lysines in the nuclear import of histones. The biased partial exclusion of H4-4A was dependent upon Cac1p, the largest subunit of yeast chromatin assembly factor 1 (CAF-1), as well as upon the karyopherin Kap123p, but was independent of Cac2p, another CAF-1 component, and other chromatin assembly proteins (Hir3p, Nap1p, and Asf1p). We conclude that N-terminal lysines of histone H4 are important for efficient histone nuclear import. In addition, our data support a model whereby Cac1p and Kap123 cooperate to ensure that only appropriately acetylated histone H4 proteins are imported into the nucleus.

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