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1 A sequence of basic residues in the porcine circovirus type 2 capsid protein 2 is crucial for its co-expression and co-localization with the replication 3 protein 4 Liping Huanga,b,+, Nicolaas Van Rennea,+, Changming Liub, Hans J. Nauwyncka,* 5 6 Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 7 133, B-9820 Merelbeke, Belgiuma; Division of Swine Infectious Diseases, State Key 8 Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese 9 Academy of Agricultural Sciences, Maduan Street 427, Harbin 150001, Chinab 10 11 Category: Animal: DNA viruses 12 13 Running Title: Function analysis of 97RIRKVK102 in PCV2 Capsid Protein 14 15 + L.H. and N.V.R. contributed equally to this work 16 17 18 * Address correspondence to Hans J. Nauwynck, [email protected] Tel.: +32 9 264 73 73 19 20 Number of words in Abstract: 149 21 Number of words in Text: 3,862 22 Number of Figures: 6 23 Number of Table: 0 1 24 Abstract 25 Porcine circovirus type 2 (PCV2) encodes two major proteins: the replication protein (Rep) 26 and the capsid protein (Cap). The capsid protein displays a conserved stretch of basic 27 residues situated on the inside of the capsid which role is hitherto unknown. We have used a 28 reverse-genetics approach to investigate its function and found that mutations in these amino 29 acids hindered Cap mRNA translation and hampered Cap-Rep co-localization, yielding unfit 30 viruses. Intriguingly, co-transfection with a wild type PCV2 virus of a different genotype 31 partially rescued mutant Cap expression, showing the importance of this basic pattern for 32 efficient translation of Cap mRNA into protein. Our work shows that Cap and Rep are 33 expressed independently of each other, and that this amino acid sequence of the Cap is vital 34 for viral propagation. This study provides a method for studying unfit PCV2 virions and 35 offers new insights into the intracellular modus vivendi of PCV2. 36 37 38 39 40 41 2 42 Introduction 43 To date, two types of porcine circovirus have been described, porcine circovirus type 1 and 44 2 (PCV1 and PCV2) (Allan et al., 1998; Tischer et al., 1982). PCV1 was originally reported 45 as a contaminant of the PK-15 cell line and is considered pathogenic only in young porcine 46 fetuses (Saha et al., 2011). PCV2 is associated with several clinical manifestations 47 collectively known as porcine circovirus diseases (PCVD) with postweaning multisystemic 48 wasting syndrome (PMWS) as the most significant manifestation (Segalés, 2012). 49 PCV2 belongs to the genus Circovirus of the family Circoviridae, of which four genotypes 50 have been described so far: PCV2a, PCV2b, PCV2c and PCV2d (Xiao et al., 2015). PCV2 51 is an extremely small non-enveloped virus with an icosahedral capsid measuring only 17 nm 52 in diameter. Its genome comprises a circular single-stranded DNA of only 1766–1769 53 nucleotides (Huang et al., 2011b) which encodes two major open reading frames (ORFs): 54 ORF1 and ORF2. ORF1 encodes the non-structural replication protein Rep and its splice 55 variant Rep', which are both necessary for viral replication (Mankertz and Hillenbrand, 2001; 56 Mankertz et al., 1998a). ORF2 is translated into the capsid protein (Cap) (Nawagitgul et al., 57 2000), 60 copies of which make up the icosahedral proteinaceous shell protecting the viral 58 genome (Crowther et al., 2003). Cap is the primary immunogenic protein, which has the 59 ability to bind to the host cell receptor (Misinzo et al., 2006). 3 60 As PCV2 exploits heparin sulfate or dermatan sulfate side chains of cell surface 61 proteoglycans as attachment receptors, the putative heparin-binding motif 98IRKVKV103 was 62 initially thought to be a potential binding site (Misinzo et al., 2006). Recently however, the 63 atomic structure of PCV2 Cap revealed this motif was located on the inside of the capsid, 64 excluding a role in cell-surface attachment (Khayat et al., 2011). Moreover, it appeared to be 65 part of a larger constellation of positively charged amino acids on the inner surface of the 66 capsid, including a notable all-conserved 97RIRKVK102 sequence strongly reminiscent of the 67 putative heparin-binding motif. The manifest conservation of this pattern suggests a 68 well-defined purpose for this specific stretch of basic residues. The present study employs a 69 reverse-genetics approach to clarify its function by mutational analysis. 70 71 Results 72 Mutations in the basic residues of 97RIRKVK102 decrease Cap expression and interfere 73 with subcellular Cap localization 74 In order to determine the role of the basic amino acids in the conserved 75 sequence, 76 M3(R97T,R99T,K100T,K102T) were constructed. Sequencing confirmed that all mutations 77 were introduced correctly. PCV-negative PK-15 cells were transfected with DNA of each mutants M1(R97T,R99T), 4 97 RIRKVK102 M2(K100T,K102T) and 78 mutant and the parental clone, and subsequently immunostained at different time points 79 after transfection. The numbers of PCV2-positive cells are shown in Fig. 1a. For 80 M1(R97T,R99T), M2(K100T,K102T) and the parental clone, Caps were detected starting 81 from 12 hours post-transfection (hpt), and the numbers of positive cells rapidly increased to 82 a maximum at 36 hpt. For M3(R97T,R99T,K100T,K102T) however, not a single 83 Cap-positive cell was ever detected. In the first 36 hours post-transfection, the number of 84 PCV2-positive cells was comparable between M1(R97T,R99T), M2(K100T,K102T) and 85 the parental clone. However at 72 hpt, the number of positive cells markedly decreased for 86 these mutant viruses, suggesting a drop in viral fitness in secondary rounds of replication. 87 Despite similar numbers of positive cells at 36 hpt, immunostaining showed a strikingly 88 different Cap expression-pattern (Fig. 1b). Cap staining intensity of M1(R97T;R99T) was 89 weaker than the parental clone, and signals for M2(K100T,K102T) were even less than 90 those of M1(R97T,R99T). M3(R97T,R99T,K100T,K102T) signal was completely absent. 91 This prompted the question if the introduced mutations caused conformational changes 92 hindering antibody binding, or whether the protein itself was expressed at a lower level. 93 Western blotting of transfected PK-15 cell lysates confirmed there was a reduction in the 94 amount of Cap for M1(R97T,R99T) and M2(K100T,K102T), and a complete absence of 95 Cap for M3(R97T,R99T,K100T,K102T) (Fig. 2a). On the contrary, Rep immunostaining 5 96 never displayed any difference between wild type and mutants, not even for 97 M3(R97T,R99T,K100T,K102T) whose Cap expression was completely abolished 98 3a). Taken together, these results demonstrate that the mutants were only hampered in Cap 99 but not Rep expression, and that Cap expression occurs independently of Rep. (Fig. 100 In addition to expression alterations, a difference in subcellular distribution was observed 101 for Cap among the different mutants and the parental clone at 36 hpt (Fig. 1b). Of the 102 parental clone-transfected cells, 3.2% showed Cap presence in both nucleus and cytoplasm, 103 and another 5.1% had a positive Cap signal in the nucleus only. For M1(R97T,R99T) and 104 M2(K100T,K102T), the number of cells that showed Cap in both nucleus and cytoplasm 105 dropped dramatically (1.2 and 0.8% respectively), but this decrease was completely 106 compensated by an increase in Cap presence in the nucleus only (8.2% and 8.8%). Cap was 107 never observed in the cytoplasm only. This finding shows that mutations in the 108 97 109 rather than maintaining a cytoplasmic stage in its lifecycle. 110 To determine whether the decrease in Cap expression in the cells coincided with a decrease 111 of viral capsids released from the transfected PK-15 cells, capture-ELISA kinetics of 112 transfected cell supernatants were studied (Fig. 2b). Throughout a time course of 72h, 113 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, 6 114 parental clone. As expected, OD450nm values for M3(R97T,R99T;K100T,K102T) were 115 negative. Moreover, the data are perfectly in line with the intracellular capsid production, 116 and indicate that the conserved stretch of basic residues has no role in viral egress once the 117 virions are assembled. 118 119 Mutations in the 97RIRKVK102 hamper Cap-Rep co-localization 120 To determine the potential co-localization between Cap and Rep, PK-15 cells were 121 transfected at 24 hpt and stained with Cap-specific mAb 12E12 (Red) and Rep-specific 122 mAb F210 D6 (Green). M1(R97T,R99T) and especially M2(K100T,K102T) displayed a 123 striking lack of Cap-Rep co-localization (Fig. 3a). The co-localization events were 124 quantified by CoLocolizer Pro 2.7.1 using Manders’ coefficient correlation. This analysis 125 revealed a Cap-Rep co-localization rate of 81%, 56% and 24% for the parental clone, 126 M1(R97T,R99T) and M2(K100T,K102T) respectively. For M3(R97T,R99T,K100T,K102T) 127 a co-localization coefficient could not be calculated since Cap was not expressed (Fig. 3b). 128 129 Mutations in the 97RIRKVK102 drastically impact viral fitness 130 To investigate whether the mutant capsids released in the supernatant were still infectious, 131 we determined the viral titers of the supernatants collected at different time points after 7 132 transfection (Fig. 4a). A time-dependent rise of viral titers was observed starting from 24 133 hpt for M1(R97T,R99T), M2(K100T,K102T) and the parental clone. The highest titers were 134 found at 60 hpt: 3.9 log10TCID50/ml for M1(R97T,R99T), 3.3 log10TCID50/ml for 135 M2(K100T,K102T) and 5.3 log10TCID50/ml for the parental clone. The titers of the mutants 136 were lower than that of parental clone, reflecting the dramatic decrease in Cap expression 137 observed by immunostaining, western blot and ELISA. M3(R97T,R99T,K100T,K102T) 138 supernatants were always negative. 139 In order to test the fitness of the progeny virus, PCV2-transfected cell supernatants were 140 passaged on PCV-negative PK-15 cells. A gradual decrease in viral titers reaching a level of 141 non-detection was observed from passages two to seven in case of the mutants, while a 142 gradual increase in titers was observed for the parental clone (Fig. 4b). PCV2 genomic 143 sequences obtained from passage two, five and seven for parental virus, and passage two 144 and four for M1(R97T,R99T) and passage two for M2(K100T,K102T) were all 100% 145 identical to the genome in the pCR-BluntII-TOPO vector. 146 Taken together, this shows that Caps with mutations in the conserved basic sequence can 147 reach the assembly stage and properly exit the cell for secondary rounds of infection. 148 However, the impact on protein expression is so enormous that overall viral fitness is 149 seriously compromised. Without this pattern intact, the virus is doomed to disappear from the 8 150 gene pool, recapitulating the selection pressure observed in vivo. 151 152 The decrease in mutant Cap expression is exclusively due to an altered amino acid 153 sequence which results in a decrease in Cap mRNA translation 154 Next, we quantified Cap mRNA using RT-qPCR to see if the decrease in Cap expression of 155 the mutant viruses was caused by a decrease in Cap mRNA transcription. All samples 156 derived from the transfected PK-15 cells were positive at 12 hpt (Fig. 5a). Notwithstanding 157 the differences in Cap mRNA at 36 hpt of the mutants compared to the parental clone, the 158 decrease was not strong enough to fully explain the drastic decrease or even absence of 159 protein expression. At 72 hpt, which corresponds with the next replication cycle, Cap 160 mRNA copy number was approximately 80% lower than the parental clone, which can be 161 explained by the reduced viral fitness of the mutants in subsequent replication cycles. This 162 clearly shows that the dramatic reduction in Cap protein expression at 36 hpt is not solely 163 due to a decrease in Cap mRNA. 164 In order to completely exclude that nucleotide changes in the viral DNA or the Cap mRNA 165 would interfere with translation processes inside the cell, a new synonymous mutant, M4, 166 was constructed. M4 has four silent nucleotide changes, which do not alter the amino acid 167 sequence of Cap. We hypothesized that, if changes on the nucleotide level would influence 9 168 transcription or translation, M4 and M3(R97T,R99T,K100T,K102T) fitness would be evenly 169 affected, since both have four nucleotide substitutions in exactly the same region. 170 Interestingly, M4 displayed an exactly similar phenotype as the parental clone, yielding 171 infectious, fit viruses (Fig. 5b and 5c). This excludes an effect of the nucleotide sequence in 172 itself, and confirms that the dramatic effects on viral viability are entirely due to the altered 173 amino acids in the mutants. 174 The inability of mutant Caps to be properly expressed in spite of ample mRNA presence 175 could be explained by proteasomal degradation of newly expressed Cap proteins. However, 176 PK-15 cells treated with proteasome inhibitor MG132 for 2h at 24hpt did not rescue proper 177 Cap expression (data not shown). The picture that emerges is that mutant Cap mRNA 178 transcripts are hampered in translation, rather than Cap proteins being degraded once 179 expressed. 180 181 Co-transfection with a fit genotype PCV2a virus partially rescues mutant Cap 182 translation through an unknown mechanism 183 Our findings led us to believe that the conserved basic sequence was critical in getting Cap 184 mRNA efficiently translated into Cap. If so, co-transfection of the 48285-based mutants 185 (genotype PCV2b) with the PCV2a-LG strain should rescue mutant Cap expression as a 10 97 RIRKVK102 pattern. First, we verified that mouse mAb 8G12 only 186 result of its intact 187 reacted to PCV2a-LG Cap and did not cross-react with PCV2b mutants. Similarly, mouse 188 mAb 6E9 only detected 48285-based mutant capsids, and did not cross react with 189 PCV2a-LG Cap (data not shown). By staining mutant-transfected cells with mAb 6E9 we 190 obtained identical results as with mAb 38C1 (Fig. 6 left column). As expected, 191 co-transfection with PCV2a-LG did not have any effect on LG expression (Fig. 6 right 192 column). Intriguingly, this procedure partially rescued mutant Cap translation, which was 193 particularly visible in M3(R97T,R99T,K100T,K102T)-LG co-transfection (Fig. 6 middle 194 column). This highlights once more the importance of an intact 97RIRKVK102 alignment for 195 efficient Cap translation. 196 197 Discussion 198 Being the smallest of all known mammal viruses with limited genomic coding capacity, 199 porcine circoviruses are truly fascinating biological systems. Despite their reliance on 200 proofreading-competent host polymerases for replication, their genomes mutate 201 exceptionally rapidly while keeping an extremely low genetic diversity within the different 202 PCV2 genotypes (Firth et al., 2009). In other words, most nucleotide substitutions 203 disappear immediately under intense purifying selection, and as a consequence, PCV2 11 204 genomes are allowed only marginal leeway to evolve. This should not come as a surprise, 205 since every part of its genome is exploited on multiple levels: the stem-loop and adjacent 206 motifs need intense conservation for (i) binding and cleavage by the replication complex 207 (Steinfeldt et al., 2001), (ii) the Cap mRNA splicing acceptor site (Mankertz et al., 1998b) 208 and (iii) the larger part of the Rep promoter sequence (Mankertz and Hillenbrand, 2002). 209 The region clockwise of the stem-loop cannot be tampered with for its DNA encodes (i) the 210 replication protein Rep and its frame-shifted splice variant Rep’ (Mankertz and Hillenbrand, 211 2001) as well as (ii) ORF3 (Liu et al., 2005) and (iii) ORF4 (He et al., 2013) on the amino 212 acid level. On a nucleotide level, this stretch also contains sequences for (iv) the Rep’ 213 mRNA splicing donor and acceptor sites (Mankertz and Hillenbrand, 2001), and (v and vi) 214 the Cap mRNA splicing donor site and the Cap promoter (Mankertz and Hillenbrand, 2002; 215 Mankertz et al., 1998b). The genomic part counterclockwise to the stem loop is supposed to 216 have more freedom for variation because it only encodes the Cap on amino acid level 217 (Nawagitgul et al., 2000), and the smaller part of the Rep promoter (Mankertz and 218 Hillenbrand, 2002) on the nucleotide level. This understanding makes any conserved 219 sequence of ORF2 an interesting target for investigation. 220 Since cryo-EM reconstruction of PCV2 VLPs demonstrated that the all-conserved 221 98 IRKVKV103 motif originally identified by Misinzo (2006) was situated on the inside of the 12 222 capsid, and took part in a larger constellation of positively charged amino acids comprising 223 the basic sequence 224 negatively charged viral ssDNA strand upon encapsidation. That this stretch of basic residues 225 was also implicated in Cap mRNA translation (Fig.2 and Fig.5) came as a complete surprise 226 and the reason for it still mystifies us. It is hard to craft an explanation without falling into a 227 chicken or egg causality dilemma. One might think that Cap mRNA needs the Cap to be 228 efficiently exported from the nucleus, but for Cap to come into existence an efficient viral 229 mRNA export would be required first. Likewise, the Cap might act as a molecular switch 230 turning on Cap translation machinery, but this would require prior translation of Cap! We 231 tried to solve this catch-22 situation by co-transfecting with a wild typePCV2 strain, but even 232 then we see a preferential translation of wild-type PCV2 (Fig. 6). This argues in favor of a 233 vision where the basic amino acid sequence is not so much a biological switch enhancing 234 translation, but rather takes part in a molecular interface interacting with another factor. One 235 can imagine that the basic amino acids of the capsid protein have to be shielded from 236 aspecific interactions with other proteins and nucleic acids, before being imported to the 237 nucleus where they are deposited on viral DNA with Swiss-watch precision. In many ways, 238 they resemble histones safeguarded and escorted by histone chaperones. Interestingly, an 239 interaction between Cap and histone chaperones NAP1 and NPM has been described 97 RIRKVK102, we understood these basic amino acids stabilize the 13 240 (Finsterbusch et al., 2009). It is tempting to think that the removal of the positively charged 241 amino 242 M3(R97T,R99T,K100T,K102T) Caps abolishes their interaction with co-translational 243 chaperones, causing any further Cap mRNA translation to grind to a halt and leaving nascent 244 Cap polypeptides stuck on the negatively charged ribosome. In this respect, the partial 245 rescue of mutant Cap translation we see by co-transfecting with a wild type PCV2a virus (Fig. 246 6) may be caused by a mutant Cap-wild type Cap interaction which releases the mutant Cap 247 from the ribosome, and allows translation to continue, albeit still inefficiently. PCV1 capsid 248 proteins display a virtually identical 98RIRKAK103 sequence, and it would be interesting to 249 see if M3(R97T,R99T,K100T,K102T) would still be rescued after co-transfection with a 250 PCV1 Cap expression construct. PCV1 Caps are expected to have inferior binding 251 capabilities with PCV2 Caps because of their antigenic differences in the capsid protein. 252 PCV1 should therefore not be able to restore M3(R97T,R99T,K100T,K102T) expression if 253 rescue is indeed dependent on an interaction with wild type Cap. In addition, an unbiased 254 siRNA screen targeting histone chaperones could identify which chaperones, if any, are 255 involved in PCV2 Cap translation. 256 As a final remark, it is conceivable that the replication protein itself exerts some additional 257 chaperone activity. Rep may just guide Cap subunits to the right sub-nuclear compartment acid pattern in M1(R97T,R99T), 14 M2(K100T,K102T) and 258 for further assembly, or even more speculative, Rep might simply drive genome 259 encapsidation. As a matter of fact, all circovirus replication proteins share a common 260 superfamily 3 helicase domain (Roux et al., 2013) which assemble into ring-shaped 261 multimeres responsible for unwinding dsDNA by means of an ATPase-powered molecular 262 motor (Hickman and Dyda, 2005). Should this activity be performed in close proximity to 263 capsid proteins, it is not improbable that Rep subunits form replicative complexes displacing 264 the viral strand towards adjacent capsid subunits, thus facilitating the process of genome 265 packaging in complete harmony with replication. Hypothetical as this may be, also in the 266 closely related beak and feather disease virus (BFDV), an elegant relationship exists between 267 the two viral proteins, for Rep nuclear localization depends entirely on a cytoplasmic 268 interaction with Cap, after which both shuttle to the nucleus (Heath et al., 2006). These 269 findings all support a vision for circoviruses in which an intimate relationship exists between 270 Cap and Rep which has been underestimated until now, and can serve as a working model for 271 future experimental studies. 272 In conclusion, this study shows that the conserved stretch of basic residues 273 comprising 97RIRKVK102 of the PCV2 Cap is crucial for its expression. Mutations in this 274 pattern resulted in (i) a reduced translation of Cap, (ii) hindered Cap-Rep 275 co-localization and (iii) finally unfit PCV2 virions. Additionally, co-transfection with a 15 276 wild type PCV2 virus partially rescues mutant Cap expression, highlighting the importance 277 of these amino acids for an efficient translation of Cap mRNA into protein. We speculate 278 that 97RIRKVK102 is part of a molecular interface interacting with co-translational 279 chaperones, however, future experiments need to be carried out to confirm this hypothesis. 280 281 282 Material and methods 283 Virus, cells and virus titration 284 PCV2 strain 48285 (Accession number: AF055394) clone 48285-24, PK-15 cells and 285 culture conditions were described earlier (Saha et al., 2012b). PCV2 strain LG (Accession 286 number: HM038034) clone pMD18/PCV2a-LG was described earlier (Huang et al., 2011b). 287 Virus titration was performed as previously described (Meerts et al., 2005). 288 289 Monoclonal antibodies against PCV2 290 Cap-binding mouse monoclonal antibodies (mAb) 38C1, 12E12 and 6E9 were generated in 291 our laboratory and described earlier (Saha et al., 2012a). MAb 8G12 was generated against 292 PCV2a-LG with a procedure described by Huang et al. (2011a). Rep-binding mAb F210 293 D6 was described previously (McNeilly et al., 2001). For western blotting, mAb 2E8 was 16 294 generated against PCV2-Cap derived from PCV2a strain LG (Huang et al., 2011a) and is 295 directed against the linear epitope 26–36aa of PCV2-Cap (Guo et al., 2011). 296 297 Construction of mutants 298 A site-directed mutagenesis was performed by targeting the basic amino acids of 299 97 300 by an uncharged hydrophilic threonine (T) using a Quickchange® site-directed mutagenesis 301 kit (Stratagene, La Jolla, USA) based on clone 48285-24. R at positions 97 and 99 were 302 substituted by T using primers M1-FW (5’-atactacacaataacaaaggttaaggttgaat-3’) and 303 M1-REV (5’-attcaaccttaacctttgttattgtgtagtat-3’) resulting in the mutant M1(R97T,R99T). 304 Mutant M2(K100T,K102T) was constructed by mutating K into T at positions 100 and 102 305 using 306 (5’-attcaaccgtaaccgttcttattctgtagtat-3’). 307 constructed by replacing R or K with T at all four positions 97, 99, 100 and 102 using 308 primers 309 (5’-attcaaccgtaaccgttgttattgtgtagtat-3’). Mutant M4 was constructed by changing the codons 310 (aga ata aga aag gtt aag) of 311 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’) 97 and and was M3-REV RIRKVK102 into (agg ata agg aaa gtt aaa) using primers (5’-atactacaggataaggaaagttaaagttgaat-3’) 17 and M4-REV (5’- 312 attcaactttaactttccttatcctgtagtat-3’), resulting in a synonymous mutant with unaltered amino 313 acid sequence. 314 315 Transfections 316 Parental and mutated plasmids were digested with Hind Ⅲ and subsequently purified 317 genomic DNA fragments were self-ligated for 20 mins at room temperature using T4 DNA 318 ligase (Invitrogen, Merelbeke, Belgium). 800 ng of DNA was then transfected onto 319 PCV-negative PK-15 cells with lipofectamine 2000 (Invitrogen, Merelbeke, Belgium) 320 according to the manufacturer's instructions. Empty-plasmid transfected PK-15 cells were 321 included as a mock. 322 323 Immunoflurescence assays and co-localization quantification 324 Methanol-fixed transfected cells were stained with a previously described indirect 325 immunofluorescence assay (Saha et al., 2010). MAb 38C1 and fluorescein isothiocyanate 326 (FITC) labeled goat anti-mouse IgG (Molecular Probes, Eugene, USA) were used as the 327 primary and secondary antibodies, respectively. Images were made with Leica TCS SP2 328 confocal microscope (Leica Microsystems GmbH, Mannheim, Germany). For double 329 immunofluorescence staining for Cap and Rep, transfected PK-15 cells were first incubated 18 330 with anti-Cap mAb 12E12 (1:2) and then with Alexa Fluor 594 goat-anti mouse IgG2b 331 (Molecular Probes; 1:200). Afterwards, the cells were stained for PCV2 Rep by incubation 332 with anti-Rep mAb F210 D6 (1:500). Subsequently, a 1:200 dilution of FITC-labeled 333 goat-anti mouse IgG1 (Molecular Probes) was added. All antibodies were incubated for 1 h 334 at 37°C. The cells were washed three times with PBS after incubation with primary and 335 secondary antibodies. Specificity of the staining for different protein was demonstrated 336 using an irrelevant, isotype-matched mAb II1H/4-5G (Costers et al., 2010) and 13D12 337 (Nauwynck and Pensaert, 1995) and by the complete absence of Cap- and Rep-specific 338 fluorescence in mock-transfected PK-15 cells. The co-localization of Cap and Rep was 339 analyzed using the software CoLocalizer Pro 2.7.1 as described previously (Zinchuk and 340 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 342 varies from 0 to 1. A coefficient value of 0 to 0.6 indicates absence of co-localization while 343 a value of 0.6 to 1.0 indicates co-localization. 344 345 Western blot analysis 346 Western-blot analysis was used to quantify the amount of Cap produced by the different 347 mutants and the parental clone. Briefly, 19 M1(R97T,R99T), M2(K100T,K102T), 348 M3(R97T,R99T,K100T,K102T), parental clone and mock-transfected PK-15 cells were 349 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. 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Curr Protoc Cell Biol Chapter 4, Unit 4 19. 475 476 477 478 479 480 481 482 483 484 485 486 487 488 25 489 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