PK-15cells transfected with porcine CD163 by PiggyBac transposon system are susceptible to porcine reproductive and respiratory syndrome virus

Wang, Xiangpeng; Wei, Ruifang; Li, Qiongyi; Liu, Hongliang; Huang, Baicheng; Gao, Jiming; Yang, Mei; Wang, Chengbao; Hsu, Walter H.; Hiscox, Julian A.; Zhou, En‐Min

doi:10.1016/j.jviromet.2013.06.035

Cited by 40 publications

(26 citation statements)

References 43 publications

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“…We mutated these residues to the neutral alanine and expressed each single-site-mutated pCD163 receptor in PK-15 cells. This cell line was chosen because native PK-15 cells are nonpermissive to PRRSV, while after transfection with pCD163 they support the virus infection and thus are ideal cells for the assay (59). Thereafter we demonstrated that Arg561 is an important residue for PRRSV infection and probably takes effect on pCD163 binding to PRRSV during virus invasion.…”

Section: Pcd163 Srcr5 Structure For Prrsv Infectionmentioning

confidence: 88%

The Crystal Structure of the Fifth Scavenger Receptor Cysteine-Rich Domain of Porcine CD163 Reveals an Important Residue Involved in Porcine Reproductive and Respiratory Syndrome Virus Infection

Jiang

Qiao

et al. 2017

J Virol

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Porcine reproductive and respiratory syndrome (PRRS) has become an economically critical factor in swine industry since its worldwide spread in the 1990s. Infection by its causative agent, PRRS virus (PRRSV), was proven to be mediated by an indispensable receptor, porcine CD163 (pCD163), and the fifth scavenger receptor cysteine-rich domain (SRCR5) is essential for virus infection. However, the structural details and specific residues of pCD163 SRCR5 involved in infection have not been defined yet. In this study, we prepared recombinant pCD163 SRCR5 in Drosophila melanogaster Schneider 2 (S2) cells and determined its crystal structure at a high resolution of 2.0 Å. This structure includes a markedly long loop region and shows a special electrostatic potential, and these are significantly different from those of other members of the scavenger receptor cysteine-rich superfamily (SRCR-SF). Subsequently, we carried out structure-based mutational studies to identify that the arginine residue at position 561 (Arg561) in the long loop region is important for PRRSV infection. Further, we showed Arg561 probably takes effect on the binding of pCD163 to PRRSV during virus invasion. Altogether the current work provides the first view of the CD163 SRCR domain, expands our knowledge of the invasion mechanism of PRRSV, and supports a molecular basis for prevention and control of the virus.IMPORTANCE PRRS has caused huge economic losses to pig farming. The syndrome is caused by PRRSV, and PRRSV infection has been shown to be mediated by host cell surface receptors. One of them, pCD163, is especially indispensable, and its SRCR5 domain has been further demonstrated to play a significant role in virus infection. However, its structural details and the residues involved in infection are unknown. In this study, we determined the crystal structure of pCD163 SRCR5 and then carried out site-directed mutational studies based on the crystal structure to elucidate which residue is important. Our work not only provides structural information on the CD163 SRCR domain for the first time but also indicates the molecular mechanism of PRRSV infection and lays a foundation for future applications in prevention and control of PRRS.KEYWORDS CD163, Drosophila S2 cells, PRRSV, SRCR5, crystal structure P orcine reproductive and respiratory syndrome (PRRS) was first reported in 1987 in the United States (1) and has become a critical economic factor in the swine industry since its worldwide spread in the 1990s (2, 3). PRRS is characterized by

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Section: Pcd163 Srcr5 Structure For Prrsv Infectionmentioning

confidence: 88%

The Crystal Structure of the Fifth Scavenger Receptor Cysteine-Rich Domain of Porcine CD163 Reveals an Important Residue Involved in Porcine Reproductive and Respiratory Syndrome Virus Infection

Jiang

Qiao

et al. 2017

J Virol

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“…The HEK293-APN cell line (stably expressing pAPN) was generated by the piggyBac (PB) transposon system [29]. pAPN was amplified by PCR including a FLAG tag in the forward primer (F: CATAGAAGATTCTAGACACCATGGATTACA-AGGACGACGATGACAAGgccaagggattctacatttc, R: ATTTAAATTCGAATTCttagctgtgctctatgaacca) and then cloned into the pB513B vector to generate pB513B-APN (System Biosciences, Mountain View, USA) [29]. Then, HEK293 cells were co-transfected with 3 μg pB513B-APN and 1 μg helper vector expressing PB transposase (System Biosciences, Mountain View, USA).…”

Section: Cells Virus Reagent and Plasmidsmentioning

confidence: 99%

Trypsin promotes porcine deltacoronavirus mediating cell-to-cell fusion in a cell type-dependent manner

Yang

Meng

Qin

et al. 2020

Emerging Microbes & Infections

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Porcine deltacoronavirus (PDCoV) is a newly emerging threat to the global porcine industry. PDCoV has been successfully isolated using various medium additives including trypsin, and although we know it is important for viral replication, the mechanism has not been fully elucidated. Here, we systematically investigated the role of trypsin in PDCoV replication including cell entry, cell-to-cell membrane fusion and virus release. Using pseudovirus entry assays, we demonstrated that PDCoV entry is not trypsin dependent. Furthermore, unlike porcine epidemic diarrhea virus (PEDV), in which trypsin is important for the release of virus from infected cells, PDCoV release was not affected by trypsin. We also demonstrated that trypsin promotes PDCoV replication by enhancing cell-to-cell membrane fusion. Most importantly, our study illustrates two distinct spreading patterns from infected cells to uninfected cells during PDCoV transmission, and the role of trypsin in PDCoV replication in cells with different virus spreading types. Overall, these results clarify that trypsin promotes PDCoV replication by mediating cell-to-cell fusion transmission but is not crucial for viral entry. This knowledge can potentially contribute to improvement of virus production efficiency in culture, not only for vaccine preparation but also to develop antiviral treatments.

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“…Since then, several attachment factors have been studied extensively as potential PRRSV receptors and CD163 and CD169 were identified the most likely candidates involved. However, only CD163 has been shown capable of conferring PRRSV permissiveness to cell lines unsusceptible to PRRSV, even in the absence of CD169 (e.g., [17,83,113,114,116,123,124]). It was shown that PRRSV permissivity was conferred by CD163 independent of the PRRSV genotype involved (1 [EU] or 2 [US]) [17,61].…”

Section: Cd163 and Prrsvmentioning

confidence: 99%

Genetic resistance - an alternative for controlling PRRS?

Reiner

2016

Porc Health Manag

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PRRS is one of the most challenging diseases for world-wide pig production. Attempts for a sustainable control of this scourge by vaccination have not yet fully satisfied. With an increasing knowledge and methodology in disease resistance, a new world-wide endeavour has been started to support the combat of animal diseases, based on the existence of valuable gene variants with regard to any host-pathogen interaction. Several groups have produced a wealth of evidence for natural variability in resistance/susceptibility to PRRS in our commercial breeding lines. However, up to now, exploiting existing variation has failed because of the difficulty to detect the carriers of favourable and unfavourable alleles, especially with regard to such complex polygenic traits like resistance to PRRS. New hope comes from new genomic tools like next generation sequencing which have become extremely fast and low priced. Thus, research is booming world-wide and the jigsaw puzzle is filling up – slowly but steadily. On the other hand, knowledge from virological and biomedical basic research has opened the way for an “intervening way”, i.e. the modification of identified key genes that occupy key positions in PRRS pathogenesis, like CD163. CD163 was identified as the striking receptor in PRRSV entry and its knockout from the genome by gene editing has led to the production of pigs that were completely resistant to PRRSV – a milestone in modern pig breeding. However, at this early step, concerns remain about the acceptance of societies for gene edited products and regulation still awaits upgrading to the new technology. Further questions arise with regard to upcoming patents from an ethical and legal point of view. Eventually, the importance of CD163 for homeostasis, defence and immunity demands for more insight before its complete or partial silencing can be answered. Whatever path will be followed, even a partial abolishment of PRRSV replication will lead to a significant improvement of the disastrous herd situation, with a significant impact on welfare, performance, antimicrobial consumption and consumer protection. Genetics will be part of a future solution.

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PK-15cells transfected with porcine CD163 by PiggyBac transposon system are susceptible to porcine reproductive and respiratory syndrome virus

Cited by 40 publications

References 43 publications

The Crystal Structure of the Fifth Scavenger Receptor Cysteine-Rich Domain of Porcine CD163 Reveals an Important Residue Involved in Porcine Reproductive and Respiratory Syndrome Virus Infection

The Crystal Structure of the Fifth Scavenger Receptor Cysteine-Rich Domain of Porcine CD163 Reveals an Important Residue Involved in Porcine Reproductive and Respiratory Syndrome Virus Infection

Trypsin promotes porcine deltacoronavirus mediating cell-to-cell fusion in a cell type-dependent manner

Genetic resistance - an alternative for controlling PRRS?

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