scholarly journals Duck Plague Virus pUL48 Protein Activates the Immediate-Early Gene to Initiate the Transcription of the Virus Gene

2021 ◽  
Vol 12 ◽  
Author(s):  
Tong Zhou ◽  
Dengjian Fan ◽  
Mingshu Wang ◽  
Anchun Cheng ◽  
Ying Wu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nuclear Localization ◽  
Transcriptional Activation ◽  
Nuclear Localization Signal ◽  
Activation Function ◽  
Immediate Early ◽  
Luciferase Reporter ◽  
Nuclear Entry ◽  
Duck Plague Virus ◽  
Localization Signal

Duck plague caused by the duck plague virus (DPV) is an infectious disease that seriously harms the waterfowl breeding industry. The VP16 protein of α herpesvirus can bind to specific cis-acting elements upstream of the promoter of the immediate-early (IE, α) gene to promote the transcription of the IE gene, so it is also called the trans-inducer of IE gene (α-TIF). However, no studies on DPV α-TIF have been reported. This study investigated the DPV pUL48, a homolog of HSV-1 VP16, transcriptional activation region, target sequence, and viral protein affecting its transcriptional activation using a dual-luciferase reporter gene detection system, and pUL48 was identified as the α-TIF of DPV. (1) The regulation of pUL48 on DPV different gene promoters showed that pUL48 could activate all the promoters of IE genes (ICP4, ICP22, and ICP27) but not the promoters of early and late genes. (2) The activity of pUL48 to ICP4 and ICP22 promoters with different upstream lengths showed that pUL48 activated ICP4 and ICP22 promoters by acting on TAATGA (T) TAT element upstream of ICP4 promoter and TAATTATAT element upstream of ICP22 promoter, respectively. (3) Transcriptional activation of IE gene by truncated proteins of different lengths at the N-terminal of pUL48 was detected. The results showed that the transcriptional activation domain of pUL48 was amino acids 1–60 at the N-terminal, and amino acids 1–20 was its core region. In addition, it was found that pUL14, pUL46, and pUL47 significantly promoted the transcriptional activation of pUL48. The effects of loss of pUL47 and its nuclear localization signal on the nuclear entry and transcriptional activation function of pUL48 were further examined. The results showed that pUL47 could promote the nuclear entry of pUL48 through its nuclear localization signal at positions 40–50 and 768–777 amino acids, thus, enhancing the transcriptional activation function of pUL48 and synergistic promotion of viral gene transcription.

No Enhancement of Nuclear Entry by Direct Conjugation of a Nuclear Localization Signal Peptide to Linearized DNA

2003 ◽  
Vol 14 (6) ◽  
pp. 1197-1202 ◽  
Author(s):  
Mitsuhide Tanimoto ◽  
Hiroyuki Kamiya ◽  
Noriaki Minakawa ◽  
Akira Matsuda ◽  
Hideyoshi Harashima
Keyword(s):  
Nuclear Localization ◽  
Signal Peptide ◽  
Nuclear Localization Signal ◽  
Nuclear Entry ◽  
Localization Signal

scholarly journals Control of NF–κB transcriptional activation by signal induced proteolysis of IκBα

1999 ◽  
Vol 354 (1389) ◽  
pp. 1601-1609 ◽  
Author(s):  
R. T. Hay ◽  
L. Vuillard ◽  
J. M. P. Desterro ◽  
M. S. Rodriguez
Keyword(s):  
Nuclear Localization ◽  
Transcriptional Activation ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Cytoplasmic Process ◽  
Leptomycin B ◽  
Rapid Degradation ◽  
Localization Signal ◽  
Export Signal

In unstimulated cells the transcription factor NF–κB is held in the cytoplasm in an inactive state by IκB inhibitor proteins. Ultimately activation of NF–κB is achieved by ubiquitination and proteasome–mediated degradation of IκBα and we have therefore investigated factors which control this proteolysis. Signal–induced degradation of IκBα exposes the nuclear localization signal of NF–κB, thus allowing it to translocate into the nucleus and activate transcription from responsive genes. An autoregulatory loop is established when NF–κB induces expression of the IκBα gene and newly synthesized IκBα accumulates in the nucleus where it negatively regulates NF–κB–dependent transcription. As part of this post–induction repression, the nuclear export signal on IκBα mediates transport of NF–κB–IκBα complexes from the nucleus to the cytoplasm. As nuclear export of IκBα is blocked by leptomycin B this drug was used to examine the effect of cellular location on susceptibility of IκBα to signal–induced degradation. In the presence of leptomycin B, IκBα is accumulated in the nucleus and in this compartment is resistant to signal–induced degradation. Thus signal–induced degradation of IκBα is mainly, if not exclusively a cytoplasmic process. An efficient nuclear export of IκBα is therefore essential for maintaining a low level of IκBα in the nucleus and allowing NF–κB to be transcriptionally active upon cell stimulation. We have detected a modified form of IκBα, conjugated to the small ubiquitin–like protein SUMO–1, which is resistant to signal–induced degradation. SUMO–1 modified IκBα remains associated with NF–κB and thus overexpression of SUMO–1 inhibits the signal–induced activation of NF–κB–dependent transcription. Reconstitution of the conjugation reaction with highly purified proteins demonstrated that in the presence of a novel E1 SUMO–1 activating enzyme, Ubch9 directly conjugated SUMO–1 to IκBα on residues K21 and K22, which are also used for ubiquitin modification. Thus, while ubiquitination targets proteins for rapid degradation, SUMO–1 modification acts antagonistically to generate proteins resistant to degradation.

scholarly journals The N-Terminal Domain of IκBα Masks the Nuclear Localization Signal(s) of p50 and c-Rel Homodimers

1998 ◽  
Vol 18 (5) ◽  
pp. 2640-2649 ◽  
Author(s):  
Matthew Latimer ◽  
Mary K. Ernst ◽  
Linda L. Dunn ◽  
Marina Drutskaya ◽  
Nancy R. Rice
Keyword(s):  
Amino Acids ◽  
Transcription Factors ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Mutational Analysis ◽  
Inhibitor Protein ◽  
N Terminus ◽  
Localization Signal ◽  
Terminal Section ◽  
Rel Homology Domain

ABSTRACT Members of the Rel/NF-κB family of transcription factors are related to each other over a region of about 300 amino acids called the Rel Homology Domain (RHD), which governs DNA binding, dimerization, and binding to inhibitor. At the C-terminal end of the RHD, each protein has a nuclear localization signal (NLS). The crystal structures of the p50 and RelA family members show that the RHD consists of two regions: an N-terminal section which contains some of the DNA contacts and a C-terminal section which contains the remaining DNA contacts and controls dimerization. In unstimulated cells, the homo- or heterodimeric Rel/NF-κB proteins are cytoplasmic by virtue of binding to an inhibitor protein (IκB) which somehow masks the NLS of each member of the dimer. The IκB proteins consist of an ankyrin-repeat-containing domain that is required for binding to dimers and N- and C-terminal domains that are dispensable for binding to most dimers. In this study, we examined the interaction between IκBα and Rel family homodimers by mutational analysis. We show that (i) the dimerization regions of p50, RelA, and c-Rel are sufficient for binding to IκBα, (ii) the NLSs of RelA and c-Rel are not required for binding to IκBα but do stabilize the interaction, (iii) the NLS of p50 is required for binding to IκBα, (iv) only certain residues within the p50 NLS are required for binding, and (v) in a p50-IκBα complex or a c-Rel-IκBα complex, the N terminus of IκBα either directly or indirectly masks one or both of the dimer NLSs.

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 The Novel Nuclear Targeting and BFRF1-Interacting Domains of BFLF2 Are Essential for Efficient Epstein-Barr Virus Virion Release

2019 ◽  
Vol 94 (3) ◽  
Author(s):  
Yu-Ching Dai ◽  
Yen-Tzu Liao ◽  
Yi-Ting Juan ◽  
Yi-Ying Cheng ◽  
Mei-Tzu Su ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Epstein Barr Virus ◽  
The Novel ◽  
Nuclear Targeting ◽  
Barr Virus ◽  
Nuclear Egress ◽  
Localization Signal ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) genomic DNA is replicated and packaged into procapsids in the nucleus to form nucleocapsids, which are then transported into the cytoplasm for tegumentation and final maturation. The process is facilitated by the coordination of the viral nuclear egress complex (NEC), which consists of BFLF2 and BFRF1. By expression alone, BFLF2 is distributed mainly in the nucleus. However, it colocalizes with BFRF1 at the nuclear rim and in cytoplasmic nuclear envelope-derived vesicles in coexpressing cells, suggesting temporal control of the interaction between BFLF2 and BFRF1 is critical for their proper function. The N-terminal sequence of BFLF2 is less conserved than that of alpha- and betaherpesvirus homologs. Here, we found that BFLF2 amino acids (aa) 2 to 102 are required for both nuclear targeting and its interaction with BFRF1. Coimmunoprecipitation and confocal analysis indicated that aa 82 to 106 of BFLF2 are important for its interaction with BFRF1. Three crucial amino acids (R47, K50, and R52) and several noncontinuous arginine and histidine residues within aa 59 to 80 function together as a noncanonical nuclear localization signal (NLS), which can be transferred onto yellow fluorescent protein (YFP)-LacZ for nuclear targeting in an importin β-dependent manner. Virion secretion is defective in 293 cells harboring a BFLF2 knockout EBV bacmid upon lytic induction and is restored by trans-complementation of wild-type BFLF2, but not NLS or BFRF1-interacting defective mutants. In addition, multiple domains of BFRF1 were found to bind BFLF2, suggesting multiple contact regions within BFRF1 and BFLF2 are required for proper nuclear egress of EBV nucleocapsids. IMPORTANCE Although Epstein-Barr virus (EBV) BFRF1 and BFLF2 are homologs of conserved viral nuclear egress complex (NEC) in all human herpesviruses, unique amino acid sequences and functions were identified in both proteins. In this study, the nuclear targeting and BFRF1-interacting domains were found within the N terminus of BFLF2. We showed that amino acids (aa) 82 to 106 are the major region required for BFLF2 to interact with BFRF1. However, the coimmunoprecipitation (Co-IP) data and glutathione transferase (GST) pulldown experiments revealed that multiple regions of both proteins contribute to reciprocal interactions. Different from the canonical nuclear localization signal (NLS) in other herpes viral homologs, BFLF2 contains a novel importin-dependent nuclear localization signal, including R47, K50, and R52 and several neighboring discontinuous arginine and histidine residues. Using a bacmid complementation system, we show that both the nuclear targeting and the novel nuclear localization signal within aa 82 to 106 of BFLF2 are required for virion secretion.

scholarly journals The C-Terminal Region of PTHrP, in Addition to the Nuclear Localization Signal, Is Essential for the Intracrine Stimulation of Proliferation in Vascular Smooth Muscle Cells

Endocrinology ◽  
2001 ◽  
Vol 142 (9) ◽  
pp. 4096-4105 ◽  
Author(s):  
F. de Miguel ◽  
N. Fiaschi-Taesch ◽  
J. C. López-Talavera ◽  
K. K. Takane ◽  
T. Massfelder ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Muscle Cells ◽  
Nuclear Entry ◽  
C Terminus ◽  
Localization Signal

Abstract PTHrP is secreted by most cell types. In addition to a paracrine/autocrine role, PTHrP has “intracrine” actions, entering the nuclear compartment under the direction of a classic bipartite nuclear localization signal. In vascular smooth muscle cells, nuclear entry stimulates mitogenesis. In the current study, we sought to more precisely define the regions of PTHrP required for the activation of mitogenesis in vascular smooth muscle cells. PTHrP deletion mutants missing large regions [i.e. the signal peptide, N terminus (1–36), mid region (38–86), nuclear localization signal, C terminus (108–139), or combinations of the above] were expressed in A-10 vascular smooth muscle cells. The consequences on nuclear localization and proliferation were examined. Deletion of the nuclear localization signal prevented nuclear entry and slowed proliferation. Deletion of the highly conserved N terminus or mid region had no impact on nuclear localization or on proliferation. Deletion of the C terminus had no deleterious effect on nuclear localization but dramatically reduced proliferation. Thus, the nuclear localization signal is both necessary and sufficient for nuclear localization of PTHrP. In contrast, activation of proliferation in vascular smooth muscle cells requires both an intact nuclear localization signal and an intact C terminus. Whereas the nuclear localization signal is required for nuclear entry, the C terminus may serve a trans-activating function to stimulate mitogenesis once inside the nucleus of vascular smooth muscle cells.

scholarly journals Conserved Regions in the Epstein-Barr Virus Leader Protein Define Distinct Domains Required for Nuclear Localization and Transcriptional Cooperation with EBNA2

2000 ◽  
Vol 74 (21) ◽  
pp. 9953-9963 ◽  
Author(s):  
RongSheng Peng ◽  
Jie Tan ◽  
Paul D. Ling
Keyword(s):  
Amino Acids ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Epstein Barr Virus ◽  
Intracellular Localization ◽  
Barr Virus ◽  
Conserved Regions ◽  
Localization Signal ◽  
Epstein Barr ◽  
Stimulation Of

ABSTRACT Epstein-Barr virus (EBV) EBNA-LP is a latent protein whose function is not fully understood. Recent studies have shown that EBNA-LP may be an important EBNA2 cofactor by enhancing EBNA2 stimulation of the latency C and LMP-1 promoters. To further our understanding of EBNA-LP function, we have introduced a series of mutations into evolutionarily conserved regions and tested the mutant proteins for the ability to enhance EBNA2 stimulation of the latency C and LMP-1 promoters. Three conserved regions (CR1 to CR3) are located in the repeat domains that are essential for the EBNA2 cooperativity function. In addition, three serine residues are also well conserved in the repeat domains. Clustered alanine mutations were introduced into CR1 to CR3, and the conserved serines were also changed to alanine residues in an EBNA-LP with two repeats, which is the minimal protein able to cooperate with EBNA2. Mutations introduced into CR1a had no effect on EBNA-LP function, while mutations introduced into CR1b resulted in EBNA-LP with slightly decreased activity. Mutations in CR1c and CR2 resulted in proteins that no longer localized exclusively to the nucleus and also had no EBNA2 cooperation activity. Mutations introduced into conserved serines S5/71 resulted in proteins with slightly higher activity, while mutations introduced into conserved serines S35/101 or in CR3 (which contains S60/126) resulted in EBNA-LP proteins with substantially reduced activity. The potential karyophilic signals within EBNA-LP CR1c and CR2 were also examined by introducing oligonucleotides encoding these positively charged amino acid groupings into a cytoplasmic test protein, herpes simplex virus ΔIE175, and by examining the intracellular localization of the resulting proteins. This assay identified a strong nuclear localization signal between EBNA-LP amino acids 43 and 50 (109 to 117 in the second W repeat) comprising CR2, while EBNA-LP amino acids 29 to 36 (91 to 98 in the second W repeat) were unable to function independently as a nuclear localization signal. However, a combination of amino acids 29 to 50 resulted in more efficient nuclear localization than with amino acids 43 to 50 alone. These results indicate that EBNA-LP has a bipartite nuclear localization signal and that efficient nuclear localization is essential for EBNA2 cooperativity function. Interestingly, EBNA-LP with only a single repeat localized exclusively to the cytoplasm, providing an explanation for why this isoform has no activity. In addition, two conserved serine residues which are distinct from nuclear import functions are important for EBNA2 cooperativity function.

scholarly journals Degradation of Saccharomyces cerevisiae Transcription Factor Gcn4 Requires a C-Terminal Nuclear Localization Signal in the Cyclin Pcl5

2009 ◽  
Vol 8 (4) ◽  
pp. 496-510 ◽  
Author(s):  
Katrin Streckfuss-Bömeke ◽  
Florian Schulze ◽  
Britta Herzog ◽  
Eva Scholz ◽  
Gerhard H. Braus
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Ubiquitin Proteasome System ◽  
Cyclin Dependent Kinase ◽  
General Amino Acid Control ◽  
Nuclear Domain ◽  
Ubiquitin Proteasome ◽  
Localization Signal

ABSTRACT Pcl5 is a Saccharomyces cerevisiae cyclin that directs the phosphorylation of the general amino acid control transcriptional activator Gcn4 by the cyclin-dependent kinase (CDK) Pho85. Phosphorylation of Gcn4 by Pho85/Pcl5 initiates its degradation via the ubiquitin/proteasome system and is regulated by the availability of amino acids. In this study, we show that Pcl5 is a nuclear protein and that artificial dislocation of Pcl5 into the cytoplasm prevents the degradation of Gcn4. Nuclear localization of Pcl5 depends on the β-importin Kap95 and does not require Pho85, Gcn4, or the CDK inhibitor Pho81. Pcl5 nuclear import is independent on the availability of amino acids and is mediated by sequences in its C-terminal domain. The nuclear localization signal is distinct from other functional domains of Pcl5. This is corroborated by a C-terminally truncated Pcl5 variant, which carries the N-terminal nuclear domain of Pho80. This hybrid is still able to fulfill Pcl5 function, whereas Pho80, which is another Pho85 interacting cyclin, does not mediate Gcn4 degradation.

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