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.

SUMOylation- and GAR1-dependent regulation of dyskerin nuclear and subnuclear localization

The nuclear and subnuclear compartmentalization of the telomerase-associated protein and H/ACA ribonucleoprotein component dyskerin is an important though incompletely understood aspect of H/ACA ribonucleoprotein function. Four SUMOylation sites were previously identified in the C-terminal Nuclear/Nucleolar Localization Signal (N/NoLS) of dyskerin. We found that a cytoplasmic localized C-terminal truncation variant of dyskerin lacking most of the C-terminal N/NoLS represents an under-SUMOylated variant of dyskerin compared to wildtype dyskerin. We demonstrate that mimicking constitutive SUMOylation of dyskerin using a SUMO3-fusion construct can drive nuclear accumulation of this variant, and that the SUMO site K467 in this N/NoLS is particularly important for the subnuclear localization of dyskerin to the nucleolus in a mature H/ACA complex assembly- and SUMO-dependent manner. We also characterize a novel SUMO-interacting motif in the mature H/ACA complex component GAR1 that mediates the interaction between dyskerin and GAR1. Mislocalization of dyskerin, either in the cytoplasm or excluded from the nucleolus, disrupts dyskerin function and leads to reduced interaction of dyskerin with the telomerase RNA. These data indicate a role for dyskerin C-terminal N/NoLS SUMOylation in regulating the nuclear and subnuclear localization of dyskerin, which is essential for dyskerin function as both a telomerase-associated protein and as an H/ACA ribonucleoprotein.

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

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.

SUMOylation- and GAR1-dependent regulation of dyskerin nuclear and subnuclear localization

SummaryDyskerin, a telomerase-associated protein and H/ACA ribonucleoprotein complex component plays an essential role in human telomerase assembly and activity. The nuclear and subnuclear compartmentalization of dyskerin and the H/ACA complex is an important though incompletely understood aspect of H/ACA ribonucleoprotein function. The posttranslational modification, SUMOylation, targets a wide variety of proteins, including numerous RNA-binding proteins, and most identified targets reported to date localize to the nucleus. Four SUMOylation sites were previously identified in the C-terminal Nuclear/Nucleolar Localization Signal (N/NoLS) of dyskerin, each located within one of two lysine-rich clusters. We found that a cytoplasmic localized C-terminal truncation variant of dyskerin lacking most of the C-terminal N/NoLS and both lysine-rich clusters represents an under-SUMOylated variant of dyskerin compared to wildtype dyskerin. We demonstrate that mimicking constitutive SUMOylation of dyskerin using a SUMO3-fusion construct can drive nuclear accumulation of this variant, and that the SUMO site K467 in this N/NoLS is particularly important for the subnuclear localization of dyskerin to the nucleolus in a mature H/ACA complex assembly- and SUMO-dependent manner. We also characterize a novel SUMO-interacting motif in the mature H/ACA complex component GAR1 that mediates the interaction between dyskerin and GAR1. Mislocalization of dyskerin, either in the cytoplasm or excluded from the nucleolus, disrupts dyskerin function and leads to reduced interaction of dyskerin with the telomerase RNA. These data indicate a role for dyskerin C-terminal N/NoLS SUMOylation in regulating the nuclear and subnuclear localization of dyskerin, which is essential for dyskerin function as both a telomerase-associated protein and as an H/ACA ribonucleoprotein involved in rRNA and snRNA biogenesis.

The GAR domain integrates functions that are necessary for the proper localization of fibrillarin (FBL) inside eukaryotic cells

Fibrillarin (FBL) is an essential nucleolar protein that participates in pre-rRNA methylation and processing. The methyltransferase domain of FBL is an example of an extremely well-conserved protein domain in which the amino acid sequence was not substantially modified during the evolution from Archaea to Eukaryota. An additional N-terminal glycine–arginine-rich (GAR) domain is present in the FBL of eukaryotes. Here, we demonstrate that the GAR domain is involved in FBL functioning and integrates the functions of the nuclear localization signal and the nucleolar localization signal (NoLS). The methylation of the arginine residues in the GAR domain is necessary for nuclear import but decreases the efficiency of nucleolar retention via the NoLS. The presented data indicate that the GAR domain can be considered an evolutionary innovation that integrates several functional activities and thereby adapts FBL to the highly compartmentalized content of the eukaryotic cell.

Identification and Characterization of the Nucleolar Localization Signal of Autographa californica Multiple Nucleopolyhedrovirus LEF5

ABSTRACT Autographa californica multiple nucleopolyhedrovirus (AcMNPV) late expression factor 5 (LEF5) is highly conserved in all sequenced baculovirus genomes and plays an important role in production of infectious viral progeny. In this study, nucleolar localization of AcMNPV LEF5 was characterized. Through transcriptome analysis, we identified two putative nucleolar proteins, Spodoptera frugiperda nucleostemin (SfNS) and fibrillarin (SfFBL), from Sf9 cells. Immunofluorescence analysis demonstrated that SfNS and SfFBL were localized to the nucleolus. AcMNPV infection resulted in reorganization of the nucleoli of infected cells. Colocalization of LEF5 and SfNS showed that AcMNPV LEF5 was localized to the nucleolus in Sf9 cells. Bioinformatic analysis revealed that basic amino acids of LEF5 are enriched at residues 184 to 213 and may contain a nucleolar localization signal (NoLS). Green fluorescent protein (GFP) fused to NoLS of AcMNPV LEF5 localized to the nucleoli of transfected cells. Multiple-point mutation analysis demonstrated that amino acid residues 197 to 204 are important for nucleolar localization of LEF5. To identify whether the NoLS in AcMNPV LEF5 is important for production of viral progeny, a lef5-null AcMNPV bacmid was constructed; several NoLS-mutated LEF5 proteins were reinserted into the lef5-null AcMNPV bacmid with a GFP reporter. The constructs containing point mutations at residues 185 to 189 or 197 to 204 in AcMNPV LEF5 resulted in reduction in production of infectious viral progeny and occlusion body yield in bacmid-transfected cells. Together, these data suggested that AcMNPV LEF5 contains an NoLS, which is important for nucleolar localization of LEF5, progeny production, and occlusion body production. IMPORTANCE Many viruses, including human and plant viruses, target nucleolar functions as part of their infection strategy. However, nucleolar localization for baculovirus proteins has not yet been characterized. In this study, two nucleolar proteins, SfNS and SfFBL, were identified in Sf9 cells. Our results showed that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection resulted in redistribution of the nucleoli of infected cells. We demonstrated that AcMNPV late expression factor 5 (LEF5) could localize to the nucleolus and contains a nucleolar localization signal (NoLS), which is important for nucleolar localization of AcMNPV LEF5 and for production of viral progeny and yield of occlusion bodies.

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

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.