Identification of Nuclear Localization Signals in the ORF2 Protein of Porcine Circovirus Type 3

Abstract

Porcine circovirus type 3 (PCV3) contains two major open reading frames (ORFs) and the ORF2 gene encodes the major structural capsid protein. In this study, nuclear localization of ORF2 was demonstrated by fluorescence observation and subcellular fractionation assays in ORF2-transfected PK-15 cells. The subcellular localization of truncated ORF2 indicated that the 38 N-terminal amino acids were responsible for the nuclear localization of ORF2. The truncated and site-directed mutagenesis of this domain were constructed, and the results demonstrated that the basic amino acid residues at positions 8-32 were essential for the strict nuclear localization. The basic motifs 8 RRR-R-RRR 16 and 16 RRRHRRR 22 were further shown to be the key functional nucleolar localization signals that guide PCV3 ORF2 into nucleoli. Furthermore, sequence analysis showed that the amino acids of PCV3 nuclear localization signals were highly conserved. Overall, this study provides insight into the biological and functional characteristics of the PCV3 ORF2 protein.case of PCVs, the N-terminus of ORF2 is rich in basic amino acids and displays nuclear localization signals (NLSs) [14][15][16]. The NLS is a short stretch of amino acids that mediates the transport of nuclear proteins into the nucleus. NLS motifs play a key role in this mechanism. NLS sequences are often composed of basic amino acids and can be classified as either monopartite or bipartite motifs [17,18]. The nuclear targeting of PCV2 ORF2 is directed by the bipartite motifs situated at the N-terminus of the proteins [14,15]. NLSs of PCV1 ORF2 show high homologies to classical monopartite or bipartite NLS, which is essential for the complete nuclear import of PCV1 ORF2. The N-terminus of PCV1 and PCV2 ORF2 contain several conserved basic amino acid stretches [15,19], and shares 70.7% in nucleotide identity and 82.9% in amino acid similarity.Verification of NLS and demonstration of intracellular distribution facilitate understanding of viral protein function. The amino acids of PCV3 ORF2 are markedly different from those of the other PCVs. This led us to study the functional motifs in nuclear targeting of PCV3 ORF2. In this study, a series of recombinant plasmids expressing PCV3 ORF2 fused to EGFP were constructed, and we identified the main motifs of the NLSs mediating nuclear localization of PCV3 ORF2.

Nucleolar Phosphoprotein NPM1 Interacts With Porcine Circovirus Type 3 Cap Protein and Facilitates Viral Replication

“…NPM1 has been demonstrated to interact with PCV2 Cap protein and contribute to PCV2 replication ( Zhou et al, 2020 ). As reported in previous studies, in which PCV3 Cap was found to have a nucleolar localization signal ( Mou et al, 2019 ), the present study also demonstrated that PCV3 Cap interacts and colocalizes with nucleolar phosphoprotein NPM1 ( Zhou et al, 2021 ). Thus, we focused on the interaction between the PCV3 Cap protein and NPM1 and the contribution of NPM1 to PCV3 replication in subsequent experiments.…”

“…Without encoding DNA polymerase in circoviruses, the replication machinery of the host cell is needed for de novo DNA synthesis ( Heath et al, 2006 ). All PCVs possess a nuclear localization signal (NLS) at the N-terminus of the Cap protein ( Liu et al, 2001 ; Shuai et al, 2008 ; Mou et al, 2019 ). Although the amino acids of the PCV3 Cap protein are significantly different from those of PCV1 and PCV2, their motifs within the NLSs share some similarities ( Liu et al, 2001 ; Shuai et al, 2008 ; Mou et al, 2019 ).…”

Molecular based detection, genetic characterization and phylogenetic analysis of porcine circovirus 4 from Korean domestic swine farms

“…Of the putative Cap, a nuclear localization signal (NLS) was predicted in the N-terminus of the putative Cap of E115 strain (Figure 4). Based on the alignment with other members of Circovirus genus, Figure 4 showed that the arginine-rich region of E115 strain was aligned with the basic motifs of the experimentally confirmed NLS of PCV1, PCV2 and PCV3 (Liu, Tikoo, & Babiuk, 2001;Mou, Wang, Pan, & Chen, 2019;Shuai et al, 2008). Additionally, motif screening (Figure 5) indicated that the putative Cap of E115 strain contained several tyrosine-based Y-x-x-φ motifs and P-x-x-P motifs (x represents any amino acid and φ denotes a large hydrophobic residue of either F, I, L or V) which were related to clathrin-mediated endocytosis (Sobhy, 2016).…”

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 ORF2-encoded capsid protein (Cap) is necessary for virion packaging and participates in genome replication by interacting with Rep protein and is also the main viral immunogen and acts as a key regulator of viral replication as well as the virus-host interaction [27][28][29][30][31][32][33][34]. The N-terminus of PCV3 Cap is rich in basic amino acids, and the amino acid residues 1-34 was identified as nuclear localization signal (NLS), which is essential for nuclear transport of PCV3 Cap [35]. Besides, PCV3 Cap interacts with signal transducer and activator of transcription 2 (STAT2) to inhibit type I interferon signaling [36].…”