Download Mutational analysis of the glycosaminoglycan

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A sequence of basic residues in the porcine circovirus type 2 capsid protein
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is crucial for its co-expression and co-localization with the replication
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protein
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Liping Huanga,b,+, Nicolaas Van Rennea,+, Changming Liub, Hans J. Nauwyncka,*
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Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan
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133, B-9820 Merelbeke, Belgiuma; Division of Swine Infectious Diseases, State Key
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Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese
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Academy of Agricultural Sciences, Maduan Street 427, Harbin 150001, Chinab
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Category: Animal: DNA viruses
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Running Title: Function analysis of 97RIRKVK102 in PCV2 Capsid Protein
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+
L.H. and N.V.R. contributed equally to this work
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*
Address correspondence to Hans J. Nauwynck, [email protected]
Tel.: +32 9 264 73 73
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Number of words in Abstract: 149
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Number of words in Text: 3,862
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Number of Figures: 6
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Number of Table: 0
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Abstract
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Porcine circovirus type 2 (PCV2) encodes two major proteins: the replication protein (Rep)
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and the capsid protein (Cap). The capsid protein displays a conserved stretch of basic
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residues situated on the inside of the capsid which role is hitherto unknown. We have used a
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reverse-genetics approach to investigate its function and found that mutations in these amino
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acids hindered Cap mRNA translation and hampered Cap-Rep co-localization, yielding unfit
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viruses. Intriguingly, co-transfection with a wild type PCV2 virus of a different genotype
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partially rescued mutant Cap expression, showing the importance of this basic pattern for
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efficient translation of Cap mRNA into protein. Our work shows that Cap and Rep are
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expressed independently of each other, and that this amino acid sequence of the Cap is vital
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for viral propagation. This study provides a method for studying unfit PCV2 virions and
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offers new insights into the intracellular modus vivendi of PCV2.
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Introduction
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To date, two types of porcine circovirus have been described, porcine circovirus type 1 and
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2 (PCV1 and PCV2) (Allan et al., 1998; Tischer et al., 1982). PCV1 was originally reported
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as a contaminant of the PK-15 cell line and is considered pathogenic only in young porcine
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fetuses (Saha et al., 2011). PCV2 is associated with several clinical manifestations
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collectively known as porcine circovirus diseases (PCVD) with postweaning multisystemic
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wasting syndrome (PMWS) as the most significant manifestation (Segalés, 2012).
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PCV2 belongs to the genus Circovirus of the family Circoviridae, of which four genotypes
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have been described so far: PCV2a, PCV2b, PCV2c and PCV2d (Xiao et al., 2015). PCV2
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is an extremely small non-enveloped virus with an icosahedral capsid measuring only 17 nm
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in diameter. Its genome comprises a circular single-stranded DNA of only 1766–1769
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nucleotides (Huang et al., 2011b) which encodes two major open reading frames (ORFs):
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ORF1 and ORF2. ORF1 encodes the non-structural replication protein Rep and its splice
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variant Rep', which are both necessary for viral replication (Mankertz and Hillenbrand, 2001;
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Mankertz et al., 1998a). ORF2 is translated into the capsid protein (Cap) (Nawagitgul et al.,
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2000), 60 copies of which make up the icosahedral proteinaceous shell protecting the viral
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genome (Crowther et al., 2003). Cap is the primary immunogenic protein, which has the
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ability to bind to the host cell receptor (Misinzo et al., 2006).
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As PCV2 exploits heparin sulfate or dermatan sulfate side chains of cell surface
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proteoglycans as attachment receptors, the putative heparin-binding motif 98IRKVKV103 was
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initially thought to be a potential binding site (Misinzo et al., 2006). Recently however, the
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atomic structure of PCV2 Cap revealed this motif was located on the inside of the capsid,
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excluding a role in cell-surface attachment (Khayat et al., 2011). Moreover, it appeared to be
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part of a larger constellation of positively charged amino acids on the inner surface of the
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capsid, including a notable all-conserved 97RIRKVK102 sequence strongly reminiscent of the
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putative heparin-binding motif. The manifest conservation of this pattern suggests a
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well-defined purpose for this specific stretch of basic residues. The present study employs a
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reverse-genetics approach to clarify its function by mutational analysis.
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Results
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Mutations in the basic residues of 97RIRKVK102 decrease Cap expression and interfere
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with subcellular Cap localization
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In order to determine the role of the basic amino acids in the conserved
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sequence,
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M3(R97T,R99T,K100T,K102T) were constructed. Sequencing confirmed that all mutations
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were introduced correctly. PCV-negative PK-15 cells were transfected with DNA of each
mutants
M1(R97T,R99T),
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97
RIRKVK102
M2(K100T,K102T)
and
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mutant and the parental clone, and subsequently immunostained at different time points
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after transfection. The numbers of PCV2-positive cells are shown in Fig. 1a. For
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M1(R97T,R99T), M2(K100T,K102T) and the parental clone, Caps were detected starting
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from 12 hours post-transfection (hpt), and the numbers of positive cells rapidly increased to
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a maximum at 36 hpt. For M3(R97T,R99T,K100T,K102T) however, not a single
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Cap-positive cell was ever detected. In the first 36 hours post-transfection, the number of
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PCV2-positive cells was comparable between M1(R97T,R99T), M2(K100T,K102T) and
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the parental clone. However at 72 hpt, the number of positive cells markedly decreased for
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these mutant viruses, suggesting a drop in viral fitness in secondary rounds of replication.
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Despite similar numbers of positive cells at 36 hpt, immunostaining showed a strikingly
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different Cap expression-pattern (Fig. 1b). Cap staining intensity of M1(R97T;R99T) was
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weaker than the parental clone, and signals for M2(K100T,K102T) were even less than
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those of M1(R97T,R99T). M3(R97T,R99T,K100T,K102T) signal was completely absent.
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This prompted the question if the introduced mutations caused conformational changes
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hindering antibody binding, or whether the protein itself was expressed at a lower level.
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Western blotting of transfected PK-15 cell lysates confirmed there was a reduction in the
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amount of Cap for M1(R97T,R99T) and M2(K100T,K102T), and a complete absence of
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Cap for M3(R97T,R99T,K100T,K102T) (Fig. 2a). On the contrary, Rep immunostaining
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never displayed any difference between wild type and mutants, not even for
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M3(R97T,R99T,K100T,K102T) whose Cap expression was completely abolished
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3a). Taken together, these results demonstrate that the mutants were only hampered in Cap
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but not Rep expression, and that Cap expression occurs independently of Rep.
(Fig.
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In addition to expression alterations, a difference in subcellular distribution was observed
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for Cap among the different mutants and the parental clone at 36 hpt (Fig. 1b). Of the
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parental clone-transfected cells, 3.2% showed Cap presence in both nucleus and cytoplasm,
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and another 5.1% had a positive Cap signal in the nucleus only. For M1(R97T,R99T) and
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M2(K100T,K102T), the number of cells that showed Cap in both nucleus and cytoplasm
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dropped dramatically (1.2 and 0.8% respectively), but this decrease was completely
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compensated by an increase in Cap presence in the nucleus only (8.2% and 8.8%). Cap was
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never observed in the cytoplasm only. This finding shows that mutations in the
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rather than maintaining a cytoplasmic stage in its lifecycle.
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To determine whether the decrease in Cap expression in the cells coincided with a decrease
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of viral capsids released from the transfected PK-15 cells, capture-ELISA kinetics of
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transfected cell supernatants were studied (Fig. 2b). Throughout a time course of 72h,
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OD450nm values of M1(R97T,R99T) and M2(K100T,K102T) remained lower than the
RIRKVK102 pattern result in Cap being shunted towards the nucleus of transfected cells,
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parental clone. As expected, OD450nm values for M3(R97T,R99T;K100T,K102T) were
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negative. Moreover, the data are perfectly in line with the intracellular capsid production,
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and indicate that the conserved stretch of basic residues has no role in viral egress once the
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virions are assembled.
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Mutations in the 97RIRKVK102 hamper Cap-Rep co-localization
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To determine the potential co-localization between Cap and Rep, PK-15 cells were
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transfected at 24 hpt and stained with Cap-specific mAb 12E12 (Red) and Rep-specific
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mAb F210 D6 (Green). M1(R97T,R99T) and especially M2(K100T,K102T) displayed a
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striking lack of Cap-Rep co-localization (Fig. 3a). The co-localization events were
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quantified by CoLocolizer Pro 2.7.1 using Manders’ coefficient correlation. This analysis
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revealed a Cap-Rep co-localization rate of 81%, 56% and 24% for the parental clone,
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M1(R97T,R99T) and M2(K100T,K102T) respectively. For M3(R97T,R99T,K100T,K102T)
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a co-localization coefficient could not be calculated since Cap was not expressed (Fig. 3b).
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Mutations in the 97RIRKVK102 drastically impact viral fitness
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To investigate whether the mutant capsids released in the supernatant were still infectious,
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we determined the viral titers of the supernatants collected at different time points after
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transfection (Fig. 4a). A time-dependent rise of viral titers was observed starting from 24
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hpt for M1(R97T,R99T), M2(K100T,K102T) and the parental clone. The highest titers were
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found at 60 hpt: 3.9 log10TCID50/ml for M1(R97T,R99T), 3.3 log10TCID50/ml for
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M2(K100T,K102T) and 5.3 log10TCID50/ml for the parental clone. The titers of the mutants
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were lower than that of parental clone, reflecting the dramatic decrease in Cap expression
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observed by immunostaining, western blot and ELISA. M3(R97T,R99T,K100T,K102T)
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supernatants were always negative.
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In order to test the fitness of the progeny virus, PCV2-transfected cell supernatants were
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passaged on PCV-negative PK-15 cells. A gradual decrease in viral titers reaching a level of
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non-detection was observed from passages two to seven in case of the mutants, while a
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gradual increase in titers was observed for the parental clone (Fig. 4b). PCV2 genomic
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sequences obtained from passage two, five and seven for parental virus, and passage two
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and four for M1(R97T,R99T) and passage two for M2(K100T,K102T) were all 100%
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identical to the genome in the pCR-BluntII-TOPO vector.
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Taken together, this shows that Caps with mutations in the conserved basic sequence can
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reach the assembly stage and properly exit the cell for secondary rounds of infection.
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However, the impact on protein expression is so enormous that overall viral fitness is
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seriously compromised. Without this pattern intact, the virus is doomed to disappear from the
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gene pool, recapitulating the selection pressure observed in vivo.
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The decrease in mutant Cap expression is exclusively due to an altered amino acid
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sequence which results in a decrease in Cap mRNA translation
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Next, we quantified Cap mRNA using RT-qPCR to see if the decrease in Cap expression of
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the mutant viruses was caused by a decrease in Cap mRNA transcription. All samples
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derived from the transfected PK-15 cells were positive at 12 hpt (Fig. 5a). Notwithstanding
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the differences in Cap mRNA at 36 hpt of the mutants compared to the parental clone, the
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decrease was not strong enough to fully explain the drastic decrease or even absence of
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protein expression. At 72 hpt, which corresponds with the next replication cycle, Cap
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mRNA copy number was approximately 80% lower than the parental clone, which can be
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explained by the reduced viral fitness of the mutants in subsequent replication cycles. This
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clearly shows that the dramatic reduction in Cap protein expression at 36 hpt is not solely
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due to a decrease in Cap mRNA.
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In order to completely exclude that nucleotide changes in the viral DNA or the Cap mRNA
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would interfere with translation processes inside the cell, a new synonymous mutant, M4,
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was constructed. M4 has four silent nucleotide changes, which do not alter the amino acid
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sequence of Cap. We hypothesized that, if changes on the nucleotide level would influence
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transcription or translation, M4 and M3(R97T,R99T,K100T,K102T) fitness would be evenly
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affected, since both have four nucleotide substitutions in exactly the same region.
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Interestingly, M4 displayed an exactly similar phenotype as the parental clone, yielding
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infectious, fit viruses (Fig. 5b and 5c). This excludes an effect of the nucleotide sequence in
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itself, and confirms that the dramatic effects on viral viability are entirely due to the altered
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amino acids in the mutants.
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The inability of mutant Caps to be properly expressed in spite of ample mRNA presence
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could be explained by proteasomal degradation of newly expressed Cap proteins. However,
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PK-15 cells treated with proteasome inhibitor MG132 for 2h at 24hpt did not rescue proper
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Cap expression (data not shown). The picture that emerges is that mutant Cap mRNA
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transcripts are hampered in translation, rather than Cap proteins being degraded once
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expressed.
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Co-transfection with a fit genotype PCV2a virus partially rescues mutant Cap
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translation through an unknown mechanism
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Our findings led us to believe that the conserved basic sequence was critical in getting Cap
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mRNA efficiently translated into Cap. If so, co-transfection of the 48285-based mutants
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(genotype PCV2b) with the PCV2a-LG strain should rescue mutant Cap expression as a
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RIRKVK102 pattern. First, we verified that mouse mAb 8G12 only
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result of its intact
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reacted to PCV2a-LG Cap and did not cross-react with PCV2b mutants. Similarly, mouse
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mAb 6E9 only detected 48285-based mutant capsids, and did not cross react with
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PCV2a-LG Cap (data not shown). By staining mutant-transfected cells with mAb 6E9 we
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obtained identical results as with mAb 38C1 (Fig. 6 left column). As expected,
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co-transfection with PCV2a-LG did not have any effect on LG expression (Fig. 6 right
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column). Intriguingly, this procedure partially rescued mutant Cap translation, which was
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particularly visible in M3(R97T,R99T,K100T,K102T)-LG co-transfection (Fig. 6 middle
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column). This highlights once more the importance of an intact 97RIRKVK102 alignment for
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efficient Cap translation.
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Discussion
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Being the smallest of all known mammal viruses with limited genomic coding capacity,
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porcine circoviruses are truly fascinating biological systems. Despite their reliance on
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proofreading-competent host polymerases for replication, their genomes mutate
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exceptionally rapidly while keeping an extremely low genetic diversity within the different
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PCV2 genotypes (Firth et al., 2009). In other words, most nucleotide substitutions
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disappear immediately under intense purifying selection, and as a consequence, PCV2
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genomes are allowed only marginal leeway to evolve. This should not come as a surprise,
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since every part of its genome is exploited on multiple levels: the stem-loop and adjacent
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motifs need intense conservation for (i) binding and cleavage by the replication complex
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(Steinfeldt et al., 2001), (ii) the Cap mRNA splicing acceptor site (Mankertz et al., 1998b)
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and (iii) the larger part of the Rep promoter sequence (Mankertz and Hillenbrand, 2002).
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The region clockwise of the stem-loop cannot be tampered with for its DNA encodes (i) the
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replication protein Rep and its frame-shifted splice variant Rep’ (Mankertz and Hillenbrand,
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2001) as well as (ii) ORF3 (Liu et al., 2005) and (iii) ORF4 (He et al., 2013) on the amino
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acid level. On a nucleotide level, this stretch also contains sequences for (iv) the Rep’
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mRNA splicing donor and acceptor sites (Mankertz and Hillenbrand, 2001), and (v and vi)
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the Cap mRNA splicing donor site and the Cap promoter (Mankertz and Hillenbrand, 2002;
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Mankertz et al., 1998b). The genomic part counterclockwise to the stem loop is supposed to
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have more freedom for variation because it only encodes the Cap on amino acid level
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(Nawagitgul et al., 2000), and the smaller part of the Rep promoter (Mankertz and
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Hillenbrand, 2002) on the nucleotide level. This understanding makes any conserved
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sequence of ORF2 an interesting target for investigation.
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Since cryo-EM reconstruction of PCV2 VLPs demonstrated that the all-conserved
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IRKVKV103 motif originally identified by Misinzo (2006) was situated on the inside of the
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capsid, and took part in a larger constellation of positively charged amino acids comprising
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the basic sequence
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negatively charged viral ssDNA strand upon encapsidation. That this stretch of basic residues
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was also implicated in Cap mRNA translation (Fig.2 and Fig.5) came as a complete surprise
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and the reason for it still mystifies us. It is hard to craft an explanation without falling into a
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chicken or egg causality dilemma. One might think that Cap mRNA needs the Cap to be
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efficiently exported from the nucleus, but for Cap to come into existence an efficient viral
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mRNA export would be required first. Likewise, the Cap might act as a molecular switch
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turning on Cap translation machinery, but this would require prior translation of Cap! We
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tried to solve this catch-22 situation by co-transfecting with a wild typePCV2 strain, but even
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then we see a preferential translation of wild-type PCV2 (Fig. 6). This argues in favor of a
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vision where the basic amino acid sequence is not so much a biological switch enhancing
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translation, but rather takes part in a molecular interface interacting with another factor. One
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can imagine that the basic amino acids of the capsid protein have to be shielded from
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aspecific interactions with other proteins and nucleic acids, before being imported to the
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nucleus where they are deposited on viral DNA with Swiss-watch precision. In many ways,
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they resemble histones safeguarded and escorted by histone chaperones. Interestingly, an
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interaction between Cap and histone chaperones NAP1 and NPM has been described
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RIRKVK102, we understood these basic amino acids stabilize the
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(Finsterbusch et al., 2009). It is tempting to think that the removal of the positively charged
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amino
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M3(R97T,R99T,K100T,K102T) Caps abolishes their interaction with co-translational
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chaperones, causing any further Cap mRNA translation to grind to a halt and leaving nascent
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Cap polypeptides stuck on the negatively charged ribosome. In this respect, the partial
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rescue of mutant Cap translation we see by co-transfecting with a wild type PCV2a virus (Fig.
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6) may be caused by a mutant Cap-wild type Cap interaction which releases the mutant Cap
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from the ribosome, and allows translation to continue, albeit still inefficiently. PCV1 capsid
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proteins display a virtually identical 98RIRKAK103 sequence, and it would be interesting to
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see if M3(R97T,R99T,K100T,K102T) would still be rescued after co-transfection with a
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PCV1 Cap expression construct. PCV1 Caps are expected to have inferior binding
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capabilities with PCV2 Caps because of their antigenic differences in the capsid protein.
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PCV1 should therefore not be able to restore M3(R97T,R99T,K100T,K102T) expression if
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rescue is indeed dependent on an interaction with wild type Cap. In addition, an unbiased
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siRNA screen targeting histone chaperones could identify which chaperones, if any, are
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involved in PCV2 Cap translation.
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As a final remark, it is conceivable that the replication protein itself exerts some additional
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chaperone activity. Rep may just guide Cap subunits to the right sub-nuclear compartment
acid
pattern
in
M1(R97T,R99T),
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M2(K100T,K102T)
and
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for further assembly, or even more speculative, Rep might simply drive genome
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encapsidation. As a matter of fact, all circovirus replication proteins share a common
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superfamily 3 helicase domain (Roux et al., 2013) which assemble into ring-shaped
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multimeres responsible for unwinding dsDNA by means of an ATPase-powered molecular
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motor (Hickman and Dyda, 2005). Should this activity be performed in close proximity to
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capsid proteins, it is not improbable that Rep subunits form replicative complexes displacing
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the viral strand towards adjacent capsid subunits, thus facilitating the process of genome
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packaging in complete harmony with replication. Hypothetical as this may be, also in the
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closely related beak and feather disease virus (BFDV), an elegant relationship exists between
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the two viral proteins, for Rep nuclear localization depends entirely on a cytoplasmic
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interaction with Cap, after which both shuttle to the nucleus (Heath et al., 2006). These
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findings all support a vision for circoviruses in which an intimate relationship exists between
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Cap and Rep which has been underestimated until now, and can serve as a working model for
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future experimental studies.
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In conclusion, this study shows that the conserved stretch of basic residues
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comprising 97RIRKVK102 of the PCV2 Cap is crucial for its expression. Mutations in this
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pattern resulted in (i) a reduced translation of Cap, (ii) hindered Cap-Rep
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co-localization and (iii) finally unfit PCV2 virions. Additionally, co-transfection with a
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wild type PCV2 virus partially rescues mutant Cap expression, highlighting the importance
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of these amino acids for an efficient translation of Cap mRNA into protein. We speculate
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that 97RIRKVK102 is part of a molecular interface interacting with co-translational
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chaperones, however, future experiments need to be carried out to confirm this hypothesis.
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Material and methods
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Virus, cells and virus titration
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PCV2 strain 48285 (Accession number: AF055394) clone 48285-24, PK-15 cells and
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culture conditions were described earlier (Saha et al., 2012b). PCV2 strain LG (Accession
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number: HM038034) clone pMD18/PCV2a-LG was described earlier (Huang et al., 2011b).
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Virus titration was performed as previously described (Meerts et al., 2005).
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Monoclonal antibodies against PCV2
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Cap-binding mouse monoclonal antibodies (mAb) 38C1, 12E12 and 6E9 were generated in
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our laboratory and described earlier (Saha et al., 2012a). MAb 8G12 was generated against
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PCV2a-LG with a procedure described by Huang et al. (2011a). Rep-binding mAb F210
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D6 was described previously (McNeilly et al., 2001). For western blotting, mAb 2E8 was
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generated against PCV2-Cap derived from PCV2a strain LG (Huang et al., 2011a) and is
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directed against the linear epitope 26–36aa of PCV2-Cap (Guo et al., 2011).
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Construction of mutants
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A site-directed mutagenesis was performed by targeting the basic amino acids of
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by an uncharged hydrophilic threonine (T) using a Quickchange® site-directed mutagenesis
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kit (Stratagene, La Jolla, USA) based on clone 48285-24. R at positions 97 and 99 were
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substituted by T using primers M1-FW (5’-atactacacaataacaaaggttaaggttgaat-3’) and
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M1-REV (5’-attcaaccttaacctttgttattgtgtagtat-3’) resulting in the mutant M1(R97T,R99T).
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Mutant M2(K100T,K102T) was constructed by mutating K into T at positions 100 and 102
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using
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(5’-attcaaccgtaaccgttcttattctgtagtat-3’).
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constructed by replacing R or K with T at all four positions 97, 99, 100 and 102 using
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primers
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(5’-attcaaccgtaaccgttgttattgtgtagtat-3’). Mutant M4 was constructed by changing the codons
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(aga ata aga aag gtt aag) of
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M4-FW
RIRKVK102 in the Cap of PCV2 strain 48285. Arginine (R) or lysine (K) was substituted
primers
M2-FW
M3-FW
(5’-atactacagaataagaacggttacggttgaat-3’)
Mutant
M2-REV
M3(R97T,R99T,K100T,K102T)
(5’-atactacacaataacaacggttacggttgaat-3’)
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and
and
was
M3-REV
RIRKVK102 into (agg ata agg aaa gtt aaa) using primers
(5’-atactacaggataaggaaagttaaagttgaat-3’)
17
and
M4-REV
(5’-
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attcaactttaactttccttatcctgtagtat-3’), resulting in a synonymous mutant with unaltered amino
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acid sequence.
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Transfections
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Parental and mutated plasmids were digested with Hind Ⅲ and subsequently purified
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genomic DNA fragments were self-ligated for 20 mins at room temperature using T4 DNA
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ligase (Invitrogen, Merelbeke, Belgium). 800 ng of DNA was then transfected onto
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PCV-negative PK-15 cells with lipofectamine 2000 (Invitrogen, Merelbeke, Belgium)
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according to the manufacturer's instructions. Empty-plasmid transfected PK-15 cells were
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included as a mock.
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Immunoflurescence assays and co-localization quantification
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Methanol-fixed transfected cells were stained with a previously described indirect
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immunofluorescence assay (Saha et al., 2010). MAb 38C1 and fluorescein isothiocyanate
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(FITC) labeled goat anti-mouse IgG (Molecular Probes, Eugene, USA) were used as the
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primary and secondary antibodies, respectively. Images were made with Leica TCS SP2
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confocal microscope (Leica Microsystems GmbH, Mannheim, Germany). For double
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immunofluorescence staining for Cap and Rep, transfected PK-15 cells were first incubated
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with anti-Cap mAb 12E12 (1:2) and then with Alexa Fluor 594 goat-anti mouse IgG2b
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(Molecular Probes; 1:200). Afterwards, the cells were stained for PCV2 Rep by incubation
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with anti-Rep mAb F210 D6 (1:500). Subsequently, a 1:200 dilution of FITC-labeled
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goat-anti mouse IgG1 (Molecular Probes) was added. All antibodies were incubated for 1 h
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at 37°C. The cells were washed three times with PBS after incubation with primary and
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secondary antibodies. Specificity of the staining for different protein was demonstrated
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using an irrelevant, isotype-matched mAb II1H/4-5G (Costers et al., 2010) and 13D12
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(Nauwynck and Pensaert, 1995) and by the complete absence of Cap- and Rep-specific
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fluorescence in mock-transfected PK-15 cells. The co-localization of Cap and Rep was
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analyzed using the software CoLocalizer Pro 2.7.1 as described previously (Zinchuk and
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Zinchuk, 2008). Background correction of the images was performed prior to coefficient
341
calculation. The co-localization rates were measured based on Manders’ coefficient, which
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varies from 0 to 1. A coefficient value of 0 to 0.6 indicates absence of co-localization while
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a value of 0.6 to 1.0 indicates co-localization.
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Western blot analysis
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Western-blot analysis was used to quantify the amount of Cap produced by the different
347
mutants
and
the
parental
clone.
Briefly,
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M1(R97T,R99T),
M2(K100T,K102T),
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M3(R97T,R99T,K100T,K102T), parental clone and mock-transfected PK-15 cells were
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harvested at 36 hpt. Sodium dodecyl sulfate polyacrylamide gel electrophoresis
350
(SDS-PAGE) and Western-blot were performed as described previously (Lefebvre et al.,
351
2008). Anti-PCV2-Cap mAb 2E8 (Guo et al., 2011; Huang et al., 2011a), a
352
PCV2-irrelevant mAb 1C11 (Nauwynck and Pensaert, 1995), HRP-conjugated α-tubulin
353
(Cell signaling technology, Danvers, MA, USA) and HRP-conjugated sheep anti-mouse
354
IgG (H+L) (Invitrogen) were used as the primary antibody, negative control, loading
355
control and secondary antibody, respectively. Antigen-antibody complexes were visualized
356
using an enhanced chemiluminescence assay (Amersham Biosciences, NJ, USA).
357
358
Capture-ELISA
359
To quantify PCV2-Cap in the supernatants collected at different time points after
360
transfection, a capture-ELISA was performed as previously described (Huang et al., 2011a)
361
with minor modifications. Purified mAb 38C1 (5 μg/ml), biotin-labeled 38C1 (1:1000) and
362
Streptavidin-biotinylated horse-radish peroxidase (HRP) complex (1:1000; GE Healthcare,
363
United Kingdom) were used as coating, detecting and secondary antibody, respectively. The
364
color was developed and the reaction was stopped as described (Saha et al., 2012a). The
365
optical density (OD) at 450 nm was measured by a microplate reader (Bio-Rad, Hercules,
20
366
CA, USA). The capture-ELISA was performed in triplicate. The mean OD450 nm value of
367
the mock plus three standard deviations were calculated to use as a cut-off value of the
368
capture-ELISA.
369
370
RT-qPCR
371
Total RNA was extracted from transfected cells using the RNeasy Mini Kit (Qiagen, Hilden,
372
Germany). For each sample, 500 ng of RNA was reverse transcribed into cDNA using
373
SuperScriptTM First-Strand Synthesis System (Invitrogen, Merelbeke, Belgium).
374
The
375
(5’-gctccacactccatcagtatgac-3’) used for quantifying Cap mRNA transcript were adapted
376
from (Yu et al., 2007) with modifications based on sequence data of PCV2 strain 48285.
377
The reverse primer was designed to span the splice junction to ensure that PCV2 DNA was
378
not amplified. qPCR was performed with Applied Biosystems StepOnePlusTM Real-Time
379
PCR system (Genetic Systems Company, Foster City, California) using SensiMixTM SYBR
380
mastermix (Bioline, London, UK).
forward
primer
(5’-agatgccatttttccttctc-3’)
and
reverse
primer
381
382
Acknowledgements
383
We would like to thank Carine Boone and Lieve Sys for their excellent technical assistance.
21
384
This study was supported by the National Natural Science Foundation of China (Grant No.
385
31302110).
22
386
References
387
Allan, G. M., McNeilly, F., Kennedy, S., Daft, B., Clarke, E. G., Ellis, J. A., Haines, D. M., Meehan, B. M.
388
& Adair, B. M. (1998). Isolation of porcine circovirus-like viruses from pigs with a wasting disease
389
in the USA and Europe. Journal of veterinary diagnostic investigation 10, 3-10.
390
Costers, S., Lefebvre, D. J., Van Doorsselaere, J., Vanhee, M., Delputte, P. L. & Nauwynck, H.J. (2010).
391
GP4 of porcine reproductive and respiratory syndrome virus contains a neutralizing epitope that is
392
susceptible to immunoselection in vitro. Archive of virology 155, 371-378.
393
Crowther, R. A., Berriman, J. A., Curran, W. L., Allan, G. M. & Todd, D. (2003). Comparison of the
394
structures of three circoviruses: chicken anemia virus, porcine circovirus type 2, and beak and
395
feather disease virus. Journal of virology 77, 13036-13041.
396
Finsterbusch, T., Steinfeldt, T., Doberstein, K., Rodner, C. & Mankertz, A. (2009). Interaction of the
397
replication proteins and the capsid protein of porcine circovirus type 1 and 2 with host proteins.
398
Virology 386, 122-131.
399
Firth, C., Charleston, M. A., Duffy, S., Shapiro, B. & Holmes, E. C. (2009). Insights into the evolutionary
400
history of an emerging livestock pathogen: porcine circovirus 2. Journal of virology 83,
401
12813-12821.
402
403
404
405
Guo, L., Lu, Y., Huang, L., Wei, Y. & Liu, C. (2011). Identification of a new antigen epitope in the nuclear
localization signal region of porcine circovirus type 2 capsid protein. Intervirology 54, 156-163.
He, J., Cao, J., Zhou, N., Jin, Y., Wu, J. & Zhou, J. (2013). Identification and functional analysis of the
novel ORF4 protein encoded by porcine circovirus type 2. Journal of virology 87, 1420-1429.
406
Heath, L., Williamson, A. L. & Rybicki, E. P. (2006). The capsid protein of beak and feather disease virus
407
binds to the viral DNA and is responsible for transporting the replication-associated protein into the
408
nucleus. Journal of virology 80, 7219-7225.
409
410
Hickman, A.B. & Dyda, F. (2005). Binding and unwinding: SF3 viral helicases. Current opinion in
structural biology 15, 77-85.
411
Huang, L., Lu, Y., Wei, Y., Guo, L. & Liu, C. (2011a). Development of a blocking ELISA for detection of
412
serum neutralizing antibodies against porcine circovirus type 2. Journal of virological methods 171,
413
26-33.
414
Huang, L. P., Lu, Y. H., Wei, Y. W., Guo, L. J. & Liu, C. M. (2011b). Identification of one critical amino
415
acid that determines a conformational neutralizing epitope in the capsid protein of porcine circovirus
416
type 2. BMC Microbiology 11, 188.
417
418
Khayat, R., Brunn, N., Speir, J. A., Hardham, J. M., Ankenbauer, R. G., Schneemann, A. & Johnson, J.
E. (2011) The 2.3-angstrom structure of porcine circovirus 2. Journal of virology 85, 7856-7862.
419
Lefebvre, D. J., Costers, S., Van Doorsselaere, J., Misinzo, G., Delputte, P. L. & Nauwynck, H. J. (2008).
420
Antigenic differences among porcine circovirus type 2 strains, as demonstrated by the use of
23
421
monoclonal antibodies. Journal of general virology 89, 177-187.
422
Liu, J., Chen, I. & Kwang, J. (2005). Characterization of a previously unidentified viral protein in porcine
423
circovirus type 2-infected cells and its role in virus-induced apoptosis. Journal of virology 79,
424
8262-8274.
425
426
427
428
429
430
431
432
433
434
Liu, Q., Tikoo, S. K., Babiuk, L. A. (2001). Nuclear localization of the ORF2 protein encoded by porcine
circovirus type 2. Virology 285, 91-99.
Mankertz, A. & Hillenbrand, B. (2001). Replication of porcine circovirus type 1 requires two proteins
encoded by the viral rep gene. Virology 279, 429-438.
Mankertz, A. & Hillenbrand, B. (2002). Analysis of transcription of Porcine circovirus type 1. Journal of
general virology 83, 2743-2751.
Mankertz, A., Mankertz, J., Wolf, K. & Buhk, H. J. (1998a). Identification of a protein essential for
replication of porcine circovirus. Journal of general virology 79, 381-384.
Mankertz, J., Buhk, H. J., Blaess, G. & Mankertz, A. (1998b). Transcription analysis of porcine circovirus
(PCV). Virus Genes 16, 267-276.
435
McNeilly, F., McNair, I., Mackie, D. P., Meehan, B. M., Kennedy, S., Moffett, D., Ellis, J., Krakowka, S.
436
& Allan, G. M. (2001). Production, characterisation and applications of monoclonal antibodies to
437
porcine circovirus 2. Archive of virology 146, 909-922.
438
Misinzo, G., Delputte, P. L., Meerts, P., Lefebvre, D. J. & Nauwynck, H. J. (2006). Porcine circovirus 2
439
uses heparan sulfate and chondroitin sulfate B glycosaminoglycans as receptors for its attachment to
440
host cells. Journal of virology 80, 3487-3494.
441
Nauwynck, H. J. & Pensaert, M. B. (1995). Effect of specific antibodies on the cell-associated spread of
442
pseudorabies virus in monolayers of different cell types. Archive of virology 140, 1137-1146.
443
Nawagitgul, P., Morozov, I., Bolin, S. R., Harms, P. A., Sorden, S. D. & Paul, P. S. (2000). Open reading
444
frame 2 of porcine circovirus type 2 encodes a major capsid protein. Journal of general virology 81,
445
2281-2287.
446
Roux, S., Enault, F., Bronner, G., Vaulot, D., Forterre, P. & Krupovic, M. (2013). Chimeric viruses blur
447
the borders between the major groups of eukaryotic single-stranded DNA viruses. Nature
448
Communications 4, 2700.
449
Saha, D., Huang, L., Bussalleu, E., Lefebvre, D. J., Fort, M., Van Doorsselaere, J. & Nauwynck, H. J.
450
(2012a). Antigenic subtyping and epitopes' competition analysis of porcine circovirus type 2 using
451
monoclonal antibodies. Veterinary microbiology 157, 13-22.
452
Saha, D., Lefebvre, D. J., Ducatelle, R., Doorsselaere, J. V. & Nauwynck, H. J., (2011). Outcome of
453
experimental porcine circovirus type 1 infections in mid-gestational porcine foetuses. BMC
454
Veterinary research 7, 64.
455
Saha, D., Lefebvre, D. J., Ooms, K., Huang, L., Delputte, P. L., Van Doorsselaere, J. & Nauwynck, H. J.
456
(2012b). Single amino acid mutations in the capsid switch the neutralization phenotype of porcine
24
457
circovirus 2. Journal of general virology 93, 1548-1555.
458
Saha, D., Lefebvre, D. J., Van Doorsselaere, J., Atanasova, K., Barbe, F., Geldhof, M., Karniychuk, U. U.
459
& Nauwynck, H. J. (2010). Pathologic and virologic findings in mid-gestational porcine foetuses
460
after experimental inoculation with PCV2a or PCV2b. Veterinary microbiology 145, 62-68.
461
Segalés, J. (2012). Porcine circovirus type 2 (PCV2) infections: clinical signs, pathology and laboratory
462
463
464
465
466
diagnosis. Virus Res 164, 10-19.
Steinfeldt, T., Finsterbusch, T. & Mankertz, A. (2001). Rep and Rep' protein of porcine circovirus type 1
bind to the origin of replication in vitro. Virology 291, 152-160.
Tischer, I., Gelderblom, H., Vettermann, W. & Koch, M. A. (1982). A very small porcine virus with
circular single-stranded DNA. Nature 295, 64-66.
467
Xiao, C. T., Halbur, P. G. & Opriessnig, T. (2015). Global molecular genetic analysis of porcine circovirus
468
type 2 (PCV2) sequences confirms the presence of four main PCV2 genotypes and reveals a rapid
469
increase of PCV2d. Journal of general virology 96, 1830-1841.
470
Yu, S., Vincent, A., Opriessnig, T., Carpenter, S., Kitikoon, P., Halbur, P. G. & Thacker, E. (2007).
471
Quantification of PCV2 capsid transcript in peripheral blood mononuclear cells (PBMCs) in vitro.
472
Veterinary microbiology 123, 34-42.
473
474
Zinchuk, V. & Zinchuk, O. (2008). Quantitative colocalization analysis of confocal fluorescence microscopy
images. Curr Protoc Cell Biol Chapter 4, Unit 4 19.
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Figures
490
Fig. 1 (a) Total number of PCV2-positive cells at different time points after transfection.
491
Positive cells were counted in 25 randomly selected fields at 40X. (b) Immunofluorescence
492
stainings of mutants and parental clone by anti-Cap mAb 38C1 at 36 hpt. The data
493
represents three separate experiments performed in duplicate.
494
Fig. 2 (a) Western blot of PCV2 Cap in transfected PK-15 cell lysates at 36 hpt. (b)
495
Kinetics of viral egress as detected by Capture-ELISA of PCV2 capsids in the supernatant
496
of transfected PK-15 cells.
497
Fig. 3 (a) PK-15 cells transfected with mutants or the parental clone, immunostained with
498
anti-Cap mAb 12E12 (red) and anti-Rep mAb F210 D6 (green) at 24 hpt. The images are
499
representative of three separate experiments performed in duplicate. (b) Colocalization
500
quantification of an average of 60 cells per sample studied from three separate experiments
501
performed in duplicate.
502
Fig. 4 (a) Viral titers of the supernatants collected at different time points after transfection.
503
(b) Viral titers of the supernatants collected at different passages.
504
Fig. 5 (a) Cap mRNA copy number kinetics. Figure represents three independent single
505
experiments. (b) Immunostaining of Cap-positive parental clone and M4-transfected
506
PK15-cells. (c) Viral titers of the synonymous M4 mutant and the parental virus in the
26
507
supernatants collected at different time points after transfection. (d) Viral titers of the
508
synonymous M4 mutant and parental viruses at different passages.
509
Fig. 6 (left column) Single transfection with PCV2b-based mutants showing the typical
510
mutant Cap expression pattern. (middle column) Cotransfection of the PCV2b-based
511
mutants with a PCV2a virus strain rescues mutant Cap expression. (right column)
512
Co-transfection does not affect PCV2a-Cap expression.
513
514
27