scholarly journals Inhibition of Human Cytomegalovirus Replication via Peptide Aptamers Directed against the Nonconventional Nuclear Localization Signal of the Essential Viral Replication Factor pUL84

2009 ◽  
Vol 83 (22) ◽  
pp. 11902-11913 ◽  
Author(s):  
Nina Kaiser ◽  
Peter Lischka ◽  
Nadine Wagenknecht ◽  
Thomas Stamminger
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Mammalian Cells ◽  
Human Fibroblasts ◽  
Peptide Aptamers ◽  
Importin Α ◽  
Localization Signal

ABSTRACT The UL84 open reading frame of human cytomegalovirus encodes an essential multifunctional regulatory protein that is thought to act in the nucleus as an initiator of lytic viral replication. Nuclear trafficking of pUL84 is facilitated by a complex nonconventional nuclear localization signal (NLS) that mediates its interaction with the cellular importin-α/β pathway. Since binding of pUL84 to importin-α proteins mechanistically differs from that of cellular proteins containing a classical NLS, we assumed that specific interference with the nuclear import of pUL84 might be possible and that this could constitute a novel principle for antiviral therapy. In order to test this hypothesis, we employed peptide aptamer technology and isolated several peptide aptamers from a randomized peptide expression library that specifically bind with high affinity to the unconventional pUL84 NLS under intracellular conditions. Coimmunoprecipitation experiments confirmed these interactions in mammalian cells, and the antiviral potential of the identified peptide aptamers was determined using three independent experimental approaches. (i) Infection experiments with a recombinant human cytomegalovirus expressing green fluorescent protein demonstrated 50 to 60% decreased viral replication in primary human fibroblasts stably expressing pUL84-specific aptamers. (ii) A 50 to 70% reduction of viral plaque formation, as well as a 70 to 90% inhibition of virus release in the presence of pUL84-specific aptamers, was observed. (iii) Immunofluorescence analyses revealed a shift from an almost exclusively nuclear pUL84 staining pattern to a nucleocytoplasmic distribution upon coexpression of the identified molecules, indicating that interference with the nuclear import of pUL84 contributes to the observed antiviral activity of the identified pUL84-binding aptamer molecules.

scholarly journals Human Cytomegalovirus UL84 Localizes to the Cell Nucleus via a Nuclear Localization Signal and Is a Component of Viral Replication Compartments

2002 ◽  
Vol 76 (17) ◽  
pp. 8931-8938 ◽  
Author(s):  
Yiyang Xu ◽  
Kelly S. Colletti ◽  
Gregory S. Pari
Keyword(s):  
Amino Acids ◽  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Human Fibroblasts ◽  
Reading Frame ◽  
Early Protein ◽  
Localization Signal ◽  
Infected Cells

ABSTRACT The UL84 open reading frame encodes a protein that is required for origin-dependent DNA replication and interacts with the immediate-early protein IE2 in lytically infected cells. Transfection of UL84 expression constructs showed that UL84 localized to the nucleus of transfected cells in the absence of any other viral proteins and displayed a punctate speckled fluorescent staining pattern. Cotransfection of all the human cytomegalovirus replication proteins and oriLyt, along with pUL84-EGFP, showed that UL84 colocalized with UL44 (polymerase accessory protein) in replication compartments. Experiments using infected human fibroblasts demonstrated that UL84 also colocalized with UL44 and IE2 in viral replication compartments in infected cells. A nuclear localization signal was identified using plasmid constructs expressing truncation mutants of the UL84 protein in transient transfection assays. Transfection assays showed that UL84 failed to localize to the nucleus when 200 amino acids of the N terminus were deleted. Inspection of the UL84 amino acid sequence revealed a consensus putative nuclear localization signal between amino acids 160 and 171 (PEKKKEKQEKK) of the UL84 protein.

scholarly journals A Nonconventional Nuclear Localization Signal within the UL84 Protein of Human Cytomegalovirus Mediates Nuclear Import via the Importin α/β Pathway

2003 ◽  
Vol 77 (6) ◽  
pp. 3734-3748 ◽  
Author(s):  
Peter Lischka ◽  
Gabriele Sorg ◽  
Michael Kann ◽  
Michael Winkler ◽  
Thomas Stamminger
Keyword(s):  
Amino Acids ◽  
Dna Replication ◽  
Human Cytomegalovirus ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Complex Structure ◽  
Importin Α ◽  
Localization Signal

ABSTRACT The open reading frame UL84 of human cytomegalovirus encodes a multifunctional regulatory protein which is required for viral DNA replication and binds with high affinity to the immediate-early transactivator IE2-p86. Although the exact role of pUL84 in DNA replication is unknown, the nuclear localization of this protein is a prerequisite for this function. To investigate whether the activities of pUL84 are modulated by cellular proteins we used the Saccharomyces cerevisiae two-hybrid system to screen a cDNA-library for interacting proteins. Strong interactions were found between pUL84 and four members of the importin α protein family. These interactions could be confirmed in vitro by pull down experiments and in vivo by coimmunoprecipitation analysis from transfected cells. Using in vitro transport assays we showed that the pUL84 nuclear import required importin α, importin β, and Ran, thus following the classical importin-mediated import pathway. Deletion mutagenesis of pUL84 revealed a domain of 282 amino acids which is required for binding to the importin α proteins. Its function as a nuclear localization signal (NLS) was confirmed by fusion to heterologous proteins. Although containing a cluster of basic amino acids similar to classical NLSs, this cluster did not contain the NLS activity. Thus, a complex structure appears to be essential for importin α binding and import activity.

scholarly journals Nuclear Localization Signal and Protein Context both Mediate Importin α Specificity of Nuclear Import Substrates

2006 ◽  
Vol 26 (23) ◽  
pp. 8697-8709 ◽  
Author(s):  
Beate Friedrich ◽  
Christina Quensel ◽  
Thomas Sommer ◽  
Enno Hartmann ◽  
Matthias Köhler
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Protein Import ◽  
Binding Studies ◽  
Vitro Binding ◽  
Importin Α ◽  
Localization Signal ◽  
Experimental Approaches

ABSTRACT The “classical” nuclear protein import pathway depends on importin α and importin β. Importin α binds nuclear localization signal (NLS)-bearing proteins and functions as an adapter to access the importin β-dependent import pathway. In humans, only one importin β is known to interact with importin α, while six α importins have been described. Various experimental approaches provided evidence that several substrates are transported specifically by particular α importins. Whether the NLS is sufficient to mediate importin α specificity is unclear. To address this question, we exchanged the NLSs of two well-characterized import substrates, the seven-bladed propeller protein RCC1, preferentially transported into the nucleus by importin α3, and the less specifically imported substrate nucleoplasmin. In vitro binding studies and nuclear import assays revealed that both NLS and protein context contribute to the specificity of importin α binding and transport.

scholarly journals The potential terminase subunit of human cytomegalovirus, pUL56, is translocated into the nucleus by its own nuclear localization signal and interacts with importin α

2000 ◽  
Vol 81 (9) ◽  
pp. 2231-2244 ◽  
Author(s):  
Kyra Giesen ◽  
Klaus Radsak ◽  
Elke Bogner
Keyword(s):  
Amino Acids ◽  
Human Cytomegalovirus ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Translocation ◽  
Reporter Protein ◽  
Importin Α ◽  
Localization Signal ◽  
Carboxy Terminal

Human cytomegalovirus (HCMV) DNA-binding protein pUL56 is thought to be involved in the cleavage/packaging process of viral DNA and therefore needs to be transported into the nucleus. By using indirect immunofluorescence analysis, HCMV pUL56 (p130) was found to be localized predominantly in the nucleus of infected cells. Solitary expression of wild-type as well as epitope-tagged pUL56 also resulted in nuclear distribution after transfection, suggesting the presence of an endogenous nuclear localization signal (NLS). Deletion of a carboxy-terminal stretch of basic amino acids (aa 816–827) prevented nuclear translocation, indicating that the sequence RRVRATRKRPRR of HCMV pUL56 mediates nuclear targetting. The signal character of the NLS sequence was demonstrated by successful transfer of the NLS to a reporter protein chimera. Furthermore, sequential substitutions of pairs of amino acids by alanine in the context of the reporter protein as well as substitutions within the full-length pUL56 sequence indicated that residues at positions 7 and 8 of the NLS (R and K at positions 822 and 823 of pUL56) were essential for nuclear translocation. In order to identify the transport machinery involved, the potential of pUL56 to bind importin α (hSRP1α) was examined. Clear evidence of a direct interaction of a carboxy-terminal portion as well as the NLS of pUL56 with hSRP1α was provided by in vitro binding assays. In view of these findings, it is suggested that nuclear translocation of HCMV pUL56 is mediated by the importin-dependent pathway.

scholarly journals Identification and Functional Characterization of a Novel Nuclear Localization Signal Present in the Yeast Nab2 Poly(A)+ RNA Binding Protein

1998 ◽  
Vol 18 (3) ◽  
pp. 1449-1458 ◽  
Author(s):  
Ray Truant ◽  
Robert A. Fridell ◽  
R. Edward Benson ◽  
Hal Bogerd ◽  
Bryan R. Cullen
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Yeast Cells ◽  
Vitro Assay ◽  
Localization Signal

ABSTRACT The nuclear import of proteins bearing a basic nuclear localization signal (NLS) is dependent on karyopherin α/importin α, which acts as the NLS receptor, and karyopherin β1/importin β, which binds karyopherin α and mediates the nuclear import of the resultant ternary complex. Recently, a second nuclear import pathway that allows the rapid reentry into the nucleus of proteins that participate in the nuclear export of mature mRNAs has been identified. In mammalian cells, a single NLS specific for this alternate pathway, the M9 NLS of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), has been described. The M9 NLS binds a transport factor related to karyopherin β1, termed karyopherin β2 or transportin, and does not require a karyopherin α-like adapter protein. A yeast homolog of karyopherin β2, termed Kap104p, has also been described and proposed to play a role in the nuclear import of a yeast hnRNP-like protein termed Nab2p. Here, we define a Nab2p sequence that binds to Kap104p and that functions as an NLS in both human and yeast cells despite lacking any evident similarity to basic or M9 NLSs. Using an in vitro nuclear import assay, we demonstrate that Kap104p can direct the import into isolated human cell nuclei of a substrate containing a wild-type, but not a defective mutant, Nab2p NLS. In contrast, other NLSs, including the M9 NLS, could not function as substrates for Kap104p. Surprisingly, this in vitro assay also revealed that human karyopherin β1, but not the Kap104p homolog karyopherin β2, could direct the efficient nuclear import of a Nab2p NLS substrate in vitro in the absence of karyopherin α. These data therefore identify a novel NLS sequence, active in both yeast and mammalian cells, that is functionally distinct from both basic and M9 NLS sequences.

scholarly journals The Extended C-Terminal Region of Influenza C Virus Nucleoprotein Is Important for Nuclear Import and Ribonucleoprotein Activity

2019 ◽  
Vol 93 (9) ◽  
Author(s):  
Yun-Sang Tang ◽  
Chun-Yeung Lo ◽  
Chris Ka-Pun Mok ◽  
Paul Kay-Sheung Chan ◽  
Pang-Chui Shaw
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Functional Significance ◽  
Influenza A ◽  
Terminal Region ◽  
B Virus ◽  
Importin Α ◽  
Influenza C Virus ◽  
Localization Signal

ABSTRACTThe influenza C virus (ICV) is a human-pathogenic agent, and the infections are frequently identified in children. Compared to influenza A and B viruses, the nucleoprotein of ICV (NPC) has an extended C-terminal region of which the functional significance is ill defined. We observed that the nuclear localization signals (NLSs) found on the nucleoproteins of influenza A and B virus subtypes are absent at corresponding positions on ICV. Instead, we found that a long bipartite nuclear localization signal resides at the extended C-terminal region, spanning from R513 to K549. Our experimental data determined that the KKMK motif within this region plays important roles in both nuclear import and polymerase activity. Similar to the influenza A viruses, NPC also binds to multiple human importin α isoforms. Taken together, our results enhance the understanding of the virus-host interaction of the influenza C virus.IMPORTANCEAs a member of theOrthomyxoviridaefamily, the polymerase complex of the influenza C virus structurally resembles its influenza A and influenza B virus counterparts, but the nucleoprotein differs by possessing an extra C-terminal region. We have characterized this region in view of nuclear import and interaction with the importin α protein family. Our results demonstrate the functional significance of a previously uncharacterized region onOrthomyxoviridaenucleoprotein (NP). Based on this work, we propose that importin α binding to influenza C virus NP is regulated by a long bipartite nuclear localization signal. Since the sequence of influenza D virus NP shares high homology to that of the influenza C virus, this work will also shed light on how influenza D virus NP functions.

scholarly journals Ciliary entry of KIF17 is dependent on its binding to the IFT-B complex via IFT46–IFT56 as well as on its nuclear localization signal

2017 ◽  
Vol 28 (5) ◽  
pp. 624-633 ◽  
Author(s):  
Teruki Funabashi ◽  
Yohei Katoh ◽  
Saki Michisaka ◽  
Masaya Terada ◽  
Maho Sugawa ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Protein Trafficking ◽  
Nuclear Localization Signal ◽  
Mammalian Cells ◽  
Permeability Barrier ◽  
Extracellular Signals ◽  
Ciliary Protein ◽  
Importin Α ◽  
Localization Signal

Cilia function as cellular antennae to sense and transduce extracellular signals. A number of proteins are specifically localized in cilia. Anterograde and retrograde ciliary protein trafficking are mediated by the IFT-B and IFT-A complexes in concert with kinesin-2 and dynein-2 motors, respectively. However, the role of KIF17, a homodimeric kinesin-2 protein, in protein trafficking has not been fully understood in vertebrate cilia. In this study, we demonstrated, by using the visible immunoprecipitation assay, that KIF17 interacts with the IFT46–IFT56 dimer in the IFT-B complex through its C-terminal sequence located immediately upstream of the nuclear localization signal (NLS). We then showed that KIF17 reaches the ciliary tip independently of its motor domain and requires IFT-B binding for its entry into cilia rather than for its intraciliary trafficking. We further showed that KIF17 ciliary entry depends not only on its binding to IFT-B but also on its NLS, to which importin α proteins bind. Taking the results together, we conclude that in mammalian cells, KIF17 is dispensable for ciliogenesis and IFT-B trafficking but requires IFT-B, as well as its NLS, for its ciliary entry across the permeability barrier located at the ciliary base.

scholarly journals Importin-α Mediates the Regulated Nuclear Targeting of Serum- and Glucocorticoid-inducible Protein Kinase (Sgk) by Recognition of a Nuclear Localization Signal in the Kinase Central Domain

2003 ◽  
Vol 14 (3) ◽  
pp. 1221-1239 ◽  
Author(s):  
Anita C. Maiyar ◽  
Meredith L.L. Leong ◽  
Gary L. Firestone
Keyword(s):  
Protein Kinase ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Dependent Manner ◽  
Central Domain ◽  
Importin Α ◽  
Localization Signal ◽  
Inducible Protein

The transcriptionally regulated serum and glucocorticoid inducible protein kinase (Sgk) is localized to the nucleus in a serum-dependent manner, and a yeast two-hybrid genetic screen uncovered a specific interaction between Sgk and the importin-α nuclear import receptor. In vitro GST pull down assays demonstrated a strong and direct association of importin-α with endogenous Sgk and exogenously expressed HA-tagged Sgk, whereas both components coimmunoprecipitate and colocalize to the nucleus after serum stimulation. Consistent with an active mechanism of nuclear localization, the nuclear import of HA-Sgk in permeabilized cells required ATP, cytoplasm, and a functional nuclear pore complex. Ectopic addition of a 107 amino acid carboxy-terminal fragment of importin-α, which contains the Sgk binding region, competitively inhibited the ability of endogenous importin-α to import Sgk into nuclei in vitro. Mutagenesis of lysines by alanine substitution defined a KKAILKKKEEK sequence within the central domain of Sgk between amino acids 131–141 that functions as a nuclear localization signal (NLS) required for the in vitro interaction with importin-α and for nuclear import of full-length Sgk in cultured cells. The serum-induced nuclear import of Sgk requires the NLS-dependent recognition of Sgk by importin-α as well as the PI3-kinase–dependent phosphorylation of Sgk. Our results define a new role importin-α in the stimulus-dependent control of signal transduction by nuclear localized protein kinases.

Nuclear localization of Ku antigen is promoted independently by basic motifs in the Ku70 and Ku80 subunits

2001 ◽  
Vol 114 (1) ◽  
pp. 89-99
Author(s):  
J. Bertinato ◽  
C. Schild-Poulter ◽  
R.J. Hache
Keyword(s):  
Amino Acids ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Mammalian Cells ◽  
Full Length ◽  
Telomere Maintenance ◽  
Nuclear Localization Signals ◽  
Localization Signal ◽  
Ku Antigen

The Ku antigen is a heteromeric (Ku70/Ku80), mostly nuclear protein. Ku participates in multiple nuclear processes from DNA repair to V(D)J recombination to telomere maintenance to transcriptional regulation and serves as a DNA binding subunit and allosteric regulator of DNA-dependent protein kinase. While some evidence suggests that subcellular localization of Ku may be subject to regulation, how Ku gains access to the nucleus is poorly understood. In this work, using a combination of indirect immunofluorescence and direct fluorescence, we have demonstrated that transfer of the Ku heterodimer to the nucleus is determined by basic nuclear localization signals in each of the Ku subunits that function independently. A bipartite basic nuclear localization signal between amino acids 539–556 of Ku70 was observed to be required for nuclear import of full-length Ku70 monomer, while a short Ku80 motif of four amino acids from 565–568 containing three lysines was required for the nuclear import of full-length Ku80. Ku heterodimers containing only one nuclear localization signal accumulated in the nucleus as efficiently as wild-type Ku, while site directed mutagenesis inactivating the basic motifs in each subunit, resulted in a Ku heterodimer that was completely localized to the cytoplasm. Lastly, our results indicate that mutations in Ku previously proposed to abrogate Ku70/Ku80 heterodimerization, markedly reduced the accumulation of Ku70 without affecting heterodimer formation in mammalian cells.

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