Dataset Information

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 8RRR-R-RRR16 and 16RRRHRRR22 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.

SUBMITTER: Mou C

PROVIDER: S-EPMC6950156 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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

Mou Chunxiao C Wang Minmin M Pan Shuonan S Chen Zhenhai Z

Viruses 20191122 12

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 ...[more]

PMID: 31766638

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Project description:Nuclear targeting of capsid proteins (VPs) is important for genome delivery and precedes assembly in the replication cycle of porcine parvovirus (PPV). Clusters of basic amino acids, corresponding to potential nuclear localization signals (NLS), were found only in the unique region of VP1 (VP1up, for VP1 unique part). Of the five identified basic regions (BR), three were important for nuclear localization of VP1up: BR1 was a classic Pat7 NLS, and the combination of BR4 and BR5 was a classic bipartite NLS. These NLS were essential for viral replication. VP2, the major capsid protein, lacked these NLS and contained no region with more than two basic amino acids in proximity. However, three regions of basic clusters were identified in the folded protein, assembled into a trimeric structure. Mutagenesis experiments showed that only one of these three regions was involved in VP2 transport to the nucleus. This structural NLS, termed the nuclear localization motif (NLM), is located inside the assembled capsid and thus can be used to transport trimers to the nucleus in late steps of infection but not for virions in initial infection steps. The two NLS of VP1up are located in the N-terminal part of the protein, externalized from the capsid during endosomal transit, exposing them for nuclear targeting during early steps of infection. Globally, the determinants of nuclear transport of structural proteins of PPV were different from those of closely related parvoviruses. Importance: Most DNA viruses use the nucleus for their replication cycle. Thus, structural proteins need to be targeted to this cellular compartment at two distinct steps of the infection: in early steps to deliver viral genomes to the nucleus and in late steps to assemble new viruses. Nuclear targeting of proteins depends on the recognition of a stretch of basic amino acids by cellular transport proteins. This study reports the identification of two classic nuclear localization signals in the minor capsid protein (VP1) of porcine parvovirus. The major protein (VP2) nuclear localization was shown to depend on a complex structural motif. This motif can be used as a strategy by the virus to avoid transport of incorrectly folded proteins and to selectively import assembled trimers into the nucleus. Structural nuclear localization motifs can also be important for nuclear proteins without a classic basic amino acid stretch, including multimeric cellular proteins.

Project description:Circoviruses are the smallest DNA viruses known to infect mammalian and avian species. Although circoviruses are known to be associated with a range of clinical diseases, the details of circovirus DNA release still remain unknown. Here, we identified p32 as a key regulator for porcine circoviral nuclear egress. Upon porcine circovirus type 2 (PCV2) infection, p32 was recruited into the nucleus by the viral capsid (Cap) protein; simultaneously, protein kinase C isoform ? (PKC-?) was phosphorylated at threonine 505 by phospholipase C (PLC)-mediated signaling at the early stage of infection, which was further amplified by Jun N-terminal protein kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling at the late infection phase. p32 functioned as an adaptor to recruit phosphorylated PKC-? and Cap to the nuclear membrane to phosphorylate lamin A/C, resulting in a rearrangement of nuclear lamina and thus facilitating viral nuclear egress. Consistent with these findings, knockout (KO) of p32 in PCV2-infected cells markedly reduced the phosphorylation of PKC-? and impeded the recruitment of p-PKC-? and Cap to the nuclear membrane, hence abolishing the phosphorylation of lamin A/C and the rearrangement of nuclear lamina. As a result, p32 depletion profoundly impaired the production of cell-free viruses during PCV2 infection. We further identified the N-terminal 24RRR26 of Cap to be crucial for binding to p32, and mutation of these three arginine residues significantly weakened the replication and pathogenesis of PCV2 in vivo In summary, our findings highlight a critical role of p32 in the activation and recruitment of PKC-? to phosphorylate lamin A/C and facilitate porcine circoviral nuclear egress, and they certainly help understanding of the mechanism of PCV2 replication.IMPORTANCE Circovirus infections are highly prevalent in mammalian and avian species. Circoviral capsid protein is the only structural protein of the virion that plays an essential role in viral assembly. However, the machinery of circovirus nuclear egress is currently unknown. In this work, we identified p32 as a key regulator of porcine circovirus type 2 (PCV2) nuclear egress that forms a complex with the viral capsid (Cap) protein to enhance protein kinase C isoform ? (PKC-?) activity; this resulted in a recruitment of phosphorylated PKC-? to the nuclear membrane, which further phosphorylates lamin A/C to promote the rearrangement of nuclear lamina and facilitate viral nuclear egress. Notably, we found that the N-terminal 24RRR26 of Cap, a highly conserved motif among circovirus species, was required for interacting with p32, and that mutation of this motif markedly impeded PCV2 nuclear egress. These data indicate that p32 is a critical regulator of PCV2 nuclear egress and reveal the importance of this finding in circovirus replication.

Dataset Information

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

Publications

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

Similar Datasets

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