XU Kui,ZHOU Yan-rong,SHANG Hai-tao,XU Chang-jiangTAO Ran,HAO Wan-jun,LlU Sha-shaMU Yu-lian#,XlAO Shao-bo#,Ll Kui#

1 State Key Laboratory of Animal Nutrition/Key Laboratory of Animal Genetics,Breeding and Reproduction of Ministry of Agriculture and Rural Affairs,Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193,P.R.China

2 Shenzhen Kingsino Technology Co.,Ltd.,Shenzhen 518106,P.R.China

3 State Key Laboratory of Agricultural Microbiology/Key Laboratory of Preventive Veterinary Medicine in Hubei Province,College of Veterinary Medicine,Huazhong Agricultural University,Wuhan 430070,P.R.China

4 Precision Medicine Institute,The First Affiliated Hospital,Sun Yat-sen University,Guangzhou 510080,P.R.China

Abstract Porcine reproductive and respiratory syndrome (PRRS) is recognized as one of the most infectious viral diseases of swine. Although Cluster of differentiation 163 (CD163) is identified as an essential receptor for mediating PRRS virus(PRRSV) infection,the important residues involved in infection on CD163 are still unclear. Therefore,it is very important to identify these key residues to study the mechanism of PRRSV infection and to generate anti-PRRSV pigs. In this study,we first generated immortalized porcine alveolar macrophage (IPAM) cell lines harboring 40-residues (residues 523-562,including R561 (arginine (R) at position 561)) deletion of CD163. PRRSV infection experiments showed that these IPAM cell lines were completely resistant to PRRSV infection. We then generated cloned pigs carrying CD163-R561A (an arginine (R) to alanine (A) substitution at position 561 of CD163). PRRSV challenge experiments in porcine alveolar macrophages (PAMs) isolated from the CD163-R561A pigs showed significantly lower susceptibility to PRRSV than that of CD163-R561 PAMs. Through this study,we show that CD163 523-562 contains essential residues for mediating PRRSV infection,and that CD163 R561 significantly contributes to PRRSV infection but is not essential for infection. These functional sites can therefore serve as new targets for understanding the mechanism of PRRSV infection. Furthermore,CD163-R561A pigs can be used as an important model for improving pig germplasm with resistance against PRRSV.

Keywords: pigs,porcine alveolar macrophages,dual-sgRNA,homology-directed repair,PRRSV,CD163

1.lntroduction

Porcine reproductive and respiratory syndrome (PRRS)is a highly pathogenic infectious disease caused by PRRS virus (PRRSV),mainly characterized by sow reproductive disorders and dyspnea in pigs of all ages(Wensvoortet al.1991;Anet al.2020). PRRS has been reported in pig farms globally,threatening huge annual economic losses to the pig industry,and for which there are currently no effective management strategies (Dhakal and Renukaradhya 2019;Anet al.2020). PRRSV can infect differentiated blood monocytes,porcine alveolar macrophages (PAMs),and Cluster of differentiation 163(CD163)-positive dendritic cells (DCs) (Lunneyet al.2016;Resendizet al.2018). Heparin sulphate (HS),sialoadhesin (Sn),and CD163 proteins on the PAM cell membrane are reported to be the three relevant receptors for PRRSV (Crockeret al.1986;Jusaet al.1997;Calvertet al.2007;Breedamet al.2010;Shiet al.2015). Among them,CD163 has been found to be an essential receptor for PRRSV infection (Calvertet al.2007;Pattonet al.2009;Gorpet al.2010a).

CD163 is a～130 kD transmembrane protein,consisting of an extracellular region with 9 tandem scavenger receptor cysteine-rich (SRCR) domains,a transmembrane region,and an intracellular region (Kowalet al.2011;Wellset al.2017) (Fig.1-A). Using several porcine CD163 mutants to study the relationship between various domains and PRRSV infection,Gorpet al.(2010b)found that SRCR domain 5 (SRCR5) of porcine CD163 is essential for successful PRRSV infection. Gene-edited pigs targeting CD163 SRCR5 are resistant to PRRSV infection (Whitworthet al.2016;Yanget al.2018;Xuet al.2020). The generation of PRRSV resistant pigs by deletion or replacement of CD163 SRCR5 also indicated that CD163 SRCR5 contributes an indispensable role in PRRSV infection (Burkardet al.2017;Wellset al.2017).

In addition to serving as a PRRSV receptor,CD163 has a variety of other biological functions (Onofreet al.2009;Gorpet al.2010a). Complete inactivation of CD163 may affect the normal physiological health of pigs.Several studies have explored the structure of CD163 in order to obtain resistance to PRRSV while minimizing the impact on the normal physiological function of CD163.For example,the CD163 SRCR5 domain was replaced with human CD163-Like SRCR8 domain to generate PRRSV-1 resistance (Wellset al.2017). Macrophages from genome-edited pigs lacking the CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function (Burkardet al.2017).Structural analysis suggests that the ligand-binding pocket(LBP) (residues 487-499) and loop 5-6 (residues 544-570) within the SRCR5 domain are involved in PRRSV infection (Graversenet al.2002;Gorpet al.2010b;Welch and Calvert 2010). Gene-edited pigs with a deletion of a 41-residue fragment (residues 481-521),containing the LBP within the SRCR5 domain,were completely resistant to PRRSV-2 infection (Guoet al.2019). However,whether the destruction or deletion of loop 5-6 can inhibit PRRSV infection has not been reported. R561 is located in the loop 5-6 region (Fig.1-A) and experiments in PK-15 cells showed that R561A cells have significantly enhanced anti-infection ability against PRRSV (Maet al.2017),but whether the precise mutation of R561 in PAMs (a target cell of PRRSV) or pigs can inhibit PRRSV infection remains unknown.

Therefore,to explore the interaction mechanism between PRRSV and CD163 SRCR5,and to protect the normal biological function of CD163 to the greatest extent possible when editing its nucleotide sequence to abolish its interaction with PRRSV,it is necessary to identify the key residues that mediate PRRSV infection.

In this study,we used a dual-single guide RNA (sgRNA)system to obtain IPAM cell lines with a 40-residue deletion(CD163Δ523-562) that included part of the loop 5-6 region and R561,which exhibited complete resistance to PRRSVWUH3 (a subtype strain of PRRSV-2) infection. We also used homology-directed repair (HDR)-mediated recombination to obtain cloned pigs with a CD163-R561A non-synonymous substitution. CD163-R561A PAMs isolated from these pigs exhibited significantly decreased susceptibility to PRRSV-WUH3 infection compared to PAMs from wild-type (WT) pigs. These results demonstrated that CD163 residues 523-562 play an indispensable role in mediating PRRSV infection,and that residue R561,in particular,is involved in PRRSV infection.

2.Materials and methods

2.1.Materials

Fig. 1 Generation of IPAM cell lines with CD163 lacking residues 523-562 (CD163Δ523-562). A,functional domains in SRCR5 region of CD163 that participate in porcine reproductive and respiratory syndrome virus (PRRSV) infection and the map of CD163 exon 7 with residues 523-562 targeted for deletion by CRSPR-Cas9 dual-sgRNA system. The extracellular region of pig CD163 has nine tandem SRCR domains,CD163 SRCR5 is encoded by the 7th exon. The Ligand-binding pocket (LBP,yellow),the loop 5-6 region (blue),and residue R561 (red) in loop 5-6 reportedly mediates the PRRSV infection process. Exons,black boxes;SRCR domains,white ovals;sgRNA recognition sequences,green;sg-PAM sequences,black frame;predicted 120 bp/40 amino acid residues deletion,gray shading;predicted Cas9 nuclease cut sites,purple arrowhead. B,flow cytometry analysis to enrich for CD163-sg1-pX330-GFP and CD163-sg2-pX330-RFP co-transfected cells. C,PCR-based genotyping for detection of fragment-deleted colonies. Red stars indicate clones carrying the deletion used for subsequent infection experiments. D,genotype confirmation by Sanger sequencing. All three colonies were homozygous for the 120-bp deletion.

Fig. 2 Deletion of residues 523-562 from CD163 confers complete resistance to porcine reproductive and respiratory syndrome virus (PRRSV)-WUH3 infection. A,IPAM cell lines carrying either CD163 lacking residues 523-562 (CD163Δ523-562) or wild-type(WT) CD163 were infected with PRRSV-WUH3 (MOI=0.5). Indirect immunofluorescence assay (IFA) was performed to detect PRRSV replication in IPAM cell lines at 24 h post-infection (hpi) using anti-PRRSV-N protein mouse monoclonal antibody (green)and DAPI to detect nuclei (blue). B,IPAM cell lines with CD163Δ523-562 and WT CD163 IPAM cell lines were infected with PRRSVWUH3 (MOI=0.5). At 24 hpi,cells and cell supernatant were harvested,and virus titers were determined by viral plaque assay.Cells harvested in (B) were used for (C) qRT-PCR detection of relative PRRSV mRNA expression,and (D) Western blot detection of PRRSV-N protein accumulation. Data are expressed as mean±SD for triplicate samples. Statistical significance was determined by Student’s t-test: ＊＊,P＜0.01;＊＊＊,P＜0.001.

PRRSV-WUH3 (GenBank: HM853673) was isolated and preserved in our laboratory (Liet al.2009). IPAM cell lines were kindly donated by Dr.Cai Xuehui (State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences) (Wanget al.2018). PRMI-1640 medium was purchased from Hyclone (China).Dulbecco’s modified Eagle’s medium (DMEM) was purchased from GIBCO (USA). Fetal bovine serum(FBS) was purchased from GIBCO (Australia). Penicillinstreptomycin (PS) was purchased from GIBCO (USA).Collagenase IV,and DNaseI were purchased from Sigma(USA). Cell culture plates were purchased from Costar(USA). The pX330 plasmid (#42230) was purchased from Addgene (USA),pX330-GFP (VK001-02),and pX330-RFP (VK001-04) were purchased from ViewSolid Biotech (China). SYBR green real-time PCR master mix was purchased from Applied Bio-Systems (USA).NaeI endonuclease was purchased from New England Biolabs (NEB) (USA). For Western blotting,the anti-PRRSV-N antibody was made in our laboratory (Jianget al.2010). β-Actin rabbit antibody was purchased from Abclonal Technology. HRP-conjugated affinipure goat antirabbit IgG (H+L) was purchased from Proteintech (China).For indirect immunofluorescence assays,anti-PRRSV-N monoclonal antibody was made in our laboratory (Zhouet al.2017);Alexa Fluor 488-conjugated anti-mouse antibodies was purchased from Invitrogen (USA).

2.2.CRlSPR construction

The sgRNA sequences in this experiment were designed using web-based software (http://crispor.tefor.net). We selected two sgRNA sequences (CD163-sg1 andCD163-sg2) to disrupt loop 5-6 of CD163 SRCR5. The distance between the two sgRNA cleavage sites was 120 bp.We also selectedCD163-sg2 (the sgRNA closest to the R561 site) for R561A precise editing.CD163-sg1 andCD163-sg2 were cloned into pX330-GFP (ViewSolid Biotech,China) and pX330-RFP (ViewSolid Biotech,China) vectors,respectively,following the instructions accompanying the kits. These two plasmids were namedCD163-sg1-pX330-GFP andCD163-sg2-pX330-RFP. To prevent the insertion of fluorescent reporter genes into the pig genome,CD163-sg2 was also cloned into the pX330 vector (Addgene,USA) according to the method described by Conget al.(2013),and the resulting plasmid was namedCD163-sg2-pX330. Based on theCD163-sg2 sequence,we also designed an singlestranded oligodeoxyribonucleotides (ssODN) donor sequence,designated CD163-R561A-ssODN. When HDR was activated,the donor sequence replaced the WT sequence,and R561 was successfully replaced with A561 (alanine (A) at position 561). At the same time,aNaeI restriction site was also introduced into the donor for colony genotyping. All of the sgRNA sequences and ssODN sequences are listed in Table 1.

Table 1 The sgRNA sequences and ssODN sequences1)

2.3.Cell selection and genotyping

For the selection of CD163Δ523-562IPAM cell lines,IPAM cell lines were maintained in PRMI-1640 supplemented with 10% FBS,and 1% PS at 38.5°C and 5% CO2in a humidified incubator (Thermo Scientific,USA). One day before transfection,IPAM cell lines were seeded into 6-well plates. When the cells were 70-80% confluent,they were co-transfected withCD163-sg1-pX330-GFP andCD163-sg2-pX330-RFP (at a ratio of 1:1) using Lonza 2B nuclear transfection system (Lonza Amaxa,Switzerland) following the manufacturer’s protocol. At 48 h post-transfection,cells expressing both GFP and RFP were sorted into 96-well plates by flow cytometry (Beckman,Germany) for culturing cell colonies. In order to detect the genotype of each line,we used the primer pairCD163-406-F2/CD163-406-R2 (Appendix A) for PCR amplification. Several colonies that showed a single band in gel electrophoresis screens,indicating the fragment was successfully deleted,were chosen for Sanger sequencing. The homozygous cell lines from isolated colonies that had precise deletions of the 120 bp fragment were then grown as above and used for subsequent experiments.

The fetuses of Large White pigs at 35 days old were used to isolate porcine embryonic fibroblasts (PEFs) as described by Liet al.(2014). Briefly,the embryo was removed and the head,limbs,and internal organs were minced and digested with collagenase-DNase in DMEM supplemented with 10% FBS,1.0% PS (GIBCO,USA),0.5 mg mL-1Collagenase IV,and 100 kU mL-1DNase I for 4-5 h at 37°C. The cells were then cultured in DMEM,supplemented with 15% FBS at 38.5°C and 5.0% CO2in a humidified incubator. In order to screen for PEFs carrying the CD163-R561A substitution,PEFs were seeded onto 6 cm plates one day before transfection.When the cells were 70-80% confluent,they were cotransfected withCD163-sg2-pX330 (5 μg) plasmid and ssODN donor (2 μg) using NucleofectorTMfollowing the manufacturer’s protocols (Lonza Amaxa,Switzerland).At 48 h post-transfection,PEFs were seeded into 10-cm dishes at a density of 150 cells per dish. Colonies derived from single cells grew after 10 days of culture.We usedCD163-406-F2 andCD163-406-R2 primers for PCR-based screening.NaeI was used to identify the HDR allele from WT and non-homologous end joining(NHEJ) alleles. Positive clones were then sequenced to confirm the genotype. PEFs carrying the intended R561A mutation were then used in subsequent somatic cell nuclear transfer (SCNT) experiments.

2.4.Generation of CD163-R561A gene-edited pigs

Pig ovaries were collected from the local slaughterhouse of the laboratory and quickly transported to the laboratory in 0.9% NaCl (Sigma,USA) at 37°C.Cumulus-oocyte complexes (COCs) were aspirated from follicles with a vacuum pumping system. COCs were selected and cultured in maturation medium for 40 h at 38.5°C,and then digest by 0.1% hyaluronidase(Sigma,USA). The matured pig oocytes were used as nuclear transfer recipient cells,and the obtained R561A PEFs were used as nuclear transfer donor cells.The nuclear transfer donor cells were transferred into enucleated oocytes,and the reconstructed embryos were fused and activated with two successive dendritic cell pulses at 130 V for 30 μs using an electrofusion instrument (BLS,Hungary). The reconstructed embryos were cultured in the porcine zygote medium-3 (PZM-3)for overnight at 38.5°C under 5% CO2. Embryos were surgically transferred into the oviduct of a surrogates that were observed in estrus the day before. After embryo transfer,we periodically checked the pregnancy status of the recipient sows.

2.5.Off-target assay

Potential off-target editing sites were predicted using an online software toolkit (CRISPOR,http://crispor.tefor.net/),and sites with the highest off-target score were selected for further analysis. We identified 10 potential off-target sites forCD163-sg2 and designed 10 primer pairs (Appendix A) to amplify and sequence them from the genomic DNA isolated from cell line #9 for determination of off-target editing activity.

2.6.lsolation of porcine-derived PAMs

After the piglets were euthanized,the lung tissue was taken to isolate PAMs. The surface of the lung tissue was washed with phosphate buffered saline (PBS) (GIBCO,USA). PRMI-1640 medium was injected into the lungs and kneaded gently. The injected liquid was then poured out and the lavage was repeated. The collected lavage solution was dispensed into 50-mL tubes and centrifuged at 300×g for 10 min. The supernatant was discarded,and PAMs were collected and frozen at -80°C.

2.7.Virus infection of lPAM cell lines and porcine-derived PAMs

IPAM cell lines (4×104) and porcine-derived PAMs (2×105)were seeded in 48-well plates the day before infection.PRRSV-WUH3 (subtype strain of PRRSV-2,GenBank accession no.HM853673) (Liet al.2009) was selected forinvitroinfection at the multiplicity of infection (MOI)of 0.5. At 24 h post-infection (hpi),the supernatant and cells were collected. The PRRSV titers in cell culture supernatants were detected by viral plaque assays. Cells were lysed with TRIzol to extract total RNA for qRT-PCR detection of PRRSV virus copy number.

2.8.lndirect immunofluorescence assay (lFA)

IPAM cell lines infected with PRRSV were fixed with 4% paraformaldehyde (Sinopharm Chemical Reagent,China) for 15 min and immediately permeabilized with precooled methanol (Sinopharm Chemical Reagent,China) for 10 min. After blocking with 5% bovine serum albumin (BSA) (Sigma,USA),the cells were incubated with an anti-PRRSV-N MAb (1:150,made in our laboratory (Zhouet al.2017)) for 1 h at room temperature. The cells were then stained with Alexa Fluor 488-conjugated anti-mouse antibodies (1:300)(Invitrogen,USA) according to the manufacturer’s protocol. Images were collected with an Olympus IX73 Inverted Microscope (Olympus,Japan).

2.9.Viral plaque assay

Plaque assays were performed essentially as described previously (Wanget al.2013). Briefly,95% confluent Marc-145 cells grown in six-well tissue culture plates were infected for 1 h with 10-fold serial dilutions (1 000 μL)of PRRSV-containing samples. After the virus was adsorbed,the virus suspension was aspirated. After three washes with PBS,low melting point agarose was added to the cells,followed by incubation at 38.5°C under 5%CO2for a further 2 days to detect the plaque number.

2.10.qRT-PCR assay

To quantify PRRSV copy number,we extracted virus RNA from PRRSV-infected IPAM cell lines or PAMs. RNA extraction was performed using TRIzol reagent (Omega Bio-Tek,USA). The RNA was reverse transcribed into cDNA according to the instructions of the Transcriptor First Strand cDNA Synthesis Kit (Roche,Switzerland).The cDNA was then amplified with SYBR Green Real-Time PCR Master Mix (Applied Biosystems,USA) in an ABI 7500 Real-Time PCR System (Applied Biosystems,USA). The primers used for qRT-PCR (qRT-PCR-F/qRTPCR-R) are listed in Appendix A.

2.11.Western blotting

Whole cell lysates of PRRSV-infected IPAM cell lines or PAMs were used to quantify the expression levels of PRRSV nucleocapsid (PRRSV-N) protein. The protein samples were separated by 8% SDS-PAGE and transferred to a polyvinylidene fluoride membrane(Millipore,USA). The membrane was blocked with 5% skim milk (Becton Dickinson,USA) for 2 h,and then incubated with primary antibody at 4°C overnight and secondary antibody at room temperature for 2 h. Chemiluminescent signals were developed with SuperSignalTMWest Pico Plus Chemiluninescent Substrate (Thermos Scientific,USA) and captured with a Tanon-520 (Tanon,China). Anti-PRRSV-N antibody(made in our laboratory) was used to detect PRRSV-N protein (Jianget al.2010);β-actin rabbit antibody(Abclonal Technology,USA) was used to stain β-actin as a loading control. HRP-conjugated affinipure goat antirabbit IgG (H+L) (Proteintech,China) was used as the secondary antibody.

2.12.Statistical analysis

All data are presented as mean±standard deviation(SD). Data from each of groups were compared with an unpairedt-test when a normal distribution was not obtained. The significance levels were set at 0.05,0.01,and 0.001,as indicated by＊,＊＊,and＊＊＊,respectively.Statistical analysis was performed with GraphPad Prism 6(GraphPad Software,La Jolla,USA).

3.Results

3.1.Generation of lPAM cell lines with CD163 lacking residues 523-562 (CD163Δ523-562)

We selected two sgRNAs targeting sites near loop 5-6 to mediate guide CRISPR/Cas9 modification.Together,these sgRNAs direct the excision of a 120 bp sequence corresponding to 40 residues for disrupting loop 5-6 region,including R561 (Fig.1-A). Vector constructs with each respective sgRNA fused with a red or green fluorescent reporter,CD163-sg1-pX330-GFP andCD163-sg2-pX330-RFP,were co-transfected into the IPAM cell lines for enrichment by flow cytometry(Fig.1-B),and the enriched cells were then placed in 96-well plates for clonal culturing. A total of 20 colonies were harvested in this experiment,and bands indicating the presence of the deletion were found in 19 colonies(Fig.1-C). Nineteen of the 20 colonies (95%) we identified showed obvious fragment deletions. We then picked nine colonies (#3,#4,#5,#8,#10,#14,#18,#19,and #20) that were indicated by genotyping to carry the deletion for further confirmation by Sanger sequencing.Five (#3,#4,#8,#10,and #20) of these 9 colonies (60%)were homozygous for the correct fragment deletion(-120 bp/-120 bp),and 3 (#3,#10,and #20) of these 5 cell lines were selected for subsequent virus infection experiments (Appendix B;Fig.1-D).

3.2.Deletion of residues 523-562 from CD163 confers complete resistance to PRRSV-WUH3 infection

Highly pathogenic strain PRRSV-WUH3 was used to infect three monoclonal IPAM cell lines carrying the deletion of CD163 residues 523-562 (CD163Δ523-562),as well as one WT IPAM cell line as a control. Cells were infected with PRRSV at an MOI of 0.5,and immunofluorescent stain targeting PRRSV-N protein was detected to track PRRSV replication in cells. The results showed no detectable PRRSV-N protein in any of the three CD163Δ523-562cell lines,while PRRSV-N protein accumulated to obviously high levels in WT cells (Fig.2-A). To further confirm that IPAM cell lines harboring the CD163Δ523-562are resistant to PRRSV infection,we next conducted viral plaque assays,qRT-PCR relative expression assays,and western blot analysis to measure viral titers,as well as viral RNA and protein levels for comparison between WT and mutant infected IPAM cell lines. The results confirmed that PRRSV transcripts and protein were undetectable in CD163Δ523-562IPAM cell lines,but were found in WT IPAM cell lines (Fig.2-B-D). These experiments strongly suggested that the CD163Δ523-562IPAM cells lacking CD163 residues 523-562 are completely resistant to PRRSV-WUH3 infection.

3.3.Generation of cloned pigs carrying CD163-R561A non-synonymous substitution

In light of our results that showed CD163Δ523-562IPAM cell lines exhibited high resistance to PRRSV infection,and that residue R561 was previously found to be an important residue for PRRSV infection in the PK-15 cells,we thus hypothesized that pigs carrying a different residue at this site,that otherwise retained CD163 function but abolished PRRSV recognition,could be resistant to PRRSV infection. We selected sgRNA sequences closest to the R561 site for cloning into the pX330 plasmid. We also synthesized a single-stranded donor oligonucleotide(ssODN) in which the CGC codon encoding R561 was replaced with GCC (A561). Concurrently,we changed the other two bases to prevent secondary cleavage and to introduce a newNaeI (GCCGGC) restriction site,for subsequent genotyping,that identified the CRISPR HDR allele,but not WT or non-homologous end joining (NHEJ)alleles (Fig.3-A).

We then co-transfected PEFs withCD163-sg2 and CD163-R561A-ssODN,and then cultured 133 isolated colonies for further examination of the mutation site.The results of restriction fragment length polymorphism(RFLP) analysis and sequencing of the PCR products showed that a total of four colonies (#2,#6,#9,and#131) carried a precise substitution of R561,among which two (#2 and #6) were heterozygous and two (#9 and #131) were homozygous (Fig.3-B-D). Although the R561 editing site of colony #9 was accurately replaced(CGC→GCC),the base “A” at the fourth upstream position of the site was not replaced by “G”. As a result,the PCR product of the colony #9 sample could not be cleaved due to the lack of theNaeI cleavage site(GCCGGC) (Fig.3-B and C). We then performed offtarget analysis of the cell line derived from colony #9 of 10 potential off-target sites forCD163-sg2. However,Sanger sequencing of these sites revealed no off-target point mutations in the genome of cell line #9 (Appendices C and D). In light of these findings,we used cells from this line as nuclear donors for somatic cell nuclear transfer (SCNT). A total of 10 recipient sows were used and 250 reconstituted embryos were transplanted into each recipient,of which 9 sows gave birth to 17 healthy liveborns and 4 stillborns,among which 6 survived postweaning (Appendix E). No apparent disease or growth abnormalities were observed in these 6 cloned pigs from birth to weaning (Fig.3-E). Genomic DNA from ear tissue of these cloned piglets was extracted forCD163genotyping and sequencing results confirmed that all of the six cloned pigs carried the CD163-R561A mutant genotype (Fig.3-F).

3.4.PAMs isolated from CD163-R561A pigs exhibit significantly decreased susceptibility to PRRSV

PAMs derived from the CD163-R561A mutant and WT pigs were infected with PRRSV-WUH3 (MOI=0.5). At 24 hpi,samples were collected for viral plaque assays to measure virus titer,qRT-PCR relative expression analysis of viral mRNA levels,and Western blots to detect viral protein accumulation in the infected IPAM cell lines from both genotypes. We found that PRRSV titer in CD163-R561A PAMs was about 50% lower than that in WT PAMs(Fig.4-A). Moreover,qRT-PCR and western blot also indicated that viral mRNA copy number and protein levels were significantly decreased in the CD163-R561A PAMs(Fig.4-B-D). These experiments thus demonstrated that pig-derived CD163-R561A PAMs exhibit significantly decreased susceptibility to PRRSV infection. Taken together,these findings showed that the R561 residue was important for PRRSV interaction with CD163 and subsequent infection.

4.Discussion

PRRSV is an enveloped,positive-sense RNA virus.PRRSV strains genetically divided into two types,the European genotype (PRRSV-1) and the American genotype (PRRSV-2) (Kappes and Faaberg 2015).The clinical symptoms induced by the two genotypes are similar,but PRRSV-2 is more infectious and virulent. Currently,most PRRSV prevalent in China is of the PRRSV-2 (Wasilket al.2004). Due to the rapid reorganization and mutation of PRRSV genome,there are now variety types of mutations of PRRSV strains spreading worldwide. Since 2006,the outbreak of HPPRRSV in many provinces of China has caused a devastating blow to the pig industry (Martin-Vallset al.2014). HP-PRRSV is a variant strain of classic PRRSV-2 with extremely high morbidity and mortality (Tianet al.2007;Zhou and Yang 2010). The PRRSV-WUH3 strain used in this study is a HP-PRRSV isolated on a pig farm in China at the end of 2006 (Liet al.2009).

Although studies have shown that CD163 SRCR5 plays a key role in the process of PRRSV infection,for example,pigs with CD163 SRCR5 deleted can completely block the infection of both types of PRRSV(Burkardet al.2017),gene-edited pigs that replace the SRCR5 of pig CD163 with SRCR8 of human CD163-L1 are resistant to PRRSV-1 infection (Wellset al.2017).However,further identification of the domains and amino acid residues in CD163 SRCR5 that mediate PRRSV infection is necessary for studying the mechanism of PRRSV infection and breeding PRRSV-resistant pigs.The two domains of CD163 SRCR5,the ligand-binding pocket (LBP) and loop 5-6,are considered to be involved in the CD163-PRRSV interaction (Graversenet al.2002;Gorpet al.2010b;Welchet al.2010). Pigs with 41 amino acids containing the LBP domain in SRCR5 deleted are fully resistant to PRRSV-2 infection,which proves the importance of LBP (Guoet al.2019). Whether deletion or destruction of the loop 5-6 structure of SRCR5 can inhibit PRRSV infection has not been reported yet. Maet al.(2017) analyzed the structure of porcine CD163 SRCR5 and predicted multiple amino acids in LBP and loop 5-6 that may be involved in PRRSV infection. Compared with the expression of WT CD163,the expression of R561A mutant CD163 in PK-15 can significantly reduce the infectivity of PRRSV (Maet al.2017). Since PK15 is not a natural target cell of PRRSV,and many plasmid copies enter the cell during the transfection process to cause transient high expression of CD163,this cannot accurately simulate the process of PRRSV’s natural infection of cells.Therefore,it is necessary to precisely edit theCD163gene in PRRSV target cells to explore the mechanism of interaction with PRRSV. In this study,we first used the dual-sgRNA CRISPR/Cas9 system to edit the CD163 of IPAM cell lines (immortalized PRRSV’s natural target cell line),and successfully deleted 40 amino acids (residues 523-562) containing part of the loop 5-6 structure in CD163 SRCR5. The deletion of 40 amino acids did not cause a frameshift mutation in CD163,so the structure of other SRCRs was reserved. This CD163Δ523-562IPAM cell lines will be used to study the interaction between loop 5-6 and PRRSV. Furthermore,we used CRISPR/Cas9 HDR system to generate pigs carrying CD163-R561A,and R561A PAMs were isolated to detect susceptibility to PRRSV. CD163Δ523-562IPAM cell lines with deleted residues 523-562,including R561 and part of the loop 5-6 region,exhibited full resistance to PRRSV-WUH3,thus suggesting that some of these 40 residues are essential for mediating PRRSV infection. This finding is consistent with previous studies that proposed the loop 5-6 region in CD163 SRCR5 is involved in the binding of PRRSV (Welchet al.2010;Guoet al.2019). Although previous structural analysis suggested that CD163 R561,in loop 5-6,is a strong candidate residue for mediation of PRRSV infection (Maet al.2017),experimental validation of this role has not been conducted in PAMs (PRRSV target cells) or in pigs until now. In our experiments,we found that porcine-derived R561A PAMs had significantly lower susceptibility to PRRSV-infection than WT,which supported the contribution of R561 to PRRSV infection.However,R561A PAMs do not exhibit the highly resistant phenotype observed in IPAM cell lines carrying the CD163Δ523-562genotype. We thus postulated that among these 40 residues,there are likely to be other contributing residues besides R561 involved in the infection process.However,this possibility requires further detailed investigation.

In a recent publication (Stoianet al.2022),the authors constructed multiple different CD163 mutants and found that SRCR5 and PSTII,as well as SRCR6-9 domains,are required for PRRSV-2 infection. They also identified a specific pentapeptide sequence (AHFGE) present in both CD163 SRCR5 and SRCR7 domains that is critical for PRRSV infection. Additionally,the deletion of the 10 amino acids,which form the distal part of the 16 amino acid PSTII domain,resulted in the complete loss of infection. The mutation region selected for this study covers the entire coding region of CD163. In this study,we mainly focused on the loop 5-6 region of the SRCR5 domain,which was previously thought to be involved in the CD163-PRRSV interaction,and demonstrated that the disruption of loop 5-6 conferred complete resistance to PRRSV-2,and the mutation of R561 located in loop 5-6 could confer PAMs significantly reduced susceptibility to PRRSV. The CD163 mutants found in the study of Stoianet al.(2022) and our study jointly supply informative references to understanding the infection mechanism of PRRSV,and also provide multiple targets for generation of anti-PRRSV gene editing pigs.

5.Conclusion

In summary,we used two CRISPR/Cas9 editing systems to identify CD163 residues involved in PRRSV infection.We determined that CD163 residues 523-562 are essential for PRRSV infection. In contrast,the decrease in susceptibility to PRRSV by porcine-derived PAMs showed that that R561 in CD163 participates in the infection process,but is not solely necessary for infection.Our study provides a valuable reference for exploring the mechanism of CD163-mediated PRRSV infection and indicates strong candidate target sites for generation of PRRSV-resistantCD163-edited pigs.

Acknowledgements

This work was supported by the Major Scientific Research Tasks for Scientific and Technological Innovation Projects of the Chinese Academy of Agricultural Sciences(CAAS-ZDRW202006),the National Transgenic Breeding Project,China (2018ZX08009-26B),the Shenzhen Science and Technology Plan Project,China(CJGJZD20210408092402006),and the Shenzhen Key Technology Projects,China (JSGG20180507182028625).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Ethical approval

The animal work described in this study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the Institute of Animal Sciences,Chinese Academy of Agricultural Sciences.All experiments were performed in accordance with the approved guidelines for animal care and management of research projects.

Appendicesassociated with this paper are available on https://doi.org/10.1016/j.jia.2022.11.010