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Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 71�77 |
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doi:10.1111/j.1745-7270.2008.00377.x |
Mapping of the nuclear
localization signals in open reading frame 2 protein from porcine circovirus
type 1
Jiangbing
Shuai, Wei Wei, Lingli Jiang, Xiaoliang Li, Ning Chen, and Weihuan Fang*
Institute of Preventive
Veterinary Medicine, Zhejiang Provincial Key Laboratory of Preventive
Veterinary Medicine, Zhejiang University, Hangzhou 310029, China
Received: July 25,
2007�������
Accepted: August
26, 2007
*Corresponding
author: Tel/Fax, 86-571-86971242; E-mail, [email protected]
Porcine
circovirus type 1 (PCV1) contains two major open reading frames encoding the
replication-associated proteins and the major structural capsid (Cap) protein. PCV1
Cap has an N-terminus carrying several potential monopartite or bipartite
nuclear localization signals (NLS). The contribution of these partially
overlapping motifs to nuclear importing was identified by expression of mutated
PCV1 Cap versions fused to enhanced green fluorescent protein (EGFP). The
C-terminus truncated PCV1 Cap-EGFP was localized in nuclei of PK-15 cells
similar to the wild-type PCV1 Cap-EGFP, whereas truncation of the N-terminus
rendered the fusion protein distributed into cytoplasm, indicating that the
nuclear import of PCV1 Cap was efficiently mediated by its N-terminal region.
Substitutions of basic residues in stretches 9RR�RR12 or the right part of 25RRPYLAHPAFRN�RYR�W�RRK43 resulted in a diffused distribution of the fusion protein
in both nuclei and cytoplasm, indicating that the two NLSs were responsible for
restricted nuclear targeting of PCV1 Cap.
Keywords������� porcine circovirus type 1; capsid protein; nuclear
localization signal
Porcine circoviruses (PCVs), as a member of the family Circoviridae, are the smallest viruses replicating autonomously to rely on host cell-encoded proteins due to limited coding capacity [1]. Both of the genotypes, PCV1 and PCV2, show a typical ambisense genomic structure with a non-enveloped, single-stranded (ss) circular DNA [2,3]. PCV1 is non-pathogenic to pigs [4]. PCV2 is believed to be the causative agent of the infectious disease called postweaning multisystemic wasting syndrome [5,6], first described in 1991 in Canada [7]. Two major open reading frames (ORF) are oriented in opposite directions in the genome of PCVs. ORF1 encodes the replicases essential for viral DNA replication through a rolling cycle replication (RCR) mechanism and is more conserved between the two genotypes [8], whereas ORF2 is highly variable between PCV1 and PCV2 (less than 60% homology) and encodes the only structural capsid protein� (Cap) [9] that contains the dominant immunological regions� [10].
As DNA synthesis of circoviruses occurs exclusively in the nucleus, the active nuclear import of DNA molecules might necessitate the involvement of karyophilic proteins [11]. In some species of geminivirus that share the same mode of replication as PCV, the capsid protein and movement protein are found to mediate viral DNA transport [12,13]. In the case of circoviruses, like PCVs and beak and feather disease virus (BFDV), the N-terminus of Cap is rich in basic amino acids and displays nuclear localization signals (NLSs) [11,14,15], which seems to be important for ssDNA accumulation [13,16]. Proteins to be targeted into the nucleus usually contain NLS which interact with importin a [17]. NLS sequences are often composed of basic amino acids and can be classified as either monopartite or bipartite motifs [18,19]. The nuclear targeting of PCV2 Cap and BFDV capsid protein has been identified to be directed by the bipartite motifs situated at the N-terminus of the proteins [11,14]. Moreover, Finsterbusch et al. have mapped the NLS of PCV1 replication-associated protein (Rep) and found that PCV1 Cap was localized in the nucleoli during the early stage of infection, but also in the nucleoplasm and the cytoplasm later in infection, whereas Cap was restricted to the nucleoli in plasmid-transfected cells [20]. However, the functional motifs in nuclear targeting of PCV1 Cap remained unknown.
In this report, a series of recombinant plasmids expressing� PCV1 ORF2 fused to green fluorescent protein� (GFP) were constructed and analyzed for their nuclear transporting activities to gain insight into the essential motifs of PCV1 Cap.
Materials and Methods
Cells culture and virus stock
PK-15 cells free of PCVs were cultured in minimum essential� medium (MEM) supplemented with 8% fetal bovine� serum (Gibco, New York, USA) at 37 �C with 5% CO2. PCV1 stock was isolated from the persistently contaminated� PK-15 cell line. The cultures were frozen and thawed three times and cell debris was removed from supernatant� by centrifugation at 12,000 g for 30 min. The supernatant was further concentrated by ultracentrifugation� for 2 h at 150,000 g. The virus pellet was resuspended in MEM and stored at -80 �C until titration.
Immunofluorescence assay
The infectious titer of PCV1 stock was determined by immunofluorescence assay (IFA) according to the Karber method [21]. Briefly, 80% confluent PCV1-free PK-15 cells were infected with serial 10-fold dilutions of PCV1 stock on a 96-well plate (Corning, New York, USA). After 3 d of inoculation, the cells were fixed with a pre-cooled solution containing 80% acetone at -20 �C for 15 min. After washing with phosphate-buffered saline (PBS) containing� 0.05% Tween-20 (PBS-T), the cells were incubated� at 37 �C for 1 h with the polyclonal antibodies against PCV1 Cap (1:500) prepared in our laboratory. The plates were washed three times with PBS-T and incubated� with the fluorescein-isothiocyanate-conjugated goat anti-rabbit IgG (KPL, Gaithersburg, USA) at 37 �C for another 45 min. Finally, the plates were examined under� a fluorescence microscope (Olympus IX71; Olympus, Tokyo, Japan).
Construction of recombinant
plasmids and in vitro transfection
The plasmid pcDNA-EGFP was constructed by cloning the enhanced green fluorescent protein (EGFP) gene under the control of cytomegalovirus (CMV) promoter of pcDNA3.1 using primers of GFP-f/r (Table 1). The full ORF2 sequence of PCV1, coding region of N-terminal 43 residues, and coding region of C-terminal 190 residues were amplified by primer pairs Pj9/Pj10, N43-f/r, and C190-f/r respectively, and linked to the 5-end of the EGFP gene in pcDNA-EGFP, resulting in plasmids pORF21-EGFP, p21-N43, and p21-C190, respectively [Fig. 1(A)]. To introduce amino acid substitutions in predicted NLS at the N-terminus of PCV1 ORF2 [Fig. 1(B)], the plasmid pORF21-EGFP was used as the template to construct mutants p21-NLS1-mut, p21-NLS2-mut, p21-NLS4-mut, p21-NLS3-mut, p21-NLS5-mut1, and p21-NLS5-mut2 by primer-directed site mutagenesis. Six primer sets were designed to generate plasmids that encode mutated ORF2-EGFP fusion protein containing different substitutions of basic residues at the N-terminus [Fig. 1(C)].
For transient expression, purified plasmids prepared using a Qiagen plasmid mini kit (Qiagen, D�sseldorf, Germany) were transfected into PCV-free PK-15 cells with Lipofectamine 2000 (Invitrogen, Carlsbad, USA). Briefly, 90% confluent cells in 24-well plates were incubated with the mixtures of 0.8 mg each plasmid DNA and 2.5 ml Lipofectamine 2000. Twenty-four hours after transfection, cells were examined for subcellular localization of the fusion proteins under the Olympus IX71 microscope and photographed.
Transcriptional analysis
Twenty-four hours after transfection, total RNA was isolated from the transfected cells using a UNIQ-10 column total RNA purification kit (Sangon, Shanghai, China), treated with RNase-free deoxyribonuclease I (DNase I; TaKaRa, Dalian, China) and reverse-transcribed into cDNA with M-MLV reverse transcriptase (Promega, Madison, USA) using oligonucleotides GFP-r (Table 1), followed by PCR amplification using primer pairs of GFP-r/f, Pj9/Pj10, N43-f/r, and C190-f/r for corresponding fragments. The amplicons were analyzed by gel electrophoresis and further confirmed by sequencing.
Results
Localization of Cap in PCV1
infected cells
The PK-15 cells were inoculated with PCV1 at a multiplicity of infection (MOI) of 1.0. The presence and subcellular localization of Cap in PCV1 infected cells were detected by IFA 48 h post-infection. The fluorescent signals of PCV1 Cap were observed either irregularly within the entire nucleus or strictly in the nucleoli [Fig. 2(A)]. However, no detectable signal was observed in PCV-free PK-15 cells incubated with the polyclonal antibody against PCV1 Cap [Fig. 2(B)].
Mapping of NLS of PCV1 Cap
As compared with the distribution of EGFP in the whole cells [Fig. 3(A)], the fusion protein containing the N-terminal fragment of Cap expressed from p21-N43 was localized in nucleoli with the same pattern as wild-type ORF2-EGFP protein [Fig. 3(B,C)], whereas the N-terminus truncated PCV1 Cap abolished nuclear targeting of the fusion protein coded by p21-C190 [Fig. 3(D)], indicating that the N-terminal region of PCV1 Cap is not only indispensable but also sufficient to direct the nuclear import of Cap.
�Prediction of the amino acid sequence of PCV1 ORF2 revealed two bipartite motifs and three four-residue pattern NLS at the N-terminal region, termed as NLS1-5, some of which were overlapped [Fig. 1(B)]. The role of these motifs in nuclear targeting was characterized by investigation of localization of fusion proteins containing basic amino acid substitutions in predicted NLS of PCV1 Cap. Observation of intracellular localization showed that the proteins expressed by plasmids p21-NLS1-mut, p21-NLS4-mut, and p21-NLS3-mut were exclusively distributed in nucleoli of PK-15 cells [Fig. 4(A,C,D)], similar to the wild-type ORF2-EGFP protein [Fig. 3(B)], indicating that the basic residues in these positions of wild-type ORF2 are not responsible for nuclear targeting. In contrast, substitutions in plasmids p21-NLS2-mut, p21-NLS5-mut1, and p21-NLS5-mut2 resulted in the distribution of mutated fusion proteins in both nuclei and cytoplasm [Fig. 4(B,E,F)], suggesting that the residues 9RRRR12 and the last six basic residues of the N-terminal region are necessary for restricted nuclear accumulation of PCV1 Cap.
Transcriptional analysis of
genes encoding mutants of PCV1 ORF2-EGFP fusion protein
To ensure that the PCV1 Cap or its mutants coding sequences were in frame with EGFP and each was capable of yielding a fusion protein in transfected PK-15 cells, transcription of the fragments containing mutated PCV1 ORF2 linked to EGFP were evaluated by RT-PCR and sequence analysis. Results showed that both of the N- or C-terminal truncated [Fig. 5(A)] and site-mutated PCV1 ORF2 [Fig. 5(B)] were transcribed together with the EGFP gene.
Discussion
Porcine circoviruses show a very compact genomic organization, containing only two major ORFs flanking the origin of replication [2,22]. ORF2 encodes the only structural protein that is localized in the nucleus [14,20]. The nuclear targeting of a protein in eukaryotes is frequently due to some motifs consisting of basic amino acids [23]. In this study, viral Cap was observed throughout the nucleus in PCV1-infected PK-15 cells as shown by IFA. Sequence analysis indicated that the N-terminus of PCV1 ORF2 protein contains abundant basic amino acid residues [Fig. 1(B)]. Some of these stretches show homology to other identified nuclear targeting motifs like "pat 4" motif consisting of four continuous basic residues or "bipartite" motif composed of two stretches of basic amino acids segregated by non-conserved residues [17]. Although PCV1 Cap was shuttled to the nucleoplasm or even cytoplasm at later stages of infection, the Cap was restricted to the nuclei in plasmid-transfected cells [20]. Investigation of subcellular localization of two truncated PCV1 ORF2 proteins fused with EGFP showed that the 43 amino acids at the N-terminus were indispensable and sufficient to direct the accumulation of protein in nuclei, similar to the full length PCV1 Cap, whereas the C-terminal region only contains some scattered basic residues without involvement in nuclear targeting (Fig. 3).
To further define the targeting activities of the predicted motifs at the N-terminal region [Fig. 1(B)], substitutions of basic amino acids were introduced into these stretches. Disruption of two four-residue stretches, NLS1 (4PRRR7) and NLS3 (24RRRP27), had no impact on PCV1 Cap localization. In contrast, the localization performance provided evidence that another "pat 4" motif (9RRRR12, as the predicted NLS2) was involved in complete nuclear accumulation of PCV1 Cap. Interestingly, this stretch of basic amino acids also contributes to the left part of NLS4. However, amino acid residues of R14, R16, and H18 that were located in the non-conserved part of this bipartite were found to be non-essential for nuclear import. Furthermore, nuclear translocation was also affected by substitutions of basic residues in the right part of NLS5.
In conclusion, the localization of PCV1 Cap
in nuclei or nucleoplasm is mediated by 43 residues of its N-terminus, in which
two stretches 9RRRR12 and 25RRPYLAHPA�FRN�RYRWRRK43 showing high homologies to
classical monopartite or bipartite NLS have been further identified to be
essential for complete nuclear import of PCV1 Cap. The nucleotide identity and
amino acid similarity between the N-terminus of PCV1 and PCV2 ORF2 are 70.7%
and 82.9%, respectively. They contain several conserved basic amino acid
stretches. As compared with functional stretches for nuclear translocation of
PCV2 Cap (12R-H-R-P-R-S-H18 and 34H-R-Y-R-W-R-R-K41) [14], the residues R14,
R16, and H18 of PCV1 Cap have no nuclear targeting activity, instead of which a
monopartite (9RRRR12) was found to be
functional in PCV1 Cap. However, in both serotypes, the last basic residue
stretches of the N-terminus of Cap that comprise the C-terminal portion of the
bipartite NLS were conserved and functional. Disruption or loss of nuclear
localization of Cap might result in a reduced level of ssDNA in geminiviruses
[13,16]. During PCV1 infection, localization of Cap in the nucleoli and Rep in
the nucleoplasm was followed by co-localization of the two proteins in the
nucleoplasm [20]. Therefore, it is generally believed that the role of PCV Cap
is beyond encapsidation and it might contribute to replication control by way
of interactions between Cap and Rep in the nucleoplasm [24]. Thus, further
study should focus on the different efficiency in control of viral replication
by Caps between PCV1 and PCV2.
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