The Nucleolar Localization Signal of Porcine Circovirus Type 4 Capsid Protein Is Essential for Interaction With Serine-48 Residue of Nucleolar Phosphoprotein Nucleophosmin-1
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ABSTRACT:
SUBMITTER: Zhou J
PROVIDER: S-EPMC8566881 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The 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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Project description:Porcine circovirus type 3 (PCV3) is a recently discovered virus with potentially significant implications on the global swine industry. PCV3 replication involves the entry of the viral capsid (Cap) protein with nucleolar localization signals into the nucleus. Using liquid chromatography-mass spectrometry analysis, nucleolar phosphoprotein NPM1 was identified as one of the cellular proteins bound to PCV3 Cap. Co-immunoprecipitation demonstrated that PCV3 Cap interacts directly with NPM1, where the region binding with NPM1 is mapped to amino acid residues 1-38 of Cap. Upon co-transfection, the expression of Cap protein promoted the redistribution of NPM1, which translocated from the nucleus to the cytoplasm and colocalized with Cap in cultured PK15 cells. NPM1 expression was upregulated and translocated from the nucleus to the cytoplasm in PCV3-infected cells, upon siRNA-mediated depletion, or upon treatment with NPM1 inhibitor in PK15 cells with impaired PCV3 replication, as evidenced by decreased levels of viral DNA synthesis and protein expression. By contrast, the replication of PCV3 was enhanced in stably NPM1-expressing cells via a lentivirus-delivered system. Taken together, these findings indicate that NPM1 interacts with PCV3 Cap and plays a crucial role in PCV3 replication.
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Project description:Porcine circovirus 3 (PCV3) is a newly emerging virus associated with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive disorders, impacting global pig populations. Porcine circoviruses contain two major open reading frames (ORFs), and the ORF2 encodes the viral capsid protein (Cap). Cap is the most antigenic structural protein and an ideal candidate for the development of vaccines and diagnostic reagents. This study generated a monoclonal antibody (MAb) specific to PCV3 Cap, MAb CCC160, for diagnosis and pathogenesis studies of this novel virus. The MAb specifically recognized PCV3-infected swine lymph node tissue in an immunohistochemical analysis confirming its clinical diagnostic potential. In addition, a novel linear B-cell epitope recognized by MAb CCC160 was identified at the amino acid region 120-134 of Cap. Nuclear localization analysis of PCV3 Cap revealed a potential nuclear localization signal (NLS) in the middle region (aa 131-143) in addition to the dominant N-terminal NLS that is already known. A cell viability assay further demonstrated that the cytotoxicity of PCV3 Cap is correlated with its nuclear localization, indicating a crucial role of Cap in the pathogenic mechanism of PCV3. A full-length construct of PCV3 Cap was successfully expressed using a baculovirus expression system and purified recombinant proteins self-assembled into virus-like particles (VLPs). The protein constitution of the VLPs was confirmed by MAb CCC160 recognition, indicating the correct conformation and specificity of VLP and exhibiting the linear epitope aa 120-134 on the VLP surface. These results provide insights for developing diagnostic tools and potential VLP vaccines for PCV3, revealing its pathogenesis and antigenic properties.
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Project description:Porcine circovirus 2 (PCV2), considered one of the most globally important porcine pathogens, causes postweaning multisystemic wasting syndrome (PMWS). This virus is localized in the mitochondria in pigs with PMWS. Here, we identified, for the first time, a mitochondrial localization signal (MLS) in the PCV2 capsid protein (Cap) at the N-terminus. PK-15 cells showed colocalization of the MLS-EGFP fusion protein with mitochondria. Since the PCV2 Cap also contained a nuclear localization signal (NLS) that mediated entry into the nucleus, we inferred that the subcellular localization of the PCV2 Cap is inherently complex and dependent on the viral life cycle. Furthermore, we also determined that deletion of the MLS attenuated Cap-induced apoptosis. More importantly, the MLS was essential for PCV2 replication, as absence of the MLS resulted in failure of virus rescue from cells infected with infectious clone DNA. In conclusion, the MLS of the PCV2 Cap plays critical roles in Cap-induced apoptosis, and MLS deletion of Cap is lethal for virus rescue.
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Project description:Wild-type human hepatitis B virus (HBV) exhibits selective export of virions containing mature genomes. In contrast, changing an isoleucine to a leucine at amino acid 97 (I97L) of the HBV core antigen (HBcAg) causes it to release immature genomes. To elucidate the structure-function relationship of HBcAg at amino acid 97, we systematically replaced the isoleucine residue at this position with 18 other amino acids via mutagenesis. Twelve of the 18 mutants exhibited no significant phenotype, while five new mutants displayed strong phenotypes. The I97D mutant had a near lethal phenotype, the I97P mutant exhibited a significantly reduced level of virion secretion, and the I97G mutant lacked the full-length relaxed circular form of viral DNA. The tip of the spike of the capsid particle is known to contain a predominant B-cell epitope. However, the recognition of this exposed epitope by an anti-HBc antibody appeared to be affected by the I97E mutation or by histidine tagging at the C terminus of mutant HBcAg, which is presumably in the capsid interior. Surprisingly, the nuclear HBcAg of mutants I97E and I97W, produced from either a replicon or an expression vector, was found to be colocalized with nucleolin and B23 at a frequency of nearly 100% by confocal immunofluorescence microscopy. In contrast, this colocalization occurred with wild-type HBcAg only to a limited extent. We also noted that nucleolin-colocalizing cells were often binucleated or apoptotic, suggesting that the presence of HBcAg in the nucleolus may perturb cytokinesis. The mechanism of this phenomenon and its potential involvement in liver pathogenesis are discussed. To our knowledge, this is the first report of nucleolar HBcAg in culture.
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Project description:Entry of circovirus into the host cell nucleus is essential for viral replication during the early stage of infection. However, the mechanisms by which nucleolar shuttle proteins are used during viral replication is still not well understood. Here, we report a previously unidentified nucleolar localization signal in circovirus capsid protein (Cap), and that circovirus hijacks the nucleolar phosphoprotein nucleophosmin-1 (NPM1) to facilitate its replication. Colocalization analysis showed that NPM1 translocates from the nucleolus to the nucleoplasm and cytoplasm during viral infection. Coimmunoprecipitation and glutathione S-transferase pull-down assays showed that Cap interacts directly with NPM1. Binding domain mapping showed that the arginine-rich N-terminal motif 1MTYPRRRYRRRRHRPRSHLG20 of Cap, and residue serine-48 of the N-terminal oligomerization domain of NPM1, are essential for the interaction. Virus rescue experiments showed that all arginine to alanine substitution in the N-terminal arginine-rich motif of Cap resulted in diminished viral replication. Knockdown of NPM1 and substitution of serine-48 in NPM1 to glutamic acid also decreased viral replication. In addition, binding assays showed that the arginine-rich motif of Cap is a nucleolar localization signal. Taken together, our findings demonstrate that circovirus protein Cap is a nucleolus-located, and regulates viral replication by directly binding to NPM1.
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Project description:The transcription factor c-Myc has a critical role in cell proliferation and growth. The control of ribosome biogenesis by c-Myc through the regulation of transcription mediated by all three RNA polymerases is essential for c-Myc-driven proliferation. Specifically, in the nucleolus, c-Myc has been shown to be recruited to ribosomal DNA and activate RNA polymerase (pol) I-mediated transcription of ribosomal RNA (rRNA) genes. In addition, c-Myc accumulates in nucleoli upon inhibition of the proteasome, suggesting nucleolar localization also has a role in c-Myc proteolysis. Nucleophosmin (NPM), a predominantly nucleolar protein, is also critical in ribosome biogenesis and, like c-Myc, is found overexpressed in many types of tumors. Previously, we demonstrated that NPM directly interacts with c-Myc and controls c-Myc-induced hyperproliferation and transformation. Here, we show that NPM is necessary for the localization of c-Myc protein to nucleoli, whereas c-Myc nucleolar localization is independent of p53, Mdm2 and ARF. Conversely, high transient NPM expression enhances c-Myc nucleolar localization, leading to increased c-Myc proteolysis. In addition, NPM is necessary for the ability of c-Myc to induce rRNA synthesis in the nucleolus, and constitutive NPM overexpression stimulates c-Myc-mediated rRNA synthesis. Taken together, these results demonstrate an essential role for NPM in c-Myc nucleolar localization and c-Myc-mediated rDNA transcription.
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Project description:Nucleophosmin (NPM1) is an abundant nucleolar protein implicated in ribosome maturation and export, centrosome duplication and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia. Mutations at the C-terminal domain led to variant proteins that aberrantly and stably translocate to the cytoplasm. We have previously shown that NPM1 C-terminal domain binds with high affinity G-quadruplex DNA. Here, we investigate the structural determinants of NPM1 nucleolar localization. We show that NPM1 interacts with several G-quadruplex regions found in ribosomal DNA, both in vitro and in vivo. Furthermore, the most common leukemic NPM1 variant completely loses this activity. This is the consequence of G-quadruplex-binding domain destabilization, as mutations aimed at refolding the leukemic variant also result in rescuing the G-quadruplex-binding activity and nucleolar localization. Finally, we show that treatment of cells with a G-quadruplex selective ligand results in wild-type NPM1 dislocation from nucleoli into nucleoplasm. In conclusion, this work establishes a direct correlation between NPM1 G-quadruplex binding at rDNA and its nucleolar localization, which is impaired in the acute myeloid leukemia-associated protein variants.
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Project description:Spatiotemporal regulation of viral capsid assembly ensures the selection of the viral genome for encapsidation. The porcine circovirus 2 is the smallest autonomously replicating pathogenic virus, yet how PCV2 capsid assembly is regulated to occur within the nucleus remains unknown. We report that pure PCV2 capsid proteins, in the absence of nucleic acids, require acidic conditions to assemble into empty capsids in vitro. By employing constant pH replica exchange molecular dynamics, we unveil the atomistic mechanism of pH-dependency for capsid assembly. The results show that an appropriate protonation configuration for a cluster of acidic amino acids is necessary to appropriately position the GH-loop for driving the capsid assembly. We demonstrate that assembly is prohibited at neutral pH because deprotonation of these residues results in their electrostatic repulsion, shifting the GH-loop to a position incompatible with capsid assembly. We propose that encapsulation of nucleic acids overcomes this repulsion to suitably position the GH-loop. Our findings provide the first atomic resolution mechanism of capsid assembly regulation. These findings are useful for the development of therapeutics that inhibit PCV2 self-assembly.
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