The Nucleolar Localization Signal of Porcine Circovirus Type 4 Capsid Protein Is Essential for Interaction With Serine-48 Residue of Nucleolar Phosphoprotein Nucleophosmin-1

Porcine circovirus type 4 (PCV4) is an emerging etiological agent which was first detected in 2019. The nucleolar localization signal (NoLS) of PCV4 Cap protein and its binding host cellular proteins are still not elucidated. In the present study, we discovered a distinct novel NoLS of PCV4 Cap, which bound to the nucleolar phosphoprotein nucleophosmin-1 (NPM1). The NoLS of PCV4 Cap and serine-48 residue at the N-terminal oligomerization domain of NPM1 were necessary for PCV4 Cap/NPM1 interaction. Furthermore, the charge property of serine residue at position 48 of the NPM1 was crucial for its oligomerization and interaction with PCV4 Cap. In summary, our findings show for the first time that the PCV4 Cap NoLS and the NPM1 oligomerization determine the interaction of Cap/NPM1.

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The serine-48 residue of nucleolar phosphoprotein nucleophosmin-1 plays critical role in subcellular localization and interaction with porcine circovirus type 3 capsid protein

Veterinary Research ◽

10.1186/s13567-020-00876-9 ◽

2021 ◽

Vol 52 (1) ◽

Author(s):

Jianwei Zhou ◽

Juan Li ◽

Haimin Li ◽

Ying Zhang ◽

Weiren Dong ◽

...

Keyword(s):

Capsid Protein ◽

Critical Role ◽

Porcine Circovirus Type ◽

Porcine Circovirus ◽

Nucleolar Localization ◽

Nucleolar Localization Signal ◽

Localization Signal ◽

Charge Property ◽

Type 3 ◽

First Time

AbstractThe transport of circovirus capsid protein into nucleus is essential for viral replication in infected cell. However, the role of nucleolar shuttle proteins during porcine circovirus 3 capsid protein (PCV3 Cap) import is still not understood. Here, we report a previously unidentified nucleolar localization signal (NoLS) of PCV3 Cap, which hijacks the nucleolar phosphoprotein nucleophosmin-1 (NPM1) to facilitate nucleolar localization of PCV3 Cap. The NoLS of PCV3 Cap and serine-48 residue of N-terminal oligomerization domain of NPM1 are essential for PCV3 Cap/NPM1 interaction. In addition, charge property of serine-48 residue of NPM1 is critical for nucleolar localization and interaction with PCV3 Cap. Taken together, our findings demonstrate for the first time that NPM1 interacts with PCV3 Cap and is responsible for its nucleolar localization.

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Identification of nuclear/nucleolar localization signal in Aplysia learning associated protein of slug with a molecular mass of 18 kDa homologous protein

Neuroscience Letters ◽

10.1016/s0304-3940(03)00269-6 ◽

2003 ◽

Vol 343 (2) ◽

pp. 134-138 ◽

Cited By ~ 9

Author(s):

Hyoung Kim ◽

Deok-Jin Chang ◽

Jin-A Lee ◽

Yong-Seok Lee ◽

Bong-Kiun Kaang

Keyword(s):

Molecular Mass ◽

Nucleolar Localization ◽

Homologous Protein ◽

Nucleolar Localization Signal ◽

Localization Signal

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NOA36 Protein Contains a Highly Conserved Nucleolar Localization Signal Capable of Directing Functional Proteins to the Nucleolus, in Mammalian Cells

PLoS ONE ◽

10.1371/journal.pone.0059065 ◽

2013 ◽

Vol 8 (3) ◽

pp. e59065 ◽

Cited By ~ 8

Author(s):

Ivan S. de Melo ◽

Maria D. Jimenez-Nuñez ◽

Concepción Iglesias ◽

Antonio Campos-Caro ◽

David Moreno-Sanchez ◽

...

Keyword(s):

Mammalian Cells ◽

Nucleolar Localization ◽

Nucleolar Localization Signal ◽

Localization Signal

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High-level expression and immunogenicity of porcine circovirus type 2b capsid protein without nuclear localization signal expressed in Hansenula polymorpha

Biologicals ◽

10.1016/j.biologicals.2017.11.003 ◽

2018 ◽

Vol 51 ◽

pp. 18-24 ◽

Cited By ~ 5

Author(s):

Yue Xiao ◽

Peiyan Zhao ◽

Junyang Du ◽

Xin Li ◽

Wenting Lu ◽

...

Keyword(s):

Capsid Protein ◽

Nuclear Localization ◽

Nuclear Localization Signal ◽

Hansenula Polymorpha ◽

Porcine Circovirus Type ◽

Porcine Circovirus ◽

High Level Expression ◽

Localization Signal ◽

High Level

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Nonstructural Protein NSs of Schmallenberg Virus Is Targeted to the Nucleolus and Induces Nucleolar Disorganization

Journal of Virology ◽

10.1128/jvi.01263-16 ◽

2016 ◽

Vol 91 (1) ◽

Cited By ~ 11

Author(s):

Julie Gouzil ◽

Aurore Fablet ◽

Estelle Lara ◽

Grégory Caignard ◽

Marielle Cochet ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Virulence Factor ◽

Nonstructural Protein ◽

Schmallenberg Virus ◽

Nucleolar Localization ◽

Pol Ii ◽

Nucleolar Localization Signal ◽

Localization Signal ◽

Infected Cells ◽

Cellular Transcription

ABSTRACT Schmallenberg virus (SBV) was discovered in Germany in late 2011 and then spread rapidly to many European countries. SBV is an orthobunyavirus that causes abortion and congenital abnormalities in ruminants. A virus-encoded nonstructural protein, termed NSs, is a major virulence factor of SBV, and it is known to promote the degradation of Rpb1, a subunit of the RNA polymerase II (Pol II) complex, and therefore hampers global cellular transcription. In this study, we found that NSs is mainly localized in the nucleus of infected cells and specifically appears to target the nucleolus through a nucleolar localization signal (NoLS) localized between residues 33 and 51 of the protein. NSs colocalizes with nucleolar markers such as B23 (nucleophosmin) and fibrillarin. We observed that in SBV-infected cells, B23 undergoes a nucleolus-to-nucleoplasm redistribution, evocative of virus-induced nucleolar disruption. In contrast, the nucleolar pattern of B23 was unchanged upon infection with an SBV recombinant mutant with NSs lacking the NoLS motif (SBVΔNoLS). Interestingly, unlike wild-type SBV, the inhibitory activity of SBVΔNoLS toward RNA Pol II transcription is impaired. Overall, our results suggest that a putative link exists between NSs-induced nucleolar disruption and its inhibitory function on cellular transcription, which consequently precludes the cellular antiviral response and/or induces cell death. IMPORTANCE Schmallenberg virus (SBV) is an emerging arbovirus of ruminants that spread in Europe between 2011 and 2013. SBV induces fetal abnormalities during gestation, with the central nervous system being one of the most affected organs. The virus-encoded NSs protein acts as a virulence factor by impairing host cell transcription. Here, we show that NSs contains a nucleolar localization signal (NoLS) and induces disorganization of the nucleolus. The NoLS motif in the SBV NSs is absolutely necessary for virus-induced inhibition of cellular transcription. To our knowledge, this is the first report of nucleolar functions for NSs within the Bunyaviridae family.

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Internal Modification of U2 Small Nuclear (Snrna) Occurs in Nucleoli of Xenopus Oocytes

The Journal of Cell Biology ◽

10.1083/jcb.152.6.1279 ◽

2001 ◽

Vol 152 (6) ◽

pp. 1279-1288 ◽

Cited By ~ 41

Author(s):

Yi-Tao Yu ◽

Mei-Di Shu ◽

Aarthi Narayanan ◽

Rebecca M. Terns ◽

Michael P. Terns ◽

...

Keyword(s):

Binding Site ◽

Xenopus Oocytes ◽

Cajal Bodies ◽

Nucleolar Localization ◽

Small Nucleolar Rnas ◽

Modified Nucleotides ◽

Nucleolar Localization Signal ◽

Localization Analysis ◽

Localization Signal ◽

Nucleolar Rnas

U2 small nuclear (sn)RNA contains a large number of posttranscriptionally modified nucleotides, including a 5′ trimethylated guanosine cap, 13 pseudouridines, and 10 2′-O-methylated residues. Using Xenopus oocytes, we demonstrated previously that at least some of these modified nucleotides are essential for biogenesis of a functional snRNP. Here we address the subcellular site of U2 internal modification. Upon injection into the cytoplasm of oocytes, G-capped U2 that is transported to the nucleus becomes modified, whereas A-capped U2 that remains in the cytoplasm is not modified. Furthermore, by injecting U2 RNA into isolated nuclei or enucleated oocytes, we observe that U2 internal modifications occur exclusively in the nucleus. Analysis of the intranuclear localization of fluorescently labeled RNAs shows that injected wild-type U2 becomes localized to nucleoli and Cajal bodies. Both internal modification and nucleolar localization of U2 are dependent on the Sm binding site. An Sm-mutant U2 is targeted only to Cajal bodies. The Sm binding site can be replaced by a nucleolar localization signal derived from small nucleolar RNAs (the box C/D motif), resulting in rescue of internal modification as well as nucleolar localization. Analysis of additional chimeric U2 RNAs reveals a correlation between internal modification and nucleolar localization. Together, our results suggest that U2 internal modification occurs within the nucleolus.

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Properties of expression of protein capside porcine circovirus type 2 in bacterial cells

Journal of the Belarusian State University Biology ◽

10.33581/2521-1722-2021-1-48-57 ◽

2021 ◽

pp. 48-57

Author(s):

Anastasia D. Titova ◽

Kirill V. Kudzin ◽

Vladimir A. Prokulevich

Keyword(s):

Nuclear Localization ◽

Nuclear Localization Signal ◽

Porcine Circovirus Type ◽

Porcine Circovirus Type 2 ◽

Porcine Circovirus ◽

Bacterial Cells ◽

Reading Frame ◽

E Coli ◽

Localization Signal

To improve expression of the porcine circovirus type 2 (PCV2) capsid protein in E. coli cells, the corresponding gene was optimized and two variants of the open reading frame were constructed, which encoded the full-sized and shortened capsid proteins as part of the expression vector. Rare codons were replaced, and in the case of a shortened version of the gene, the region corresponding to the N-terminal domain of the protein was deleted. A comparison was made of the expression level of the studied proteins. It was established that the highest level of expression in bacterial cells is achieved by simultaneously optimizing the codons and removing the initial (N-terminal) 108 base pair (bp) portion of the gene, which contains the nuclear localization signal.

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Design and preliminary application of affinity peptide based on the structure of the porcine circovirus type II Capsid (PCV2 Cap)

PeerJ ◽

10.7717/peerj.8132 ◽

2019 ◽

Vol 7 ◽

pp. e8132 ◽

Cited By ~ 2

Author(s):

Junfang Hao ◽

Fangyu Wang ◽

Guangxu Xing ◽

Yunchao Liu ◽

Ruiguang Deng ◽

...

Keyword(s):

Molecular Docking ◽

Magnetic Beads ◽

Ph Value ◽

Porcine Circovirus Type ◽

Single Step ◽

Porcine Circovirus ◽

Ligand Density ◽

Affinity Adsorbent ◽

Affinity Peptide ◽

First Time

Background Affinity peptides, as a core part of affinity chromatography, play an important role in the purification of target molecules. Methods Here we describe the use of molecular docking technology for virtual screening of affinity peptides that specifically recognize the PCV2 Cap protein for the first time. Thirteen candidate peptides with high scores were obtained and then further characterized. Experimentally, the affinity and sensitivity of the peptides studied were identified by ELISA and LSPR, respectively. In order to investigate the purification effect of a selected peptide (L11) for the recombinant PCV2 Cap protein, it was coupled to NHS agarose magnetic beads as an affinity adsorbent (NaMB-L11); and the ligand density of the affinity adsorbent and pH value in the purification of the recombinant PCV2 Cap protein were optimized. Results Our data showed that the peptide L11- DYWWQSWE has the smallest KD = 103 nM with higher specificity for PCV2 Cap protein recognition. The NaMB-L11 affinity adsorbent yielded a purified Cap sample with 98% purity at 90% recovery in a single step. Conclusion Based on the structure, we obtained a high affinity peptide L11 binding to the PCV2 Cap protein by molecular docking technology. It not only provides a theoretical basis for the design of PCV2 Cap affinity peptide, but a new method for the purification of the PCV2 Cap protein.

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ARF Function Does Not Require p53 Stabilization or Mdm2 Relocalization

Molecular and Cellular Biology ◽

10.1128/mcb.22.1.196-206.2002 ◽

2002 ◽

Vol 22 (1) ◽

pp. 196-206 ◽

Cited By ~ 68

Author(s):

Chandrashekhar Korgaonkar ◽

Lili Zhao ◽

Modestos Modestou ◽

Dawn E. Quelle

Keyword(s):

Transcriptional Activation ◽

Growth Arrest ◽

Nucleolar Localization ◽

Wild Type ◽

Growth Inhibitory ◽

Nucleolar Localization Signal ◽

Localization Signal ◽

Reporter Assays ◽

Dependent Growth ◽

Block Growth

ABSTRACT It is generally accepted that the ARF tumor suppressor induces p53-dependent growth arrest by sequestering the p53 antagonist Mdm2 in the nucleolus. Previous mutagenic studies of murine ARF suggested that residues 1 through 14 and 26 through 37 were critical for Mdm2 binding, while the latter domain also governed ARF nucleolar localization. We show that mouse ARF residues 6 to 10 and 21 to 25 are required for ARF-induced growth arrest whereas residues 1 to 5 and 29 to 34 are dispensable. Deletion of the putative nucleolar localization signal 31RRPR34 did not prevent nucleolar localization. Surprisingly, unlike wild-type ARF, growth-inhibitory mutants D1-5 and D29-34 failed to stabilize p53 yet induced its transcriptional activation in reporter assays. This suggests that p53 stabilization is not essential for ARF-mediated activation of p53. Like wild-type ARF, both mutants also exhibited p53-independent function since they were able to arrest p53/Mdm2-null cells. Notably, other mutants lacking conserved residues 6 to 10 or 21 to 25 were unable to suppress growth in p53-positive cells despite nucleolar localization and the ability to import Mdm2. Those observations stood in apparent contrast to the ability of wild-type ARF to block growth in some cells without relocalizing endogenous Mdm2 to nucleoli. Together, these data show a lack of correlation between ARF activity and Mdm2 relocalization, suggesting that additional events other than Mdm2 import are required for ARF function.

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