s2fieldsofstudy: Biology Medicine

text:
Aberrantly elevated Wnt signaling is responsible for cementum overgrowth and dental ankylosis

Vertebrate teeth are attached to the jawbones using a variety of methods but in mammals, a fibrous connection is the norm. This fibrous periodontal ligament (PDL) allows teeth to move in the jawbones in response to natural eruptive forces, mastication, and orthodontic tooth movement. In some disease states the PDL either calcifies or is replaced by a mineralized tissue and the result is ankylosis, where the tooth is fused to the alveolar bone. To understand how the PDL maintains this fibrous state we examined a strain of mice in which tooth movement is arrested. Da β cat Ot mice express a stabilized form of β -catenin in DMP1-positive alveolar bone osteocytes and cementocytes, which results in elevated Wnt signaling throughout the periodontium. As a consequence, there is an accrual of massive amounts of cellular cementum and alveolar bone, the PDL itself calcifies and teeth become ankylosed. These data suggest that to maintain its fibrous nature, Wnt signaling must normally be repressed in the PDL space.

Introduction
The periodontium is a tripartite structure consisting of a connective tissue, the periodontal ligament (PDL) and two mineralized tissues, the cementum and alveolar bone. The PDL spans the two mineralized tissues and this arrangement permits movement of the teeth within the jawbones. For example, in response to mastication the PDL dissipates potentially damaging strains that would otherwise accumulate in the bone and tooth [1]. Fibrous tissues that serve this type of function are referred to as entheses [2,3]. In health, entheses remain fibrous in nature even though they express osteogenic proteins [4]. In some disease states, entheses mineralize and this results in a condition known as ankylosis, where a joint becomes immobile.
Biological signals are required to maintain the fibrous state of the PDL (reviewed recently in [5]). For example, the transcription factor Scleraxis actively prevents mineralization, in part by opposing the effects of the transcription factor Osterix [6]. Physical signals-both tensile and compressive-are also implicated in maintaining the fibrous nature of the PDL [3]. Whether biological signals, physical stimuli, or a combination of both are required to maintain the fibrous state of the PDL, one thing is certain: if the PDL has inappropriately undergone mineralization then teeth become immobile [7][8][9][10]. Dental ankylosis represents a pathologic condition in mammals but in stem mammals including extinct flying and aquatic reptiles and dinosaurs, dental ankylosis was the natural terminal stage in successful tooth replacement [11].
To gain insights into the mechanisms regulating the development and potentially, evolution of the periodontium, we undertook an analysis of a strain of mice in which tooth movement is defective. Daβcat Ot mice express a stabilized form of β-catenin in osteocytes [12], and exhibit defects in the movement of both incisors and molars. We combined molecular, cellular, histologic, and imaging analyses to understand how a stabilized form of β-catenin led to prolonged Wnt/β-catenin signaling in alveolar bone osteocytes and surprisingly, in cementocytes as well, and how this aberrant Wnt signal led to dental ankylosis through mineralization of the PDL.

Micro-computed tomography analysis
Micro-computed tomography (µCT) scans of murine skulls were performed on daβcat Ot control (N=3) and daβcat Ot mutant (N=3) groups. Mice were sacrificed, tissues were fixed in 70% ethanol and then scanned at 10µm resolution (Micro XCT, Xradia Inc., Pleasanton, CA). Samples were reconstructed, segmented with ScanIP then using the measure function, the length of incisors were calculated. After orienting the µCT slice planes to show longitudinal sections of the incisors, the length of root-to-tip was measurements and the average length of 3 mice were calculated.

Histology, immunohistochemistry, and histomorphometric analyses
Animals were sacrificed at the time points indicated by first anesthetizing them with an IP injection of ketamine (80 mg/kg) and Xylazine (16 mg/kg) followed by cervical dislocation. Tissues were fixed in 4% paraformaldehyde overnight then washed in PBS for 30 min and decalcified in 19% EDTA for up to 14 days in 4ºC, with shaking. After decalcification, specimens were dehydrated through an ascending ethanol series and embedded in paraffin. Eight-micron-thick longitudinal, transverse, and coronal sections were cut and collected on superfrost-plus slides. Movat's pentachrome staining was performed on slides [15], further dehydrated in an ethanol series, and then mounted with Permount. Tartrate resistant acid phosphatase (TRAP) staining was performed [16]. Immunohistochemistry was performed [17] with primary antibodies: rabbit anti-PCNA (ab18197, Abcam, Cambrdige, UK), rabbit anti-Periostin (ab14041, Abcam), rabbit anti-Osterix (ab22552, Abcam), mouse anti-Sclerostin (AF1589, R&D systems, Minneapolis, USA), mouse anti-CTNNB1 (β-catenin) antibody (610154, BD Biosciences New Jersey, USA), rabbit anti-green fluorescent protein (GFP; 2956S, Cell Signaling Technology, Danvers, USA) and secondary antibodies including biotinylated goat anti-rabbit IgG antibody (BA-1000, Vector Lab, Burlingame, USA) and biotinylated horse anti-mouse IgG antibody (BA-2000, Vector Lab). The staining was visualized by ABC peroxidase standard staining kit (32020, Thermo Fisher Scientific, Rockford, USA). Tissue sections were photographed using a Leica digital imaging system. Histomorphometry was performed using Image J software (Version 1.49v) to quantify the TRAP activity. At least 4 slides from each sample were used for quantification. The total length of incisor was divided into three part from the tip to the apex. The first 1/3 part was defined as the region of interest (ROI) for the incisive edge and the third 1/3 was used for apex quantification. To quantify the cell number in PDL area, the total DAPI +ve cell in PDL area are counted and normalized to the PDL area.

Statistical analyses
Results were presented as mean ± standard deviation. A two-tailed Student's t-test was used to determine significant differences between data sets. A p value<0.05 was considered statistically significant and all statistical analyses were performed with Microsoft Excel software (Version 15.16, USA).
This study follows ARRIVE guidelines.

Tooth eruption is perturbed in daβcat Ot mice
A functional periodontium is required for active tooth eruption [18,19], and in murine incisors this eruption process continues throughout life. In daβcat Ot mice, however, incisor eruption was arrested. Compared with µCT scans of P42 littermate controls (Fig. 1A,C), the mandibular incisors of aged-matched daβcat Ot mutant mice were not visible in the oral cavity ( Fig. 1B,D). In control littermates, the incisor tip (green asterisk) projected past the most coronal extent of the alveolar ridge (white arrow; Fig. 1E) whereas in daβcat Ot mice the incisor tip (red asterisk) was still encased in alveolar bone (white arrow; Fig. 1F; quantified in G). Upon close examination, the PDL near the incisor tip was thin and discontinuous with no obvious fibrous attachment between the bone and the lingual incisor surface (Fig. 1F).
One explanation for this phenotype could be a lack of adequate tooth structure; in other words, the eruption machinery was intact but there was insufficient tooth material to protrude into the oral cavity. The overall lengths of the maxillary incisors in controls and mutants, however was equivalent (Fig. 1H,I), yet one erupts and the other doesn't fully erupt ( Fig. 1A-D). Therefore, it was unlikely the case that the eruption defect in daβcat Ot mice was solely caused by inadequate tooth structure.
Three-dimensional volumetric reconstruction data revealed that overall, the mandibular daβcat Ot incisors were significantly shorter than controls (Fig. 1J,K). We considered cellular mechanisms of incisor eruption in rodents. For example, cell proliferation at the rodent incisor apex drives eruption [20][21][22]. Using PCNA immunostaining, we identified proliferating cells in controls (Fig. 1L) and the number of PCNA +ve cells was notably reduced in the mutants (Fig. 1M). We also noted a defect in molar eruption (Fig. 1A,B) but PCNA staining failed to reveal any proliferating cells at the apices of these teeth (data were not shown). Therefore, it was unlikely that a lack of cell proliferation was solely responsible for the widespread eruption defect and turned to consider other mechanisms that could contribute to this phenotype.

The daβcat Ot eruption defect is not due to perturbed osteoclast activity
Osteopetrosis is a genetic condition of increased bone mass caused by defects in osteoclast formation and function [23]. Osteopetrosis can arrest tooth movement [24] because the alveolar bone fails to resorb properly [25,26]. To ascertain whether the daβcat Ot eruption defect was attributable to an osteopetrotic phenotype we evaluated osteoblast and osteoclast function around the roots using ALP and TRAP activities ( Fig. 2A,B). In controls, ALP and TRAP activities was readily detectable in the alveolar bone surrounding the molar roots ( Fig. 2A), as well as around the incisor buccal and lingual alveolar bone surfaces (Fig.  2C,D). In daβcat Ot mutants, TRAP activity was evident around the alveolar bone surrounding the molar roots ( Fig. 2B) and along the thickened buccal and lingual alveolar bone surfaces (Fig. 2E,F). The extent of TRAP staining was quantified, and these analyses showed that rather than being depressed, daβcat Ot mutants had significantly higher osteoclast activity compared to controls (Fig. 2G). These data agree with previously published analyses of the daβcat Ot strain [12]. Notably, in some regions, the low density PDL space is missing and the alveolar bone appears to be fused with the tooth (Fig. 2E, yellow boxes).

Daβcat Ot mutants lack a function periodontal ligament
The daβcat Ot eruption defect was not due to an arrest in bone resorption nor was it due to a primary defect in osteoclast activity; we therefore considered other explanations for the tooth eruption defect in daβcat Ot mutants. For example, tooth movement is possible because of the fibrous nature of the PDL (Fig. 3A); we reasoned that changes in the width and/or its fibrous state might adversely affect tooth eruption. We first determined there was a narrowing of the PDL space in the mutant: at post-natal day 24 (P24), histomorphometric comparisons between the strains revealed a slightly reduced PDL space in daβcat Ot mutants (compare Fig. 3A,B; quantified in E). Histomorphometric comparisons were made again at P42. The PDL normally widens until animals reach adulthood, which was seen in the control group (compare Fig. 3A,C). In daβcat Ot mutants, however, the PDL instead had narrowed significantly (Fig. 3D, quantified in E). Closer analyses revealed aberrant mineralization within the PDL proper that appeared like "spot-welds" (arrows) within what is a normally fibrous tissue (compare Fig. 3F,G). These observations together suggested that the mineralized tissues surrounding the PDL e.g., the alveolar bone and/or cementum were either enroaching onto the PDL space, or that the PDL itself was undergoing mineralization. µCT imaging analyses verified the widespread nature of this aberrant mineralization: normally, the PDL appears as a dark "halo" around teeth ( Fig. 3H) but in daβcat Ot mutants, the radiolucent area was replaced in places by a radio-opaque tissue (arrows, Fig. 3I).
To further examine the fibrous versus mineralized state of the daβcat Ot PDL, we examined daβcat Ot control and mutant mice for the expression of Periostin. Periostin is normally expressed in the fibrous PDL ( Fig. 3J) but was minimally detectable in the daβcat Ot mutant (Fig. 3K). On tissue sections from daβcat Ot mutants, we found that in sites that still retained a fibrous PDL, the osteogenic protein Osterix was expressed very strongly (compare Fig.  3L,M). Did this change in Osterix result from a direct increase in the number of osteogenic cells in the daβcat Ot PDL? To address this question, we verified that cell proliferation in the daβcat Ot mutant PDL, just like in an adult wild-type PDL [4], was minimal (data were not shown). We then performed DAPI staining to quantify the density of cells in the PDL area in both daβcat Ot control and mutant mice. The number of DAPI +ve cells in PDL was normalized to the area. These data demonstrated that although the PDL space was narrower in the daβcat Ot group, cell density was comparable to the control group (Fig. 3N,O, quantified in P). Finally, to confirm that the Osterix +ve cells were indeed committed to an osteogenic fate, expression of a late-stage osteogenesis marker [27,28], Osteocalcin, was examined. Osteocalcin is normally absent from PDL cells (Fig. 3Q) but Osteocalcin +ve cells were present in the daβcat Ot PDL (Fig. 3R). Together, these data demonstrated that the increase in osteogenic cells within the daβcat Ot PDL was directly due to a greater percentage of PDL cells committing to an osteogenic fate.

Sustained Wnt/β-catenin signaling in the periodontium causes ankylosis
Thus far our data demonstrated that the replacement of a fibrous PDL by mineralized tissue was the primary cause of ankylosis in daβcat Ot mutants. Our next analyses set out to determine how amplified Wnt signaling in the daβcat Ot mice specifically caused dental ankylosis.
We confirmed that Wnt signaling was elevated in the daβcat Ot PDL using an antibody to phosphorylated β-catenin. Compared to controls (Fig. 4E), phosphorylated β-catenin expression was higher in daβcat Ot PDL (Fig. 4F). Sclerostin expression domains were also expanded: normally, Sclerostin is expressed in osteocytes and cementocytes (Fig. 4G) but in daβcat Ot mutants, its expression was also detected in the PDL (Fig. 4H). We returned to the DMP1 reporter strain of mice, this time focusing on how the daβcat Ot mutation affected DMP1 expression itself. In comparison to controls, DMP1 expression itself was dramatically increased in daβcat Ot mutants (Fig. 4I,J, compared with Fig. 4 B,D). Collectively, these data demonstrated that Wnt signaling was aberrantly elevated in the periodontium of daβcat Ot mice.
We examined the longer-term sequelae of this elevated Wnt signaling in the periodontium. Normally, acellular cementum covers most of the root surface, and cellular cementum is restricted to the root apices (Fig. 4K,L). In daβcat Ot mutants, however, the elevated state of Wnt signaling caused an dramatic expansion in cementum (Fig. 4M,N). At the apex of the root, cellular cementum is normally separated from the adjacent alveolar bone by an intervening fibrous PDL (Fig. 4O). In daβcat Ot mutants, however, the fibrous PDL was replaced by a solid mass of mineralized tissue (Fig. 4P). This series of events obliterated the distinction between alveolar bone and cellular cementum, and effectively fused the tooth to its socket. Thus, elevated Wnt signaling in multiple components of the periodontium results in the formation of mineralized tissue at the expense of fibrous tissue, thereby resulting in dental ankylosis.

Regulating PDL mineralization
Entheses are tissues that must remain fibrous in order to function. In some disease states, as we have shown here, the fibrous PDL enthesis mineralizes and the result is ankylosis. A wide variety of factors-both biological and physical-can influence the mineralization state of entheses. For example, the mineralization state of the cementum can be influenced by a phosphate/pyrophosphate ratio [29] and it is tempting to speculate that a similar general mechanism might underlie mineralization of the PDL. There is no direct data to support this hypothesis, however, and it must be emphasized that, rather than a generalized phenomenon, the aberrant mineralization events in the daβcat Ot PDL are not uniform. Rather, the mineralization nodes appear more like "spot-welds" within a normally fibrous tissue (Figs.  2,3). This complicates a biochemical analysis of the mutant PDL as a tissue, since the mineralization spots are surrounded by fibrous PDL.
Matrix Gla protein (MGP) has also been recognized as a potent calcification inhibitor, which also suggests a potential role in preventing PDL mineralization. In vitro data support this conjecture [30], but again, whether a similar mechanism is at work to maintain a fibrous PDL is simply not known. At least in vitro, the aberrant expression of an extracellular matrix protein such as bone sialoprotein is initially sufficient to promote mineralization, but the effect is transient [31], and so is unlikely to be the causal link in the daβcat Ot ankylosis phenotype.
Another potential clue may come from analysis of ANK, a transmembrane protein that control movement of intracellular inorganic pyrophosphate across the cell membrane [32]. Genetic modulation of ANK in humans and other vertebrates have identified a role in regulating tissue calcification [32] and potentially, mineral deposition in tendons and ligaments. There is, however, no mention of dental ankylosis in ank/ank mice [33]. Clearly, there is still much to be learned about what factors, both biological and physical, are key regulators of mineralization within the functioning PDL.

Wnt signaling is essential for the homeostasis of the periodontium
An abundance of data demonstrates that Wnt signaling is required for proper development of the periodontium. For example, if Wnt signaling is disrupted early during embryogenesis then eventually the development of the periodontium is arrested [34][35][36]. If Wnt signaling is disrupted after the periodontium has formed, then homeostasis of the periodontium is disrupted [37,38]. Wnt signaling is also required to maintain alveolar bone [39] and cementum [37]. Given the interdependence of the cementum, bone, and the PDL on one another during development and homeostasis, it is challenging to unravel just when and where endogenous Wnt signals are having their primary action. Daβcat Ot mutant mice offered rare insights into this conundrum because Wnt signaling was amplified only after the embryonic periodontium had formed [40], and then only in very specific cell niches.

Elevated Wnt/β-catenin signaling causes mineralization of the PDL
Daβcat Ot mutant mice demonstrate a progressive expansion of Wnt responsive cells that has an unusually detrimental effect on the periodontium. Wnt/β-catenin signaling is normally restricted in alveolar bone osteocytes and cementocytes, but in daβcat Ot mutant mice, Wnt signaling spreads to envelop the entire PDL (Fig. 4). From our analyses of the DMP1 reporter strain, we knew that DMP1 becomes aberrantly expressed in the daβcat Ot mutant PDL (Fig. 4B,D), a conclusion that was validated using DMP1 immunostaining (Fig. 4I,J). Precisely how this expansion in DMP1 expression occurs was not immediately obvious. Our current hypothesis is that the gradual accretion of bone and cementum constricts the PDL space in daβcat Ot mice (Fig. 3A-D). Both osteoblasts and cementoblasts normally produce and respond to Wnt signals (Fig. 4) therefore it is reasonable to speciulate that as the PDL space narrows, Wnt signals emanating from the mineralizing tissues begin to impact cells in the middle of the PDL space. The subsequent expression of DMP1 in PDL cells may therefore be a cause-rather than an instigating factor-in PDL mineralization.
While some parts of this hypothesis are still speculative, one thing is clear: as the Wnt responsive cell populations expand so, too, does the mineralized tissues. Within short order, both the volume and distribution of cementum and alveolar bone have enlarged in daβcat Ot mutants (Fig. 4), which together obliterates the fibrous PDL (Fig. 4). Other investigators have shown that stabilizing beta-catenin via an Osteocalcin-Cre reporter strain leads to an accumulation of cellular cementum deposited over the entire molar root surfaces of molar in the mutant mice, which causes a narrowing of the PDL space [41,42]. Likewise, stabilizing beta-catenin via an Col1a1-Cre reporter strain to leads to abnormal tooth eruption and excessive cementum hypoplasia [36]. Neither group, however, reported an eruption defect due to ankylosis, as we have shown here for daβcat Ot mice. Finally, there is the possibility that the ankylosis phenotype in daβcat Ot mice is that because of sustained Wnt signaling, the PDL itself undergoes calcification (Fig. 4). Whether the cementum and bone are simultaneously encroaching on the PDL space, or the PDL itself undergoes mineralization, the end result is a fusion of the cementum to the alveolar bone, which arrests tooth eruption. Collectively, these data also raise a possibility that in order to maintain its fibrous nature, Wnt signaling must normally be repressed in the PDL space. Mechanosensory studies support this relationship between repressed Wnt signaling and a fibrous fate [43,44].

A pattern of ankylosis
The pattern of ankylosis we observe in the daβcat Ot mutant may reflect the pattern of tooth eruption. For example, the daβcat Ot phenotype is based on conditional expression of a stabilized form of beta-catenin, driven by the DMP1 promoter. Since mandibular molars erupt prior to maxillary molars, we speculate that expression of stabilized beta catenin has not yet reached its maximum in the mandible by the time molars initiate their eruption. Conversely, by the time the maxillary molars erupt the expression of stabilized beta-catenin may have reached its zenith. Analyses of other strains of mice in which Wnt signaling is aberrrantly elevated in the periodontium may shed light on this hypothesis.

Ankylosis may represent a developmental atavism
In mammals, ankylosis represents a pathological condition that prevents tooth movement, and our study suggests that its etiology is related to pathologically sustained Wnt signaling. In some animals, however, ankylosis is not a pathologic condition. Rather, it represents a natural stage in odontogenesis [45]. For example, in stem mammals including extinct flying and aquatic reptiles and dinosaurs, dental ankylosis was the norm [46]. In the marine lizard Mosasauridae, cellular cementum and alveolar bone accumulated throughout the animal's life, leading to ankylosis [11,47].
This same interpretation aptly describes the daβcat Ot mutant phenotype, where accelerated accumulation of both mineralized tissues eventually causes ankylosis of the molars and incisors. In the case of daβcat Ot mutant mice, the molecular basis for the phenotype is clear; it is tempting to speculate that a similar molecular mechanism of sustained Wnt signaling in the periodontium might have been shared by the Mosasauridae tooth attachment apparatus [11].  In the PDL of P42 daβcat Ot control mice, Osteocalcin is absent. Relative to this control, (R) Osteocalcin expression is observed in daβcat Ot mutant mice in the cleft of tissue remaining between the tooth surface and alveolar bone (N=4). Abbreviations: as indicated previously and M1, first molar; M2, second molar; Scale bars: H,I=1000 μm, others =100 μm. cellular cementum at the root apex is juxtaposed to a fibrous PDL. (P) In daβcat Ot mutants, the mineralized tissue of the alveolar bone and cementum are contiguous. Abbreviations: as indicated previously. Scale bars: K,M=1000 μm, others =100 μm.


extfieldsofstudy: Biology Medicine

text:
Genetic and biological characteristics of four novel recombinant avian infectious bronchitis viruses isolated in China

Highlights • Two IBV strains were proved to be originated from multiple recombination events.• Viruses with very similar S1 gene sequences showed varying biological features.• Point mutations were observed in the RBD and HVRs of the recombinant viruses.• Point mutations likely have an effect on these differences in biological characteristics.

Introduction
Avian infectious bronchitis (IB) is an acute and highly contagious disease caused by the IB virus (IBV), which causes respiratory diseases, interstitial nephritis, and reproductive disorders in chickens of all ages (Cavanagh, 2007). The disease can induce severe economic losses, especially in terms of decreased egg production, poor eggshell quality, reduced hatchability, increased feed conversion, and carcass condemnation at slaughterhouses (Santos Fernando et al., 2017). IBV variants are continuously emerging in different regions of the world and continue to be a major problem for chicken production worldwide (Jackwood, 2012). Vaccination is considered to be the most reliable approach for IBV control; however, existing vaccines have proved to be inadequate due to the constant emergence of new virus variants (De Wit, 2000;de Wit et al., 2011). In addition, co-infection of a chicken by different IBV types carry the risk of recombination, accounting for the emergence of novel IBV strains with different antigenic properties, tissue tropism, and pathogenicity; such strains have been identified in many parts of the world (Franzo et al., 2015;Hewson et al., 2014;Hong et al., 2012;Kottier et al., 1995;Lee and Jackwood, 2000;Mardani et al., 2010;Naguib et al., 2016;Quinteros et al., 2016;Pohuang et al., 2011;Thor et al., 2011;Zanaty et al., 2016;Zhang et al., 2015), and this presents a major challenge to current IBV prevention and control strategies.
IBV belongs to the Coronaviridae family, and its virion contains a single copy of a positive-sense, single-stranded, 27.6-kb RNA, which encodes polyproteins 1a and 1b (which are further processed into nonstructural proteins 2-16), four structural proteins (the spike [S], envelope [E], membrane [M], and nucleocapsid [N] proteins), and several accessory proteins (3a, 3b, 5a, and 5b). The S protein, especially the S1 subunit formed by post-translational cleavage, is critical for antigenic neutralization, hemagglutination, and determining cell tropism (Casais et al., 2001;Cavanagh, 2007;1988;1997;Kant et al., 1992;Promkuntod et al., 2014). The high mutation rate of the S1 gene during IBV replication and evolution generates extensive genotypic, antigenic, and pathogenic variability in the progeny viruses. This implies the existence of multiple genotypes/serotypes and variants, against which the existing IB vaccines might not offer complete crossprotection (Cavanagh et al., 1997;Gelb et al., 2005), thus hampering the control of IBV infection by vaccination.
Many researchers have typically focused on analysis of the IBV S gene; most of such studies have analyzed the S1 subunit of the S protein for genotyping IBV strains, unifying IBV classification, and clarifying IBV evolution. For example, 7 genotypes and 35 distinct lineages of IBV have been defined on the basis of the sequence analysis findings of the complete S1 gene of IBV strains isolated from different countries or geographic regions worldwide Jiang et al., 2018;Valastro et al., 2016). However, the results of S1 sequence analysis might not be solely sufficient to explain the changes observed in IBV antigenicity, tissue tropism, and pathogenicity. In recent years, several studies have noted that viral replication, pathogenicity, and immune escape might be modulated by non-structural and accessory viral proteins in IBV and other coronaviruses (Armesto et al., 2009;Chen et al., 2009;Laconi et al., 2018;Zheng et al., 2008;van Beurden et al., 2018). These findings emphasize the importance of studying the characteristics of the complete genomes of coronaviruses and their associations with antigenicity, tissue tropism, and pathogenicity.
More recently, phylogenetic studies have reported that-in addition to the well-established lineages/genotypes such as GI-1, GI-7, GI-13, GI-19, GI-22, and GVI-1-a number of novel IBV variants have been isolated in China (Chen et al., 2015Han et al., 2017;Xu et al., 2016Xu et al., , 2018Zhao et al., 2017). These variants have been proved to be distinct from other reference strains on the basis of genetic characteristics and phylogenetic differences in the S1 gene (Xu et al., 2018). To provide a comprehensive outlook on the genetic characteristics of these IBV variants, we sequenced and comparatively analyzed the complete genome sequences of four novel IBV variants with those of other IBV strains in the present study. We also characterized the antigenicity, pathogenicity, and tissue tropism of these IBV strains and looked for possible associations between their genes and their phenotypic properties.

Cloning and sequencing of the complete genomes of the IBV strains
The complete genomes of strains I0718/17, I0722/17, I0724/17, and I0737/17 were cloned and sequenced in this study. Viral RNA was extracted from allantoic fluid by using an RNAiso Plus kit (Takara Bio Inc., Shiga, Japan) in accordance with the manufacturer's instructions. RT-PCR was performed using a one-step RT-PCR kit (Takara Bio Inc.) in accordance with the manufacturer's instructions. The primers and strategies used for cloning the complete genomes and the 3′/5′ termini of the four IBV strains were as previously described . Each PCR amplicon was cloned into a PMD-18 T vector system (Takara Bio Inc.), and three to five independent clones were sequenced for each amplicon.

Comparison and analysis of the sequences of S1 gene
The complete sequences of the S1 gene of the four IBV strains were assembled from both forward and reverse sequences by using the Clustal W method (available in the Bioedit software package; http:// www.mbio.ncsu.edu/bioedit). Phylogenetic trees were constructed with the MEGA 6.0 software (http://www.megasoftware.net/) by using the S1 gene sequences of 75 IBV strains, including the four novel strains described in this study and 71 reference strains. The trees were constructed by the neighbor-joining method with 1000 bootstrap replicates. The S1 gene sequences of the reference strains, which represented the 7 well-established genotypes and 30 well-established lineages, were retrieved from the GenBank database.
Alignment of the deduced amino acid sequences of the S1 subunit of the spike protein was performed on our four IBV strains by comparing with those of the vaccine strain, 4/91, which is used commonly in chicken flocks in China, and two IBV field strains, LGX/100508 and YX10, that represented the main IBV type circulating in chicken flocks in China. We compared the amino acid sequences of the three hypervariable regions (HVRs), five conformation-dependent neutralizing antigenic sites (epitopes) mapped to the S1 subunit of the spike protein (Kusters et al., 1989;Lenstra et al., 1989;Koch et al., 1990), as well as the minimal receptor-binding domain (RBD) (Promkuntod et al., 2014) in a pairwise manner.

Characteristics of the complete genome
The consensus sequence of each PCR product was assembled from both forward and reverse sequences by using MEGA 6.0 (http://www. megasoftware.net/). The complete genome sequences of the four IBV strains were carefully edited and were submitted for a BLAST search in the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for identifying homologous IBV sequences. The complete genome sequence of IBV strain 4/91 was used to compare with those of our four IBV strains. The open reading frames (ORFs) of the four IBV strains were identified by comparison with those of the 4/91 and YX10 strains. The nucleotide sequence data reported in this study were deposited in the GenBank database (I0718/17: MK032178; I0722/17: MK032179; I0724/17: MK032177; and I0737/17: MK032180).
To further confirm the recombination events previously observed in the S1 gene (Xu et al., 2018) and investigate possible recombination events in other regions of the genome, the complete genome sequences of our four IBV strains were compared with that of the 4/91 vaccine strain by using the Multiple Alignment with Fast Fourier Transformation (MAFFT) tool (version 6; http://mafft.cbrc.jp/alignment/software/ ). Then, sequence fragments that were different from those of the 4/91 strain were submitted for a BLAST search in the NCBI database for identifying homologous IBV sequences. On the basis of the search results, the complete genomic sequences of our four strains were again compared with those of the 4/91and YX10 strains (GenBank no. JX840411) by using MAFFT. The recombination events in the genomes of each of our four strains were further confirmed by bootscan analysis using the Simplot software (Lole et al., 1999). This analysis was conducted using a sliding window of 1000 nucleotides (nt) moving in 50-nt steps, with the genome sequence of strain ck/CH/LJL/101150 as the query.
To identify potential recombination switch sites, multiple sequences of the potential parental viruses were compared pairwise with those of each of our IBV strains on the basis of the MAFFT and SimPlot results.

Virus cross-neutralization tests
On the basis of sequence analysis results, strains I0718/17, I0722/ 17, I0724/17, and I0737/17 were selected for virus cross-neutralization tests with each other and with strains LGX/100508 and 4/91. Six groups of five 1-day-old SPF layers were housed in six isolators and challenged with strains LGX/100508, 4/91, I0718/17, I0722/17, I0724/17, and I0737/17 by the oculonasal route. At 3 weeks of age, the birds in the six groups were boosted with each of the corresponding viruses. At 35 days of age, all birds were bled. The blood samples were pooled per group, and the serum samples of each group were stored at −70°C until use. The serum of SPF chickens was also used in this test.
Virus cross-neutralization tests were conducted by the β method. The virus (100 EID 50 ) was added to a twofold dilution of the serum and incubated for 1 h at 37°C. Subsequently, the virus-serum mixtures were injected into the allantoic cavity of five SPF chicken embryos, which were then observed for 7 days. Virus replication was determined by amplifying the IBV N gene from allantoic fluid by RT-PCR (Liu et al., 2009). The tests were performed in the presence of appropriate controls.

Challenge viruses
On the basis of the results of genome sequence analysis, six strains of IBV (LGX/100508, 4/91, I0718/17, I0722/17, I0724/17, and I0737/ 17) were selected for further testing by pathotyping. These challenge strains were inoculated by the oculonasal route, and this procedure yielded 10 4 EID 50 per bird at the age of 1 day.

Experiment tests
The Ethical and Animal Welfare Committee of the Harbin Veterinary Research Institute, China, granted ethical approval for the animal experiments. One-day-old SPF layer chickens (N = 105) were randomly divided into seven groups (n = 15, each), and housed in isolators. Each group of chickens was challenged with one of the challenge IBV strains (LGX/100508, 4/91, I0718/17, I0722/17, I0724/ 17, or I0737/17). One group of 15 SPF birds was mock-infected with phosphate-buffered saline in the same manner, and this functioned as the negative control group. Signs, such as eye irritation and/or scratching of the inoculated eye, lethargy, mild coughing and/or "snicking," were scored as 0 (absent), 1 (mild), 2 (moderate), and 3 (severe). To determine the pathogenicity of the challenge viruses in the respiratory and urinary tracts at 5 days post-challenge (dpc) and to evaluate their tissue tropism in SPF chickens, five birds in each group were humanely sacrificed, and samples of the trachea and kidneys were carefully collected for titrating the viruses in 9-day-old SPF chicken eggs. The remaining ten birds were monitored daily for clinical signs and death for a period of 32 days. Blood samples and oropharyngeal and cloacal swabs were collected on days 4, 8,12,16,20,24,28, and 32 dpc. The serum samples were analyzed for the presence of IBVspecific antibodies by using a commercial enzyme-linked immunosorbent assay kit (IDEXX, Inc., Westbrook, ME, USA) in accordance with the manufacturer's instructions . The oropharyngeal and cloacal swabs were used for virus recovery in 9-dayold SPF chicken eggs .

Statistical analysis
Data are expressed as mean ± standard deviation. Virus titers were analyzed by a Student's t-test using GraphPad Prism for Windows version 5 (GraphPad Software, La Jolla, CA, USA). Differences were considered significant if the p value was < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001).

Results
3.1. Phylogenetic analysis and sequence comparison based on the complete S1 gene Phylogenetic analysis was performed by using the complete nucleotide sequences of the S1 gene of our four IBV strains and 71 reference IBVs strains, which were representative of 35 well-established lineages and 7 genotypes. The results demonstrated that our four IBV strains were phylogenetically clustered among IBV strains with the GI-13 genotype (Valastro et al., 2016), including the 4/91 vaccine strain and Moroccan/G/83 (El-Houadfi et al., 1986). Our results also showed that our four strains formed two phylogenetic subclusters, in which strains I0737/17, 4/91, and Moroccan/G/83 occurred in the same subcluster, and strains I0718/17, I0722/17, and I0724/17 were grouped together (Fig. 1).
The results of alignment of the complete S1 gene sequences of our four viruses with those of the 4/91 strain (the currently used vaccine strain) and the LGX/100508 and YX10 strains (GI-19-lineage viruses) revealed the presence of multiple mutations (Fig. 2). The majority of variations between I0737/17 and 4/91 were observed at the far N terminus of the S1 subunit of the S protein, in which three substitutions were located in HVRI. The nucleotide sequence of this region in strain I0737/17 was same as those in strains LGX/100508 and YX10. In contrast, the nucleotide sequences of the remaining regions in I0737/17 were nearly the same as those in strain 4/91 and different from those in strains LGX/100508 and YX10. These remaining regions included several important domains, such as the deduced minimal RBD, most parts of HVRI, all parts of HVRs II and III, and the five epitopes mapped to the S1 subunit of the S protein (Kusters et al., 1989;Koch et al., 1990). Strains I0718/17, I0722/17, and I0724/17 possessed similar nucleotide sequences and showed differences with the amino acid sequence of 4/ 91 at the N terminus of the S1 subunit, which included HVRs I and II, epitopes D and E, half of the RBD, and four critical amino acids in the RBD. The nucleotide sequences of the remaining regions in the three viruses were the same as those in 4/91 and different from those in strains LGX/100508 and YX10. In addition, strains LGX/100508 and YX10 differed only by two amino acid substitutions (F193Y and I252T).

Complete genome sequence and recombination analysis
The complete genomes of strains I0718/17, I0722/17, I0724/17, and I0737/17 are 27677, 27,666, 27,669, and 27,666 nt in length, respectively, excluding the poly-A tail. The genomes include ten ORFs and are organized as follows: 5′-UTR-1a-1b-S -3 a-3b-E-M-5a-5b-N-3′-UTR (UTR: untranslated region). The ORFs, genes, or genomic regions present in our four IBV strains and two reference strains (4/91 and YX10) are listed in Table 1. The complete genome sequences have been deposited in GenBank, and the accession number of each virus is listed in Fig. 1.
The complete genome sequences of our four strains were compared with that of strain 4/91 by using MAFFT. The results showed differences in the sequence of 1ab gene between our four IBV strains and 4/ 91 (data not shown). Therefore, the 1ab gene sequences were submitted to a BLAST search in the NCBI database. The search results demonstrated that the 1ab gene of our four strains was highly homologous with that of IBV strain YX10. Then, the complete genome sequences of our four IBV strains were compared with those of 4/91 and YX10 by using MAFFT, and the results suggested the occurrence of recombination events in the genomes of our four strains (Fig. 3).
In both bootscan analysis and pairwise comparison, strain I0717/18 exhibited a greater similarity to strain YX10 in three fragments (from 5′ UTR-nt10546;nt10,743-21,014;and nt23899-26,166) and to strain 4/ 91 in three other fragments (nt10546-10,704; 21,017-23,893; and 26,174-37,677) (Fig. 4A). The nucleotide sequence of strain I0724/17 was similar to that of strain YX10 in two fragments (nt1-10, 276 and 11,715-20,796) and strain 4/91 in two other fragments (nt10293-11,712 and 20,800-27,669) (Fig. 4B). These results implied that strains I0718/17 and I0724/17 both originated from multiple (five and three, respectively) recombination events between a 4/91-like virus and a YX10-like virus. The results of bootscan analysis of the complete genome revealed that the 5′ ends of strains I0717/22 and I0717/37 were highly similar in sequence to that of YX10 and less similar to that of 4/91; in contrast, their 3′ ends were highly related to that of 4/91 and different from that of YX10 (Supplemental Figure). These results correspond to those of the previous report (Xu et al., 2018).

Antigenic characteristics
Twofold dilutions of the serum were tested for the presence and quantity of cross-neutralizing antibodies (against homologous and heterogeneous novel IBV strains as well as the deduced parental IBV strains 4/91 and LGX/100508) against a constant virus titer of 100 EID 50 . The results are shown in Table 2. The antigenic relatedness value (r, 64.4) calculated in this experiment confirmed the presence of an antigenic relationship between strains I0718/17 and I0724/17. The value of r obtained by comparing strains I0737/17 and 4/91 was relatively high (r, 48.6) but did not reach the 50% threshold for antigenic relatedness between strains. Strains 4/91 and LGX/100508 were not cross-neutralized by LGX/100508 and 4/91 antisera, respectively. The antisera against our four novel viruses did not neutralize LGX/100508, although LGX/100508 antiserum was highly neutralizing against our four IBV strains. Although some cross-reactivity was observed among the other viruses, the r values did not reach the 50% threshold, which suggested the absence of an antigenic relationship among these strains.

Pathogenicity in SPF chickens
Ten birds in each challenge group were observed for clinical signs until 32 dpc. None of the mock-or 4/91-infected birds exhibited any signs of disease. However, SPF chickens challenged with different IBV strains exhibited variable mortality rates and clinical manifestations. These birds exhibited respiratory signs starting at 3 dpc and continuing until 12 dpc, after which all birds recovered. Birds challenged with strains I0722/17, I0724/17, and I0737/17 showed only mild clinical signs, ranging from sneezing and coughing to head shaking; these signs were limited to the respiratory tract. In contrast, SPF chickens experimentally infected with strains I0718/17 and LGX/100508 exhibited relatively severe respiratory signs represented by occasional mouth breathing. The duration and severity of the clinical responses induced by the six IBV strains were evaluated by scoring the respiratory signs (Fig. 5).
The results of analysis of the oropharyngeal and cloacal swabs collected at regular intervals showed that the IBV-challenged birds shed the challenge viruses from the respiratory tract between 4 and 24 dpc and from the cloaca between 4 and 32 dpc. Birds generally shed viruses for a longer duration from the cloaca than from the respiratory tract. In the present study, birds challenged with strains I0722/17, I0724/17, and LGX/100508 exhibited similar patterns of oropharyngeal and cloacal shedding. However, the durations of tracheal shedding of I0718/17 and both tracheal and cloacal shedding of I0737/17 were shorter than those of other viruses. At 32 dpc, the viruses were still detected in the cloacal swabs of some birds challenged with strains I0718/17, I0722/17, and I0724/17. Birds inoculated with I0724/17 shed the virus for a longer duration than did those inoculated with other viruses. No IBVs were detected in any of the swabs collected from the mock-infected birds at any time point. The results of virus recovery are presented in Fig. 6.
At 4 days of age, all birds in all challenge groups exhibited antibody titers below the cutoff values. At 12 days of age, IBV-specific antibodies were detected in nearly half of the birds in the groups challenged with strains I0718/17, I0722/17, I0724/17, and I0737/17. In the LGX/ 100508-challenge group, IBV seroconversion was detected in some and all birds at 8 and 12 days of age, respectively. No IBV-specific antibodies were detected in birds in the negative control group during the experiment. Fig. 7A and B show the virus titers observed in the trachea and kidneys of IBV-challenged birds at 5 dpc. Overall, strains I0718/17, I0722/17, I0724/17, and LGX/100508 showed similar virus titers and replication efficiency in the trachea of experimentally infected chickens; the virus titers in these groups were relatively high, exceeding 3.2 log 10 in the trachea. In contrast, birds inoculated with IBV strains I0737/17 and 4/91 showed an obviously lower production of infectious virus particles in the trachea at 5 dpc. Strains I0718/17 and LGX/ Fig. 2. Alignment of the deduced amino acid sequences of the S1 subunit of the spike protein of IBV strains LGX/100508, I0718/17, I0722/17, I0724/17, I0737/17, and 4/91. Amino acid sequences encoding the three hypervariable regions are shown in bold. The sequences of the five conformation-dependent neutralizing antigenic sites (epitopes) mapped to the S1 subunit are underlined. The minimal receptor-binding domains corresponding to the most N-terminal residues 19-272 of the spike protein of IBV M41 are boxed, and the critical amino acids 38, 43, 63, and 69 are highlighted in red. The GenBank accession numbers are the same as those in Fig. 1. IBV, infectious bronchitis virus.

Tissue tropism
100508 showed an obviously high affinity for both tracheal and kidney tissues, as reflected by their high titers in these organs. Interestingly, one of the birds infected with I0722/17 showed a relatively low viral titer in the kidneys, while a chicken challenged with I0724/17 showed a higher viral titer in the kidneys relative to the remaining birds in the same group (data not shown). In sharp contrast, in the 4/91-challenge group, chickens showed very lower viral titer in the kidneys.

Discussion
Understanding the genetic and biological characteristics related to the antigenicity, pathogenicity, tissue tropism, and shedding of IBV isolates in chickens is important for better elucidating how this virus is evolving and contributing to the emergence of IBV variants that can alter the immune status conferred by regular IBV vaccines. It also helps address the processes of virus maintenance and circulation in chicken flocks. We, therefore, studied the genetic characteristics of four IBV isolates by analyzing their full-length genome sequences and also evaluated their persistence and biological properties. It has been reported that the side effects of the 4/91 vaccine might provide the basis for recombination with other strains. This phenomenon has been shown by several studies which have traced the emergence of many novel IBV strains to recombination events between the 4/91 vaccine and field strains (Feng et al., 2018;Han et al., 2017;Jiang et al., 2017Jiang et al., , 2018Liu et al., 2013;Mo et al., 2013;Ovchinnikova et al., 2011;Zhang et al., 2015Zhang et al., , 2016Zhou et al., 2017) in cases where the 4/91 vaccine strain could not provide effective protection against the field strains , leading to coinfection of the vaccine and field strains in the same chickens and ultimately resulting in recombination. In this study, we performed complete genome sequence analysis of strains I0718/17, I0722/17, I0724/17, and I0737/17 and confirmed the occurrence of recombination events at the 5′ ends of the S1 gene between a 4/91-like virus (GI-13 lineage) and a YX10-like virus (GI-19) (Xu et al., 2018), which contributed to the emergence of the IBV strains. In addition, we also identified recombination "hotspots" in other regions of the genome in strains I0718/17 and I0724/17. We, therefore, speculate that these two strains originated from multiple recombination events between 4/91-like and YX10-like IBV strains.
A potential concern regarding these recombination events is that the changes occurring in the S1 subunit during recombination have altered the neutralizing epitopes, resulting in the emergence of novel IBV serotypes. The S1 subunit of the S protein contains HVRs associated with serotype-specific and virus-neutralizing epitopes; these HVRs are present within amino acid residues 38-67, 91-141, and 274-387 (Wang and Huang, 2000). In the present study, strains I0718/17, I0722/17, and I0724/17 were found to show a significant clustering of substitutions in HVRs I and II and epitopes D and E in comparison with 4/91 strain; these strains also showed such significant clustering of substitutions in the remaining regions of the S1 domain-including HVR III and epitopes B/C/A-in comparison with strains YX10 and LGX/ 100508 (which differ by only two amino acids in the S1 region). These regions have previously been identified as the most variable genomic regions among different IBV serotypes (Cavanagh, 2007), and point mutations in these regions might have an effect on protein structure and polarity, possibly leading to changes in protein antigenicity. Such point mutations might also account for the decreased cross-neutralization activity between the two deduced parental strains (4/91 and LGX/ 100508) and the novel IBV strains evaluated in this study (I0718/17, I0722/17, and I0724/17), as evident from the absence of antigenic relatedness between the two groups. Upon comparing the r values between strains I0718/17, I0722/17, and I0724/17, we found that strain I0718/17 is serologically related to I0724/17. However, it was surprising to find that the other IBV pairs are not serologically related to each other, although the three viruses have very similar S1 gene sequences. IBV strains differing only by a few amino acid substitutions have been reported to show variations in antigenicity (Abdel-Moneim Table 1 Comparison of putative genes/genomic regions between the four novel IBV strains and two reference strains (4/91 and YX10).     et al., 2006). However, strains I0722 and I0724/17 have identical S1 gene sequences. Similarly, we found a difference of only ten substitutions between strains I0737/17 and 4/91, nine of which were located at the N terminus of the S1 subunit (with only three being located in HVR I). The r value calculated from the cross-neutralization test was less than 50, which implied that strain I0737/17 is not serologically related to 4/91. Contrary to this result, we have previously demonstrated that replacing the three HVRs of the avirulent Beaudette strain of IBV separately or simultaneously with the corresponding regions from a GI-19-lineage nephropathogenic strain by reverse genetics does not produce any changes in the serotype of the Beaudette strain (Shan et al., 2018). These results might suggest that the amino acid sequences of the HVRs were not the only factor accounting for the variations in antigenicity of IBVs. Another potential concern regarding recombination events between vaccine and field strains is that recombination events leading to reversion to virulence might produce nephropathogenic and highly pathogenic recombinant strains. In the present study, all four IBV strains were pathogenic in comparison with the 4/91 vaccine strain, although they showed varying levels of pathogenicity in SPF chickens. Some of the strains showed a comparable affinity for replication in the kidneys as the  LGX/100508 strain. Coronavirus binding and entry into susceptible host cells requires interactions between cell-surface receptors and the viral attachment protein (S protein). As in other  coronaviruses, the S1 subunit of the S protein of IBV contains an RBD (Koch et al., 1990;Schultze et al., 1992;Spaan et al., 1988;Sturman et al., 1985). The location of the RBD in IBV genome has been mapped to the most N-terminal 19-272 amino acid residues of the S1 subunit (Promkuntod et al., 2014). Moreover, amino acids N38, H43, P63, and T69 appear to be critical for the binding of the S protein to host cells in the chicken respiratory tract (Promkuntod et al., 2014). In the present study, although strains I0737/17 and 4/91 showed a difference of ten amino acid substitutions, only four were found to be located in the RBD domain. Moreover, when tested by means of experimental infection in SPF birds, these two viruses showed no drastic differences in their affinity for replication in the trachea or their pathogenicity. Although the C-terminal sequences of the RBD of strains I0718/17, I0722/17, and I0724/17 differed greatly from that of strain LGX/100508, all these viruses showed a similar affinity for replication in the trachea of infected chickens. These results suggest that these sequence exchanges might have no effect on the affinity of the viruses for the trachea, particularly for interaction with α-2,3-sialic acid or other potential IBV receptors on the surface of cells in the respiratory tract of chickens (Shahwan et al., 2013;Winter et al., 2006). However, strain LGX/ 100508 was more pathogenic to SPF chickens than the other strains analyzed in the present study. Therefore, the point mutations observed in the RBD might have an effect on the affinity of the viruses for different tissues, rendering them highly pathogenic to SPF chickens. Interestingly, although strain I0718/17 differed from strains I0722/17 and I0724/17 by only six amino acid substitutions in the S1 protein, it showed a higher affinity for kidneys and was more pathogenic to SPF chickens. This indicates that these amino acid changes might affect receptor binding. Otherwise, the S1 protein of these IBV strains might not be the only factor responsible for their expression of pathogenicity. The binding of susceptible host cells is the first step in the viral life cycle, and host innate immune responses could also be a major contributing factor to the pathological outcome of IBV infection. In addition, we cannot exclude that the differences in pathogenicity are not due to other changes between the parental 4/91 strain and the  recombinant viruses. By using reverse genetics, a previous report showed that the replicase gene of IBV is a determinant of pathogenicity (Armesto et al., 2009) and recent studies have shown that IBV pathogenicity is also due to the accessory proteins (Laconi et al., 2018;van Beurden et al., 2018). In order to establish the mechanisms, further study by reverse genetics and animal studies is needed to verify the association between changes in the genome sequence and the pathogenicity of IBV strains. While all IBV strains appear to initially infect chickens through the respiratory tract, viremia enables the viruses to spread to secondary sites for further replication and persistence. It has been considered that the IBV strain replicates mainly in the respiratory tract, although it can also spread and replicate in a range of other tissues, such as the digestive and urogenital tracts. In this study, we found that IBV-infected birds shed the virus for a longer duration from the cloaca than from the respiratory tract; moreover, the birds shed the virus from the respiratory tract even after the clinical signs had disappeared. It is important to consider that, during an active IBV infection, even in the absence of clinical disease, and during the course of chronic and persistent infection, the gastrointestinal tract contains a significant amount of virus, and such persistently infected tissues are a likely source of reinfection in chickens, as reported previously Gough, 1978, 1977;Chong and Apostolov, 1982;Cook, 1968;Jones and Ambali, 1987). In addition, our results from analysis of oropharyngeal and cloacal swabs showed that most of the challenged birds shed their challenge viruses from the digestive tract at 20 dpc, by which time the birds were positive for anti-IBV antibodies. This suggests that these IBVs had reached a somewhat persistent status despite the presence of systemic anti-IBV immunoglobulin G. Thus, these anti-IBV antibodies present in the serum seem to exert a non-complete protective role against IBV infection in SPF chickens, as reported previously (Santos Fernando et al., 2017).
Taken together, the results of our genetic and biological analyses clearly demonstrate that our four IBV strains, which emerged from recombination events between GI-13-and GI-19-like viruses, are antigenically different from their deduced parental viruses. They also show variations in their pathogenicity and affinity for the trachea and kidneys of chickens after infection. Fig. 7. Replication of IBV strains LGX/100508, I0718/17, I0722/17, I0724/17, I0737/17, and 4/91 in chickens. Chickens were challenged with each of the IBV strains, and the trachea (A) and kidneys (B) from each bird were collected at 5 dpc for virus titration in eggs. Each bar represents the virus titer from an individual bird. dpc, days post-challenge; IBV, infectious bronchitis virus. Differences were considered significant if the p value was < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001).


extfieldsofstudy: Medicine Biology

text:
Key within-membrane residues and precursor dosage impact the allotopic expression of yeast subunit II of cytochrome c oxidase

Experimentally relocating mitochondrial genes to the nucleus for functional expression (allotopic expression) is a challenging process. The high hydrophobicity of mitochondria-encoded proteins seems to be one of the main factors preventing this allotopic expression. We focused on subunit II of cytochrome c oxidase (Cox2) to study which modifications may enable or improve its allotopic expression in yeast. Cox2 can be imported from the cytosol into mitochondria in the presence of the W56R substitution, which decreases the protein hydrophobicity and allows partial respiratory rescue of a cox2-null strain. We show that the inclusion of a positive charge is more favorable than substitutions that only decrease the hydrophobicity. We also searched for other determinants enabling allotopic expression in yeast by examining the COX2 gene in organisms where it was transferred to the nucleus during evolution. We found that naturally occurring variations at within-membrane residues in the legume Glycine max Cox2 could enable yeast COX2 allotopic expression. We also evidence that directing high doses of allotopically synthesized Cox2 to mitochondria seems to be counterproductive because the subunit aggregates at the mitochondrial surface. Our findings are relevant to the design of allotopic expression strategies and contribute to the understanding of gene retention in organellar genomes.

INTRODUCTION
Most mitochondrial proteins are encoded in the nuclear genome, translated by cytosolic ribosomes, and directed to mitochondria. However, migration of mitochondrial genes to the nucleus occurs widely in nature (Adams and Palmer, 2003;Burger et al., 2003;Timmis et al., 2004). These relocalized nucleotide sequences acquire more complex regulation and tend to exhibit lower mutation rates (Martin and Herrmann, 1998). Mitochondrial genomes mainly encode subunits of oxidative phosphorylation (OXPHOS) complexes. In general, these proteins are embedded in membranes and are highly hydrophobic, a feature that is considered to be one of the main driving forces for the retention of a genome in mitochondria (Johnston and Williams, 2016).
Experimentally relocating mitochondrial genes to the nucleus holds promise for the development of treatments of mitochondrial diseases (De Grey, 2000;DiMauro et al., 2006;Kyriakouli et al., 2008). A mutation in the mitochondrial genome, yielding a defective protein, could be overcome in principle by the import of a nucleus-encoded functional version of the protein. Multiple efforts have been undertaken to express mitochondria-encoded genes allotopically using yeast or mammalian model systems, but conflicting and inconsistent results call for careful interpretation of results and warrant further experimental testing (Oca-Cossio et al., 2003;Perales-Clemente et al., 2011).
The COX2 gene, encoding subunit 2 (Cox2) of cytochrome c oxidase (CcO), provides an example of natural migration of a mitochondrial gene to the nucleus. This gene is generally mitochondria-encoded, but in a few organisms it is found in the nuclear genome (Adams et al., 1999;Pérez-Martínez et al., 2001;Szafranski, 2017). For example, COX2 was transferred to the nucleus in some members of the legume lineage, resulting in mitochondrial and nuclear versions of COX2. The representative species of this lineage encode Cox2 in the nuclear DNA, in the mitochondrial DNA, or in both genomes, depending on which copy was activated and/or inactivated (Adams et al., 1999;Daley et al., 2002a,b). Also, the COX2 gene has been relocalized experimentally to the yeast nucleus (Supekova et al., 2010), and subsequent studies have provided supporting genetic and biochemical evidence for successful allotopic expression (Cruz-Torres et al., 2012;Elliott et al., 2012;Rubalcava-Gracia et al., 2018). The substitution of a tryptophan for an arginine in position 56 (Cox2 W56R ), within the first transmembrane segment (TMS) of allotopically produced Cox2, decreases its hydrophobicity and allows the partial respiratory rescue of a cox2-null yeast strain, with an estimated ∼60% recovery of CcO levels as compared with the wild-type strain (Cruz-Torres et al., 2012). This partial complementation can be attributed solely to diminished import, since a mitochondrial COX2 gene producing the W56R variant cannot be distinguished from wild-type (Rubalcava-Gracia et al., 2018). The decrease in hydrophobicity of the first TMS in Cox2 also occurred during the natural relocation of the COX2 gene to the nucleus in legumes (Daley et al., 2002b). Other necessary modifications for successful allotopic expression include recoding the mitochondrial gene to the nuclear genetic code and fusing a sequence encoding a mitochondrial targeting sequence (MTS) to the open reading frame. Given that the W56R substitution was found in a random mutagenesis screen of 20,000 alleles (Supekova et al., 2010), it is likely that this is the only permissible change that enables import from the cytosol to the mitochondria and at the same time maintains CcO functionality. This underscores the power of such an approach to uncover mutations allowing the allotopic production of other mitochondria-encoded proteins.
Here, we further explored the W56 position in Cox2 by testing whether other residues with similar properties to arginine could confer allotopic expression. We also tested whether naturally occurring changes in the legume Glycine max Cox2 enable yeast Cox2 to be imported from the cytosol into mitochondria and assembled into a functional CcO. Finally, we evidenced that dosage of the cytosolsynthesized Cox2 precursor is critical for the outcome of allotopic expression.

RESULTS
Other substitutions at residue 56 enable the allotopic expression of COX2 First, we tested whether other changes at position 56, besides the tryptophan-to-arginine substitution (Supekova et al., 2010) (W56R), can restore the respiratory growth of a cox2-null strain expressing an allotopic version of COX2. We substituted W56 for two residues sharing at least one physicochemical property with arginine: lysine (W56K), which also has a positive charge, and glutamine (W56Q), which has the closest hydrophobicity score to arginine (Calado-Botelho et al., 2011). Upon introduction of the allotopic constructs encoding Cox2 W56K or Cox2 W56Q into a cox2-null strain, we observed a restoration of respiratory growth. Respiratory growth of the strain with the W56K substitution was comparable to that of the strain carrying the W56R substitution, while the recovery of respiratory competence by Cox2 W56Q was significantly less ( Figure 1A). Hence, the growth behavior of yeast carrying these constructs was Cox2 W56R = Cox2 W56K > Cox2 W56Q . As a negative control, we included a strain expressing an allotopic wild-type COX2 gene (producing the Cox2 WT subunit). As expected, neither this construct producing Cox2 WT , nor the empty plasmid, was able to restore respiratory growth in the cox2-null strain ( Figure 1A).
Previous work evidenced that most of the allotopically synthesized Cox2 W56R protein accumulates as an unprocessed precursor (i.e., still carrying its MTS) detected in the mitochondrial fraction (Supekova et al., 2010;Cruz-Torres et al., 2012;Rubalcava-Gracia et al., 2018). We thus performed cellular fractionations to test the cytosolic versus mitochondrial distribution of Cox2 WT , Cox2 W56K , and Cox2 W56Q . Most of the Cox2 precursors were found associated with mitochondria ( Figure 1B), indicating that, similarly to Cox2 W56R , the limiting step occurs before their processing into mature forms in the mitochondrial matrix. Our results show that other residues at position 56 allow allotopic expression of COX2 and that the inclusion of a positive charge in the first TMS (W56K) is favorable over a substitution that only diminishes hydrophobicity (W56Q).
Mutations that allow the nuclear expression of COX2 in legumes also confer allotopic expression in yeast Next, we sought to identify other changes in Cox2 that might enable its allotopic expression in the yeast system. We reasoned that in organisms that naturally encode COX2 in the nucleus, the protein FIGURE 1: Substituting residues of properties similar to those of arginine in position 56 of allotopic Cox2 restores cellular respiration to a cox2-null strain. (A) Tenfold serial dilutions of yeast liquid cultures from strains transformed with the indicated constructs or with an empty vector (plasmid) were plated on fermentable (glucose), respiratory (ethanol + glycerol), and minimal, selective media (-ura or -arg, for verifying the presence of the transforming plasmid or the mitochondrial genome, respectively). (B) Immunoblots decorated with an anti-Cox2 antibody of cytosolic (c; 100-µg) and mitochondrial (m; 50-µg) fractions of the indicated yeast strains. The Cox2 precursor and mature forms of Cox2 are indicated. Antibodies anti-Atp2 and anti-Oxa1 were used to immunodetect the corresponding mitochondrial markers and anti-Hog1 was used for the cytosolic marker. Squared brackets indicate Cox2 variants expressed from plasmids. All the Cox2 variants contain an MTS from Oxa1. sequence must carry determinants for import into the mitochondria and subsequent assembly into a functional holoenzyme. For example, the legume Glycine max expresses the COX2 gene from both nuclear and mitochondrial genomes (Adams et al., 1999). Previous research showed that a two-residue substitution within the first TMS of Cox2 is necessary and sufficient for the mitochondrial version of Cox2 to be imported into isolated mitochondria. Accordingly, the nucleus-encoded Cox2 protein could no longer be imported into the organelle when the residues (Q and G) at these two positions were modified to the ones (L and L) present in the mitochondriaencoded Cox2 (Daley et al., 2002b; Figure 2A). The conclusion from this study is that this two-residue substitution was determinant in facilitating the import of Cox2 from the cytosol to the mitochondria.
To test whether this two-residue substitution could confer allotopic expression in the yeast system, we sought to reproduce the legume COX2 mutations in the COX2 sequence from yeast. We generated an alignment of the two legume Cox2 subunits with the one from yeast and identified that the key residues conferring import of the legume Cox2 correspond to a valine and a leucine at positions 49 and 51 in the yeast sequence ( Figure 2A). We therefore engineered the mutations corresponding to the single and double substitutions V49Q and L51G in yeast COX2. We observed that only the Cox2 V49Q/L51G variant was able to restore respiratory growth in a cox2-null strain, while the Cox2 V49Q and Cox2 L51G variants could not ( Figure 2B), demonstrating that both changes are required for successful allotopic expression of COX2. However, the respiratory growth of the Cox2 V49Q/L51G strain was less than that of the Cox2 W56R strain. To test whether the overall decrease in hydrophobicity within the first TMS of Cox2 is sufficient for restoring respiratory growth, we substituted other aliphatic residues at other positions, resulting in the construct Cox2 L47Q/V49G . To explore any additive effect, a triple COX2 mutant corresponding to W56R combined with a double substitution (L47Q/V49G/W56R) was also generated. All these constructs could restore the respiratory growth of a cox2-null strain, although to a lesser extent than the construct producing Cox2 V49Q/L51G . We also could not observe any additive effect in the strain producing the Cox2 V49Q/L51G/W56R variant. Using in-gel activity staining, we verified that CcO activity correlated with the observation that the Cox2 W56R variant exhibits increased respiratory growth compared with that in the variants containing two-residue substitutions ( Figure 2C). In contrast, another OXPHOS complex, ATPase, exhibited comparable activity for all strains.
Next, we questioned whether Cox2 V49Q/L51G follows the same biogenesis pathway as that of allotopically produced Cox2 W56R . In contrast to orthodox Cox2, synthesized in mitochondria, allotopically FIGURE 2: Mutations that allow the nuclear expression of COX2 in legumes also confer allotopic expression in yeast. (A) Sequence alignments of the first transmembrane segment (TMS1) from the yeast Cox2 (mScCox2) and the mitochondrial and nuclear Cox2 versions of Glycine max, mGmCox2 and nGmCox2, respectively. An asterisk (*) indicates positions that have a single, fully conserved residue. A colon (:) indicates conservation between groups of strongly similar properties (scoring >0.5 in the Gonnet PAM 250 matrix). A period (.) indicates conservation between groups of weakly similar properties (scoring ≤0.5 in the Gonnet PAM 250 matrix). Key residues that are critical for import of the legume Cox2 from the cytosol are boxed (see text). Two yeast Cox2 constructs were designed based on this alignment: Cox2 V49Q/L51G and Cox2 L47Q/V49G . (B) Tenfold serial dilutions of yeast cultures from the indicated strains were spotted on fermentable (glucose), respiratory (ethanol + glycerol), and minimal, selective media (-ura or -arg). (C) In-gel activity staining of complex IV and of complex V as a loading control. Isolated mitochondria (250 µg) from the indicated strains were solubilized with lauryl maltoside and resolved by BN-PAGE. (D) Strains (WT, Δcox2, and Δcox18) were plated on fermentable (glucose), respiratory (lactate), and minimal, selective media (-ura or -arg). Squared brackets indicate Cox2 variants expressed from plasmids. All the Cox2 variants contain an MTS from Oxa1.

FIGURE 3:
The allotopically synthesized proteins face the limiting step by aggregating at the mitochondrial periphery. (A) Immunoblots decorated with anti-Cox2 of cytosolic (c; 100-µg) and mitochondrial (m; 50-µg) fractions. Arrows indicate the precursor and mature forms of Cox2. The mitochondrial markers Oxa1 and the cytosolic marker Hog1 were decorated with their corresponding antibodies. (B) Isolated mitochondria (50 µg) were treated with proteinase K (PK) to degrade proteins external to the outer membrane. Parallel samples were preincubated with Triton X-100 to dissolve the membranes and to make all mitochondrial proteins accessible to protease degradation. The asterisk indicates partial degradation of Cox2, possibly due to imperfect mitochondrial preparation. (C) Carbonate extraction separating the membrane extrinsic proteins in the supernatant (S) from the integral membrane proteins in the pellet (P) from yeast expressing the indicated constructs. Atp2 was used as a soluble protein marker and Oxa1 as an integral membrane protein marker. (D) Triton X-100 extraction separating the detergent-solubilized proteins in the supernatant (S) from the (detergent-resistant) aggregated proteins in the pellet (P) from yeast expressing the indicated constructs. Cox3 was used as a solubilized membrane protein marker. Squared brackets indicate Cox2 variants expressed from plasmids. All the Cox2 variants contain an MTS from Oxa1. synthesized Cox2 W56R does not require the Cox18 translocase for the correct insertion of its second TMS into the inner membrane (Elliott et al., 2012), and it was postulated to be released laterally into this membrane directly from the TIM23 complex (Rubalcava-Gracia et al., 2018). We observed that, similarly to the Cox2 W56R strain, the Cox2 V49Q/L51G variant is able to restore respiration when expressed in a cox18-null ( Figure 2D), indicating that the Cox18 translocase is completely dispensable. This suggests that the second TMS of Cox2 V49Q/L51G is released laterally into the inner mitochondrial membrane by the TIM23 translocator.
We observed that while the mature forms of Cox2 were protected from degradation upon incubation of mitochondria-enriched fractions with proteinase K (PK), a result indicative of mitochondrial localization, the Cox2 precursors were mostly degraded by the protease (Figure 3, A and B). These results may be interpreted in two ways: either the precursors are associated with the external face of the outer mitochondrial membrane or they are retained while in transit through the TOM and TIM23 complexes with their C-termini still exposed to the cytosol (note that our anti-Cox2 antibody recognizes the C-terminus of Cox2). To distinguish between these two possibilities, we performed carbonate and Triton X-100 extractions of the mitochondrial fractions. We found that the precursors are mostly resistant to both carbonate ( Figure 3C) and detergent treatments ( Figure 3D). As expected, mature Cox2 proteins resist carbonate extraction but are solubilized into the supernatant upon Triton extraction (Figure 3, C and D). These observations suggest that most Cox2 precursors aggregate at the external mitochondrial surface, thus preventing their import.
Taken together, our data show that substitutions at a position different from that of W56 enable the successful allotopic expression of COX and that a major limiting step for importing allotopic Cox2 into mitochondria is its aggregation.

Lowering the dose of allotopic COX2 expression enhances Cox2 import and assembly in CcO
While the use of a multicopy plasmid for allotopic expression of COX2 to ensure successful complementation seemed justified a priori (Supekova et al., 2010), we wondered whether reducing the allotopic expression of COX2 would decrease the rescue of the respiratory defect proportionally. For this purpose, we opted to test allotopic expression under a condition where the same COX2 construct carried in the multicopy plasmid was used as a single copy. To achieve this, we integrated the COX2-expressing construct (under the same phosphoglycerate kinase promoter and terminator present in the multicopy plasmid) in the nuclear genome. Surprisingly, we observed a notable increase in respiratory growth in the strains expressing the nuclear-integrated constructs producing Cox2 W56R and Cox2 V49Q/L51G in comparison with the strains producing the same Cox2 variants from a multicopy plasmid ( Figure 4A). Growth of the yeast containing the singlecopy COX2 W56R construct was even comparable to that of a wild-type strain, and the strain containing the single-copy COX2 V49Q/L51G exhibited more robust respiratory growth than the one with the high-copy COX2 W56R . The strain producing Cox2 WT did not display any growth in respiratory medium, even at low doses. Immunodetection of Cox2 in mitochondria-enriched fractions of these strains showed that, at low dosage, Cox2 W56R and Cox2 V49Q/L51G only accumulated in their mature forms at levels similar to those of the WT strain ( Figure 4B). As previously observed, at high dosage, Cox2 W56R and Cox2 V49Q/L51G were mainly observed as precursors, with only a fraction converted to the FIGURE 4: Lowering the expression of allotopic COX2 in a cox2-null strain improves rescue of the respiratory growth. (A) Tenfold serial dilutions of yeast cultures from the indicated strains were plated on fermentable (glucose), respiratory (ethanol + glycerol), and minimal, selective media (-arg or -ura) and in the presence of hygromycin B (+hygB). The letter p represents expression from a multicopy plasmid, and the letter n represents expression from a single copy inserted in the nuclear genome. (B) Cox2 steady-state levels in isolated mitochondria (50 µg) from the indicated strains. Arrows indicate the precursor and mature forms of Cox2. The letters p and n indicate expression levels as in A. (C) Tenfold serial dilutions of yeast cultures from the indicated strains were plated on fermentable (glucose); respiratory (ethanol + glycerol), and minimal, selective media (-arg or -ura) and in the presence of hygromycin B (+hygB). The letter c represents expression from a centromeric plasmid. The order of the letters indicates the order in which COX2expressing constructs were introduced into yeast cells. All the Cox2 variants contain an MTS from Oxa1. (D) Biogenesis model of allotopically synthesized Cox2. On the left, insertion of Cox2 results in its correct topology. On the right, the high hydrophobicity of the first TMS leads the cytosol-synthesized Cox2 to aggregate at the mitochondrial periphery or to be prematurely released by TIM23 into an incorrect topology. TOM: translocase of the outer membrane, TIM: translocase of the inner membrane, MPP: mitochondrial processing peptidase, LP: leader peptide, MTS: mitochondrial targeting sequence, TMS: transmembrane segment.  mature form. In line with the absence of respiratory growth of the strains carrying the COX2 WT construct, the Cox2 WT protein was observed only as a precursor and, at low dosage, its steady-state level was probably extremely low, as we could not detect it under our experimental conditions ( Figure 4B). We observed the same behavior, that is, enhanced respiratory growth, lower accumulation of precursors, and higher levels of mature forms of Cox2, when the same constructs were expressed in few copies from a centromeric plasmid ( Figure 4C and unpublished data). To test the importance of high versus low doses of Cox2 for the rescue of respiratory growth, we increased the dosage by producing the high-copy Cox2 variants (p) in a strain already encoding a single-copy Cox2 in the nuclear genome (n). We observed that this strain (n+p) grew in respiratory medium to levels similar to those of the strain expressing Cox2 encoded only in the nucleus ( Figure 4C) and verified the presence of the precursors by immunoblotting (unpublished data). Unexpectedly, these increased levels of expression did not show the same growth defect of the high-copy Cox2 strain. However, when we inverted the order of production of allotopic Cox2 variants in the cells by integrating Cox2 into the nuclear genome in a strain already encoding a high-copy Cox2 (p+n), we observed that this strain showed the same level of respiratory growth as the strain expressing a high-copy Cox2 from a plasmid ( Figure 4C). The aforementioned results suggest that, although overexpression of allotopic COX2 constructs is able to restore respiration to cox2-null strains, directing high doses of these proteins to mitochondria at the same time is counterproductive. Because the limiting step resides in the import of allotopic Cox2, low-dose expression seems to have a better outcome in terms of aggregation propensity and recovery of the respiratory growth.

DISCUSSION
The experimental relocation of mitochondrial genes to the nucleus has paved the way for the development of therapies intended to treat syndromes caused by mutations in the mitochondrial genome (González-Halphen et al., 2004;Taylor and Turnbull, 2005;Kyriakouli et al., 2008;Calvo and Mootha, 2010;Gorman et al., 2016). Ongoing research has unveiled several constraining factors that limit allotopic expression (Claros et al., 1995;Oca-Cossio et al., 2003;Figueroa-Martínez et al., 2011;Björkholm et al., 2017). Because most proteins encoded in the mitogenome are integral to the membrane, the high hydrophobicity and the number of TMSs in these proteins seem to be major limiting factors for their import when synthesized in the cytosol (Claros et al., 1995;Johnston and Williams, 2016). In this work we also found that aggregation propensity, which is directly correlated with high hydrophobicity (Chiti et al., 2003;Dobson, 2004), contributes to the efficiency of importability before assembly of allotopic Cox2 into cytochrome c oxidase. Average hydrophobicities for the first TMSs of the different Cox2 variants used in this work are shown in Table 1.
First, we turned our attention to the Cox2 subunit to examine the importance of the residues enabling its allotopic production. Previous work demonstrated that substituting a tryptophan for an arginine decreases the hydrophobicity of the first TMS of Cox2 by adding a positive charge (Supekova et al., 2010). In this study, substitution of a lysine or glutamine in the same position also allowed recovery of the respiratory growth, but residues containing a positive charge (arginine and lysine) yield better rescue than a residue decreasing the hydrophobicity (glutamine). It is possible that Cox2 W56Q is poorly inserted into the mitochondrial inner membrane, because the key determinant for importability is the presence of a positive charge (R + or K + ) and not reduction of the hydrophobicity (Q) at this position (von Heijne, 1992;Andersson and von Heijne, 1994) (see Table 1). This view is supported by the fact that while a similar number of Cox2 variant precursors accumulate in the mitochondria, the mature form of Cox2 W56Q is significantly less abundant than those of Cox2 W56R and Cox2 W56K ( Figure 1B). This indicates that Cox2 W56Q is probably limited in its insertion in the inner membrane because it is more prone to aggregation. That mature Cox2 W56K and Cox2 W56R accumulate at the same level reinforces the view that a positive charge at this position is the key determinant of Cox2 importation into mitochondria when it originates from the cytosol.
Here, we designed allotopic Cox2 variants in yeast based on naturally evolving alterations in legumes. Notably, we observed that the double substitution reported as key for nuclear expression of Cox2 in Glycine max also allowed the allotopically expressed yeast COX2 to restore respiration to a cox2-null mutant. Similarly to what was observed in in vitro import assays in legumes (Daley et al., 2002b), each single-substitution V49Q or L51G was insufficient to restore respiration. In line with the cytosol-synthesized Cox2 biogenesis model (Daley et al., 2002a;Jiménez-Suárez et al., 2012;Rubalcava-Gracia et al., 2018), the first TMS (TMS1) of Cox2 is transferred to the matrix through the TIM23 translocase, while the second TMS is laterally released into the inner membrane by TIM23 ( Figure 4D). As a final step, TMS1 is inserted from the matrix in the inner membrane by Oxa1, positioning Cox2 in its correct topology ( Figure 4D). We assume that in the presence of either substitution (V49Q or L51G), the proteins aggregate at the mitochondrial periphery before import because they are too hydrophobic ( Figure 4D and Table 1). Accordingly, only the double substitution, which diminishes the overall hydrophobicity of the TMS1 of Cox2, allows its correct insertion into the inner membrane.
It should be mentioned that the legume Cox2 substitutions (V49Q/L51G) are less efficient in conferring respiratory growth than the W56R that was identified through random mutagenesis (Supekova et al., 2010). Indeed, the W56R substitution meets Goldilocks conditions: besides maintaining CcO activity intact (Rubalcava-Gracia et al., 2018), it balances the need to diminish the hydrophobicity of Cox2 TMS1, but at the same time, it maintains the ability of TMS1 to be recognized as a transmembrane domain by the inner membrane translocator Oxa1. Notably, in the mitochondrial Cox2 sequence from Glycine max, the residue corresponding to position 56 contains an arginine, indicating that a positive charge in the first TMS of Cox2 is permissible, at least in some organisms. Production of low levels of Cox2 V49Q/L51G results in the accumulation of mature Cox2 at a level similar to that of Cox2 W56R , suggesting that insertion in the inner membrane occurs to the same extent for both Cox2 variants ( Figure 4B). However, the fact that the rescue with Cox2 V49Q/L51G is significantly less efficient indicates that the V49Q/ L51G modifications impact the biogenesis of Cox2 or the activity of the yeast enzyme. Furthermore, we observed that the combination of W56R substitution with the V49Q and L51G changes did not further improve respiratory growth, and resulted in lower CcO activity, as observed in Figure 2C. We assume that, despite the fact that these mutations enable respiratory growth, they may also affect CcO activity. Another possibility is that the hydrophobicity of the first TMS of the Cox2 L47Q/V49G/W56R subunit is lowered to such an extent ( Table 1) that it is not promptly recognized as a transmembrane domain and is therefore only partially routed for insertion into the inner membrane by the Oxa1 translocator.
Our experiments indicate that allotopically expressed proteins face a limiting step by aggregating before their import into mitochondria. When we lowered the COX2 expression levels by integrating the COX2 gene into the nuclear genome, we observed a notable increase in the rescue of the cox2-null strain, which correlated with lower levels of precursor and corresponding enhanced levels of mature Cox2. It is conceivable that excess cytosol-synthesized Cox2 aggregates at the mitochondrial surface, limiting the import of allotopic Cox2 ( Figure 4D). These aggregates do not affect the import of other mitochondria-targeted proteins, as the precursors of Oxa1 and Atp2 were not observed to accumulate in our mitochondrial preparations ( Figures 1B, 3A, and 4B). We noted that aggregation can also be avoided if large doses of Cox2 are produced in a strain already synthesizing small doses of allotopic Cox2. We explain this observation by the importing competence of cox2-null mitochondria. If large doses of the precursors of Cox2 are directly produced in a cox2-null mutant, most precursors aggregate and only a fraction assemble into CcO, resulting in difficulties in restoring respiration and in building up the electrochemical gradient required for further protein import. At small doses, aggregation of Cox2 is prevented and its assembly into CcO is enhanced, giving rise to an optimal transmembrane gradient that facilitates further import of cytosolic proteins. These results suggest that expression levels of the allotopic gene (from a centromeric plasmid or a high-copy plasmid) define the levels of importability versus aggregation of Cox2. It is also conceivable that cytosolic chaperones and other aggregation-response proteins might play a role in preventing the aggregation of Cox2 precursors.
Aggregation propensity is directly related to hydrophobicity and has been an underinvestigated factor in the study of allotopic expression. Highly hydrophobic proteins imported from the cytosol must avoid aggregating at the mitochondrial periphery to cross both mitochondrial membranes. All reports of allotopic expression in either yeast or cell cultures use high-level expression plasmids to ensure a high accumulation of the desired protein products inside the mitochondria Manfredi et al., 2002;Ojaimi et al., 2002;Bokori-Brown and Holt, 2006;Bonnet et al., 2008;Figueroa-Martínez et al., 2011;Cwerman-Thibault et al., 2015;Boominathan et al., 2016). Here, we found that a high level of the allotopically expressed Cox2 protein may be detrimental to the mitochondrial import system.
In summary, we provide a proof of principle that mimicking the modifications found in an organism that naturally transferred its mitochondrial gene to the nucleus can result in successful allotopic expression. This strategy includes expressing a gene with adequate mutations in a low copy number to ensure that moderate protein quantities are produced for mitochondrial import. Our results underscore that dosage, a previously unexplored important parameter, may be critical for allotopic expression in mammals, especially in human cell lines. The relevance of our finding is high, since allotopic expression still represents a promising strategy for developing treatments for patients with mitochondrial diseases. Our data also stress the relevance of studying organisms that naturally and atypically encode hydrophobic mitochondrial OXPHOS proteins in the nuclear genome.
The yeast allotopic COX2 W56R gene was recoded for expression from the nucleus and a sequence encoding the MTS of Oxa1 was added at the 5′ end, as previously described (Supekova et al., 2010). The construct was chemically synthesized (GeneScript, Piscataway, NJ) and cloned at the NotI site in the pFL61 vector, which contains a phosphoglycerate kinase (PGK) promoter and terminator (Minet et al., 1992).
Additional COX2 mutations (WT, W56K, W56Q, V49Q, L51G, V49Q/L51G, L47Q/V49G, L47Q/V49G/W56R) were constructed using the pFL61 plasmid containing the COX2 W56R insert via site-directed mutagenesis, according to the manufacturer's instructions (QuikChange II site-directed mutagenesis kit, Agilent Technologies). The presence of the site-directed mutations was confirmed by DNA sequencing. For the expression of the constructs from centromeric plasmids, the fragments containing the COX2 constructs flanked by the PGK promoter and terminator were cloned from pFL61 into pRS306H upon digestion with HindIII and SmaI restriction enzymes. Nuclear localization was achieved by integration transformation at the URA3 nuclear locus using the pRS306H plasmid and hygromycin B resistance as a selection (Taxis and Knop, 2006).
Yeast cells were transformed in the presence of lithium acetate and salmon sperm DNA as described (Gietz and Schiestl, 2007). At least 12 transformed individual colonies were grown in Petri dishes containing fresh SD medium and then were replica-plated into YPEG to ensure that the selected colonies had respiratory growth similar to the rest of the transformants.

Isolation of yeast mitochondria
Mitochondria were isolated from 100-200 ml of liquid SGal cultures as previously reported (Herrmann et al., 1994). Briefly, cells were harvested at logarithmic growth phase (O.D. 600 0.8-1.0), washed with H 2 O at 4500 rpm in a Beckman Coulter JA-25.5 rotor (2500 × g) for 5 min, resuspended in TD buffer (100 mM Tris, 10 mM dithiothreitol), and centrifuged at 4500 rpm (2500 × g) for 5 min. The cells were resuspended in 10 ml of zymoliase buffer (1.2 mM sorbitol, 20 mM KH 2 PO 4 [pH 7.4], 3-5 mg zymoliase/g of wet weight), incubated for 1 h in a shaker at 30°C, and centrifuged at 4500 rpm (2500 × g) for 5 min at 4°C. The pellet was resuspended in 1 ml of Dounce buffer (0.6 M sorbitol, 10 mM Tris [pH 7.4], 1 mM EDTA, 0.2% bovine serum albumin, 1 mM phenylmethylsulfonyl fluoride [PMSF]) using a homogenizer and pestle (30 strokes). Samples were transferred to 1.5-ml tubes and centrifuged at 4500 rpm in a microcentrifuge (2000 × g) for 5 min at 4°C. The supernatants were transferred to new tubes and centrifuged at 12,000 rpm (13,500 × g) for 10 min at 4°C. The supernatant (cytosolic fraction) was collected in new tubes and the pellet (mitochondrial fraction) was resuspended in 100 µl of SH buffer (0.6 M sorbitol, 20 mM HEPES, pH 7.4). Protein concentration was determined by a modified Lowry method (Markwell et al., 1978). Mitochondria and cytosolic fractions were frozen and stored at -70°C until use.

Proteinase K protection assays
For protease treatment of mitochondria, 50 µg of mitochondria was gently resuspended in 100 µl of SH buffer and incubated at 20°C for 30 min in the presence of 100 µg/ml Proteinase K; parallel reactions were made in the presence of 1% Triton X-100. PMSF was added to all suspensions (at 4 mM final concentration), which were then incubated on ice for 10 min and centrifuged at 14,000 rpm (18,400 × g) for 10 min. Most of the supernatant in each tube was discarded; the pellet was resuspended with 4 µl of 6× loading buffer (375 mM Tris-HCl [pH 6.8], 6% SDS, 48% glycerol, 9% β-mercaptoethanol) + 4 mM PMSF and incubated for 1 min in boiling water before being loaded onto 12% SDS-tricine-PAGE gels.

Sodium carbonate and Triton X-100 extractions
Integral membrane proteins were separated from peripheral or soluble proteins by carbonate extraction. Aggregated proteins were separated from membrane proteins by Triton X-100 extraction. Briefly, 50 µg of mitochondria was incubated with 100 µl of cold 0.1 M Na 2 CO 3 (pH 11.0) or with 9.4 µl of 1% Triton X-100 (2 mg Triton X-100/mg protein) for 30 min on ice. The samples were centrifuged at 90,000 × g in a TLA 55 rotor for 30 min at 4°C. Supernatants and pellets were incubated with 500 µl of 12.5% TCA for 15 min on ice, followed by centrifugation for 15 min at 14,000 rpm (18,400 × g) at 4°C. Samples were washed by adding 500 µl of cold 100% acetone, centrifuged for 10 min at 14,000 rpm (18,400 × g) at 4°C, and dried for ∼10 min at room temperature. The dried pellets were resuspended in 15 µl of 1× loading buffer and incubated for 1 min in boiling water before loading onto 12% SDS-tricine-PAGE gels.

Gel electrophoresis and in-gel enzymatic activities
Denaturing gel electrophoresis was carried out via SDS-tricinepolyacrylamide gels with 12% acrylamide (Schagger, 1994a). Sample preparation and blue native polyacrylamide gel electrophoresis (BN-PAGE) were carried out as described (Schagger, 1994b). Briefly, mitochondria were washed twice in 250 mM sorbitol, 50 mM Bis-Tris (pH 7.0), and centrifuged at 12,000 rpm (13,500 × g) for 10 min at 4°C. The pellet was resuspended in sample buffer (750 mM aminocaproic acid, 50 mM Bis-Tris, pH 7.0) and solubilized with 2 g of lauryl maltoside per g of protein for 30 min with gentle stirring and centrifuged at 13,200 rpm (16,400 × g) at 4°C for 12 min. The supernatants were loaded on 5-15% polyacrylamide gradient gels. The stacking gel contained 4% (wt/vol) polyacrylamide. In-gel activities were carried out following established procedures for CcO activity  and ATPase activity (Zerbetto et al., 1997;Wittig and Schägger, 2005). Gels were stained with Coomassie solution (50% ethanol, 10% acetic acid, 0.1% Coomassie Brilliant Blue G) or otherwise transferred and then subjected to immunodetection.

In silico protein sequence analysis
A TMS1 of 24 residues was considered for the yeast Cox2. The hydrophobicity calculations were performed using the "biological" hydrophobicity scale (Calado-Botelho et al., 2011).