Involvement of sialoadhesin in entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) shows a very restricted tropism for cells of the monocyte/macrophage lineage. It enters cells via receptor-mediated endocytosis. A monoclonal antibody (MAb) that is able to block PRRSV infection of porcine alveolar macrophages (PAM) and that recognizes a 210-kDa protein (p210) was described previously (MAb41D3) (X. Duan, H. Nauwynck, H. Favoreel, and M. Pensaert, J. Virol. 72:4520-4523, 1998). In the present study, the p210 protein was purified from PAM by immunoaffinity using MAb41D3 and was subjected to internal peptide sequencing after tryptic digestion. Amino acid sequence identities ranging from 56 to 91% with mouse sialoadhesin, a macrophage-restricted receptor, were obtained with four p210 peptides. Using these peptide data, the full p210 cDNA sequence (5,193 bp) was subsequently determined. It shared 69 and 78% amino acid identity, respectively, with mouse and human sialoadhesins. Swine (PK-15) cells resistant to viral entry were transfected with the cloned p210 cDNA and inoculated with European or American PRRSV strains. Internalized virus particles were detected only in PK-15 cells expressing the recombinant sialoadhesin, demonstrating that this glycoprotein mediated uptake of both types of strains. However, nucleocapsid disintegration, like that observed in infected Marc-145 cells as a result of virus uncoating after fusion of the virus with the endocytic vesicle membrane, was not observed, suggesting a block in the fusion process. The ability of porcine sialoadhesin to mediate endocytosis was demonstrated by specific internalization of MAb41D3 into PAM. Altogether, these results show that sialoadhesin is involved in the entry process of PRRSV in PAM.     

重组猪肺表面活性蛋白A在体外可抑制PRRSV感染宿主细胞

【目的】研究重组猪肺表面活性蛋白A(SP-A)在体外对猪繁殖与呼吸综合征病毒(PRRSV)感染的抑制作用。【方法】采用PCR方法从含有猪SP-A基因的质粒中扩增SP-A基因,并将其插入到含有人CD5信号肽序列的真核表达载体pcDNA3.1A-CD5中,构建成SP-A基因的真核分泌型表达载体pcDNA-CD5-SPA/MH。将重组表达载体通过磷酸钙介导转染HEK293T细胞进行瞬时表达,通过Western blot方法鉴定表达产物,采用Ni-NTA琼脂糖凝胶亲和层析法从培养基中分离和纯化重组SP-A蛋白,通过ELISA方法检测SP-A蛋白与PRRSV的结合活性。将SP-A蛋白与PRRSV孵育,然后感染MARC-145细胞和猪肺泡巨噬细胞,感染72 h后测定病毒滴度,分析重组SP-A蛋白对PRRSV感染的抑制作用。【结果】结果表明构建的真核表达载体能够介导SP-A基因在HEK293T细胞中进行分泌表达;表达的重组猪SP-A蛋白能够与PRRSV进行剂量依赖性结合;用重组猪SP-A蛋白与PRRSV进行孵育,然后感染MARC-145细胞和猪肺泡巨噬细胞,结果显示SP-A处理的PRRSV感染细胞后的病变程度明显低于对照组。感染72 h后,SP-A处理组的PRRSV在MARC-145细胞和猪肺泡巨噬细胞的滴度明显低于SP-A非处理组。【结论】重组猪SP-A在体外对PRRSV的感染有明显的抑制作用,揭示SP-A具有抗PRRSV的活性。     

Identification of a Putative Receptor for Porcine Reproductive and Respiratory Syndrome Virus on Porcine Alveolar Macrophages

To identify the receptor which may determine the macrophage tropism of porcine reproductive and respiratory syndrome virus (PRRSV), monoclonal antibodies (MAbs) against porcine alveolar macrophages (PAM) were produced. Two MAbs (41D3 and 41D5) which completely blocked PRRSV infection of PAM were further characterized. It was found that they reduce the attachment of PRRSV to PAM and immunoprecipitate a 210-kDa membrane protein from PAM. This protein was detected on the cell membranes of PAM but not of PRRSV-nonpermissive cells. A colocalization was found between the reactive sites of MAb 41D3 and PRRSV on PAM membranes. All PRRSV-infected cells in tissues of experimentally infected pigs reacted with MAb 41D3. Taken together, all these data suggest that the identified 210-kDa membrane protein is a putative receptor for PRRSV on porcine macrophages.     

Indirect ELISA with recombinant GP5 for detecting antibodies to porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome is caused by the PRRS virus (PRRSV), which has six structural proteins (GP2, GP3, GP4, GP5, M and N). GP5 and N protein are important targets for serological detection by enzyme-linked immunosorbent assay (ELISA) and other methods. Toward this goal, we developed an indirect ELISA with recombinant GP5 antigens and this method was validated by comparison to the LSI PRRSV-Ab ELISA kit. The results indicated that the optimal concentration of coated recombinant antigen was 0.2 μg/well for a serum dilution of 1:40. The rate of agreement with the LSI PRRSV-Ab kit was 88.7% (266/300). These results support the potential use of recombinant GP5 as an antigen for indirect ELISA to detect PRRSV antibodies in pigs.     

体外共表达的猪繁殖与呼吸综合征病毒GP5和M蛋白可形成异源二聚体

为了探讨猪繁殖与呼吸综合征病毒(PRRSV)ORF5基因编码的GP5蛋白和ORF6编码的M蛋白体外共表达特性,分别构建了PRRSV ORF5、ORF6单基因或双基因共表达的真核表达质粒pCI-ORF5、pCI-ORF6和pCI-ORF5/ORF6,转染BHK-21细胞,Western blot检测证实共表达的GP5和M蛋白能够形成异源二聚体。同时,以绿色荧光蛋白(EGFP)和红色荧光蛋白(RFP)为示踪,发现当ORF5-EGFP和ORF6-RFP共表达时,能促进GP5蛋白从内质网向高尔基体转运,提示GP5-M异源二聚体的形成可能与GP5蛋白的翻译后修饰、转运、定位有关。     

表达猪繁殖与呼吸综合征病毒GP5和M融合蛋白的重组腺病毒的构建及其对小鼠的免疫原性

利用口蹄疫病毒(FMDV)2A蛋白具有自我裂解的功能,将其作为连接肽将猪繁殖与呼吸综合征病毒(PRRSV)的GP5和M蛋白编码基因串联,置于复制缺陷型腺病毒载体的表达盒中,通过一次转录和翻译,可以同时实现2个蛋白的表达,以发挥GP5蛋白的病毒中和优势和M蛋白的细胞免疫优势作用。分别利用RT-PCR、间接免疫荧光和Western blotting等方法,对获得的重组腺病毒(rAd-GP5-2A-M)进行检测,结果均证明GP5-2A-M蛋白不仅获得了正确表达,而且能自我裂解为GP5和M蛋白。以单独表达GP5(rAd-GP5)、M(rAd-M)和融合表达GP5-M(rAd-GP5-M)的重组腺病毒为对照,研究该重组腺病毒在小鼠体内诱导免疫应答情况,结果表明,虽然4种重组腺病毒均能诱导小鼠产生特异性抗体和细胞免疫反应,但重组腺病毒rAd-GP5-2A-M所诱导产生的体液免疫和细胞免疫应答水平最高。本研究结果提示,利用具有自动裂解功能的FMDV2A多肽构建PRRSV复合基因工程疫苗是一种切实可行的新策略,也为构建其他动物病毒病的基因工程疫苗提供了新思路。     

Porcine arterivirus infection of alveolar macrophages is mediated by sialic acid on the virus

Recently, we showed that porcine sialoadhesin (pSn) mediates internalization of the arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) in alveolar macrophages (Vanderheijden et al., J. Virol. 77:8207-8215, 2003). In rodents and humans, sialoadhesin, or Siglec-1, has been described as a macrophage-restricted molecule and to specifically bind sialic acid moieties. In the current study, we investigated whether pSn is a sialic acid binding protein and, whether so, whether this property is important for its function as a PRRSV receptor. Using untreated and neuraminidase-treated sheep erythrocytes, we showed that pSn binds sialic acid. Furthermore, pSn-specific monoclonal antibody 41D3, which blocks PRRSV infection, inhibited this interaction. PRRSV attachment to and infection of porcine alveolar macrophages (PAM) were both shown to be dependent on the presence of sialic acid on the virus: neuraminidase treatment of virus but not of PAM blocked infection and reduced attachment. Enzymatic removal of all N-linked glycans on the virus with N-glycosidase F reduced PRRSV infection, while exclusive removal of nonsialylated N-linked glycans of the high-mannose type with endoglycosidase H had no significant effect. Free sialyllactose and sialic acid containing (neo)glycoproteins reduced infection, while lactose and (neo)glycoproteins devoid of sialic acids had no significant effect. Studies with linkage-specific neuraminidases and lectins indicated that alpha2-3- and alpha2-6-linked sialic acids on the virion are important for PRRSV infection of PAM. From these results, we conclude that pSn is a sialic acid binding lectin and that interactions between sialic acid on the PRRS virion and pSn are essential for PRRSV infection of PAM.     

携带CD163报告载体的猪诱导多能干细胞株的建立

猪肺泡巨噬细胞(porcine alveolar macrophage,PAM)是包括猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV)在内的多种高致病病毒的受体细胞,是研究病毒与宿主互作机制的重要模型。然而PAM来源有限,难以满足当前需求。利用猪诱导性多能干细胞(induced pluripotent stem cells, iPSCs)向巨噬细胞定向诱导是解决PAM细胞数量不足的有效方法。CD163是PAM细胞的重要标记,也是PRRSV等病毒的主要受体。建立实时报告CD163激活程度的报告系统对于建立并优化猪iPSCs向PAM的诱导分化体系具有指导意义。本研究利用CRISPR/Cas9介导的基因编辑系统,设计靶向CD163终止密码子的sgRNA并构建相应的打靶载体,将其导入到猪PAM中的检测报告系统。进一步将该报告系统导入猪iPSCs中,通过碱性磷酸酶染色、免疫荧光染色和EDU染色等手段来检测其安全性。将猪内源CD163的报告载体系统转染至原代PAM中,检测到了红色荧光的表达,证明了该载体系...     

毕赤酵母表达猪干扰素—γ基因及其抑制蓝耳病毒效果

为了研究和应用猪重组干扰素-γ（rPoIFN-γ)预防和治疗病毒性疫苗,将大白猪PoIFN-γ基因插入到酵母整合载体pHIL-S1,构建了重组GS115工程菌（pHIL-S1/rPoIFN-γ)。经过SDS－PAGE,Western blot分析和抗滤泡性口炎病毒（VSV）活性测定,证实了rPoIFN-γ分子量为18kD,在GS115中的表达量为18％。其抗VSV活性为450－540u/mL。用rPoIFN-γ处理猪肺巨噬细胞系Marc-145后,经细胞病变抑制法（CPE50）测定,rPoIFN-γ可以抑抗蓝耳病病毒（PRRSV）感染。结果显示酵母表达的rPoIFN-γ是有应用价值的抗病毒生物工程制剂。     

Immune evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involves both glycoprotein 5 as well as glycoprotein 3

Passive administration of porcine reproductive and respiratory syndrome virus (PRRSV) neutralizing antibodies (NAbs) can effectively protect pigs against PRRSV infection. However, after PRRSV infection, pigs typically develop a weak and deferred NAb response. One major reason for such a meager NAb response is the phenomenon of glycan shielding involving GP5, a major glycoprotein carrying one major neutralizing epitope. We describe here a type II PRRSV field isolate (PRRSV-01) that is highly susceptible to neutralization and induces an atypically rapid, robust NAb response in vivo. Sequence analysis shows that PRRSV-01 lacks two N-glycosylation sites, normally present in wild-type (wt) PRRSV strains, in two of its envelope glycoproteins, one in GP3 (position 131) and the other in GP5 (position 51). To determine the influence of these missing N-glycosylation sites on the distinct neutralization phenotype of PRRSV-01, a chimeric virus (FL01) was generated by replacing the structural genes of type II PRRSV strain FL12 cDNA infectious clone with those from PRRSV-01. N-glycosylation sites were reintroduced into GP3 and GP5 of FL01, separately or in combination, by site-directed mutagenesis. Reintroduction of the N-glycosylation site in either GP3 or GP5 allowed recovery of in vivo and in vitro glycan shielding capacity, with an additive effect when these sites were reintroduced into both glycoproteins simultaneously. Although the loss of these glycosylation sites has seemingly occurred naturally (presumably by passage through cell cultures), PRRSV-01 virus quickly regains these glycosylation sites through replication in vivo, suggesting that a strong selective pressure is exerted at these sites. Collectively, our data demonstrate the involvement of an N-glycan moiety located in GP3 in glycan shield interference.     

CD163 expression confers susceptibility to porcine reproductive and respiratory syndrome viruses

Direct functional screening of a cDNA expression library derived from primary porcine alveolar macrophages (PAM) revealed that CD163 is capable of conferring a porcine reproductive and respiratory syndrome virus (PRRSV)-permissive phenotype when introduced into nonpermissive cells. Transient-transfection experiments showed that full-length CD163 cDNAs from PAM, human U937 cells (histiocytic lymphoma), African green monkey kidney cells (MARC-145 and Vero), primary mouse peritoneal macrophages, and canine DH82 (histocytosis) cells encode functional virus receptors. In contrast, CD163 splice variants without the C-terminal transmembrane anchor domain do not provide PRRSV receptor function. We established several stable cell lines expressing CD163 cDNAs from pig, human, and monkey, using porcine kidney (PK 032495), feline kidney (NLFK), or baby hamster kidney (BHK-21) as the parental cell lines. These stable cell lines were susceptible to PRRSV infection and yielded high titers of progeny virus. Cell lines were phenotypically stable over 80 cell passages, and PRRSV could be serially passed at least 60 times, yielding in excess of 10(5) 50% tissue culture infective doses/ml.     

Exostosin glycosyltransferase 1 reduces porcine reproductive and respiratory syndrome virus infection through proteasomal degradation of nsp3 and nsp5

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a serious threat to the swine industry worldwide. Exostosin glycosyltransferase 1 (EXT1), an enzyme involved in the biosynthesis of heparin sulfate, has also been reported to be a host factor essential for a wide variety of pathogens. However, the role of EXT1 in PRRSV infection remains uncharted. Here, we identified that PRRSV infection caused an increase of EXT1 expression. EXT1 knockdown promoted virus infection, whereas its overexpression inhibited virus infection, suggesting an inhibitory function of EXT1 to PRRSV infection. We found that EXT1 had no effects on the attachment, internalization, or release of PRRSV but did restrict viral RNA replication. EXT1 was determined to interact with viral nonstructural protein 3 (nsp3) and nsp5 via its N-terminal cytoplasmic tail and to enhance K48-linked polyubiquitination of these two nsps to promote their degradation. Furthermore, the C-terminal glycosyltransferase activity domain of EXT1 was necessary for nsp3 and nsp5 degradation. We also found that EXT2, a EXT1 homolog, interacted with EXT1 and inhibited PRRSV infection. Similarly, EXT1 effectively restricted porcine epidemic diarrhea virus and porcine enteric alphacoronavirus infection in Vero cells. Taken together, this study reveals that EXT1 may serve as a broad-spectrum host restriction factor and suggests a molecular basis for the potential development of therapeutics against PRRSV infection.     

3′非翻译区靶向人工微小RNA对PRRSV在猪肺巨噬细胞中复制的抑制作用（英文）北大核心CSCD

【目的】研究重组腺病毒(rAd)传送的3′非翻译区(UTR)靶向amiR3UTR对猪繁殖与呼吸综合征病毒(PRRSV)在猪肺巨噬细胞(PAM)中复制的抑制作用。【方法】用表达amiR3UTR或对照amiRcon的腺病毒载体转染AAV-293细胞,获得rAd-amiR3UTR-GFP和rAd-amiRcon-GFP,用定量RT-PCR检测amiR3UTR在rAd转导细胞中的表达,用定量RT-PCR、Western blotting和病毒滴定检测amiR3UTR对PRRSV复制的抑制作用。【结果】原代PAM及其细胞系3D4/163均能被rAd-amiR3UTR-GFP转导,但前者转导效率很低;rAd-amiR3UTR-GFP转导细胞能有效表达amiR3UTR,且表达具有剂量和时间依赖性;rAd表达的amiR3UTR能显著抑制不同毒株PRRSV在PAM细胞中的复制,且抑制作用具有剂量依赖性。【结论】amiR3UTR能抑制不同毒株PRRSV在PAM中的复制,其rAd有望作为抗PRRSV新策略进行深入研究。     

The polymorphism analysis of CD169 and CD163 related with the risk of porcine reproductive and respiratory syndrome virus (PRRSV) infection

Porcine reproductive and respiratory syndrome virus (PRRSV) could infect porcine alveolar macrophages (PAM), and the CD169 and CD163 are identified as critical receptors on the surface of PAM, but whether the single nucleotide polymorphisms (SNPs) of these genes could influence the infection is remain unclear. In this study, we identified totally 6 SNPs for CD169 (G1640T, C1654A, C4175T) and CD163 (G2277A, A2552G and C2700A), and evaluated their associations with PRRSV infection using two classified methods in a 524 pig population to investigate the effects of mutations on the PRRSV receptors. The pigs with genotypes of AA of CD169-C1654A, CT of CD169-C4175T and AA of CD163-A2552G appeared to resistant to the PRRSV infection by the combination of two classified results. The results provided fundamental molecular investigation to promote pig breeding with disease resistance. However, the identification of functional changes induced by SNPs and molecular mechanism were need further research.     

Indirect ELISA with Recombinant GP5 for Detecting Antibodies to Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome is caused by the PRRS virus (PRRSV),which has six structural proteins (GP2,GP3,GP4,GP5,M and N). GP5 and N protein are important targets for serological detection by enzyme-linked immunosorbent assay (ELISA) and other methods. Toward this goal,we developed an indirect ELISA with recombinant GP5 antigens and this method was validated by comparison to the LSI PRRSV-Ab ELISA kit. The results indicated that the optimal concentration of coated recombinant antigen was...     

Construction and immunogenicity of recombinant pseudorabies virus expressing the modified GP5m protein of porcine reproduction and respiratory syndrome virus

Pseudorabies virus (PRV),an alpha-herpesvirus,has been developed as a live viral vector for animal vaccines.However,the PRV recombinant virus TK-/gE-/GP5+expressing GP5 of porcine reproductive and respiratory syndrome virus (PRRSV),based on the PRV genetically depleted vaccine strain TK-/gE-/LacZ+,scarcely stimulated the vaccinated animals to produce neutralizing antibodies against PRRSV.To develop a booster-specific immune response of such PRV recombinants,the ORF5m gene (the modified ORF5 gene having better immune responses)was substituted for the ORF5 gene and introduced into PRV TK-/gE-/LacZ+,resulting in a PRV recombinant named TK-/gE-/GPSm+,which expressed the modified GPSm protein.The recombinant virus was confirmed using PCR,Southern blotting and Western blotting.TK-/gE-/GPSm+and TK-/gE-/GP5+expressing the authentic GP5 protein were inoculated into Balb/c mice to evaluate their immune responses.The results indicated that the protecting neutralization antibodies (the 3/6 vaccinated mice obtained 1:16)and cell immune responses induced by TK-/gE-/GPSm+against PRRSV were higher than that induced by TK-/gE-/GP5+.Thus,the development of the new PRV recombinant expressing the modified GP5m protein as a candidate vaccine established the basis for the study of bivalent genetic engineering vaccines against PRRSV and PRV.     

Role of lipid rafts in porcine reproductive and respiratory syndrome virus infection in MARC-145 cells

Lipid rafts play an important role in the life cycle of many viruses. Cholesterol is a critical structural component of lipid rafts. Although the porcine reproductive and respiratory syndrome virus (PRRSV) has restricted cell tropism for cells of the monocyte/macrophage lineage, a non-macrophage cell MARC-145 was susceptible to PRRSV because of the expression of virus receptor CD163 on the cell surface, therefore MARC-145 cells is used as model cell for PRRSV studies. In order to determine if cholesterol is involved in PRRSV infection in MARC-145 cells, we used three pharmacological agents: methyl-β cyclodextrin (MβCD), mevinolin, and filipin complex to deplete cholesterol in MARC-145. Although these agents act by different mechanisms, they all significantly inhibited PRRSV infection. The inhibition could be prevented by addition of exogenous cholesterol. Cell membrane cholesterol depletion after virus infection had no effect on PRRSV production and cholesterol depletion pre-infection did not reduce the virus attachment, suggesting cholesterol is involved in virus entry. Further results showed that cholesterol depletion did not change expression levels of the PRRSV receptor CD163 in MARC-145, had no effect on clathrin-mediated endocytosis, but disturbed lipid-raft-dependent endocytosis. Collectively, these studies suggest that cholesterol is critical for PRRSV entry, which is likely to be mediated by a lipid-raft-dependent pathway.