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.

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.

Amino acid at position 176 was essential for porcine reproductive and respiratory syndrome virus (PRRSV) non-structural protein 1α (nsp1α) as an inhibitor to the induction of IFN-ß.

Previous studies have shown that porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 1α (nsp1α) was the interferon (IFN) antagonist. However, the mechanism was unclear. In the present study, deletion of the carboxyl-terminal extension (CTE) (167-180 amino acid (aa)) made nsp1α lose its inhibitory ability to the induction of IFN-ß. And a series of C-terminal truncated mutants for nsp1α showed that 1-176 aa of nsp1α was able to inhibit the induction of IFN-ß and deleting or mutating the amino acid F176 made nsp1α not inhibit the induction of IFN-ß. In conclusion, the CTE and the amino acid F176 were critical for nsp1α as the IFN antagonist and the region representing 167-176 was the minimal subunit of the CTE for nsp1α to retain its suppressive activity to the induction of IFN-ß.

Surface IgM on DT40 cells may be a component of the putative receptor complex responsible for the binding of infectious bursal disease virus.

To investigate the host-pathogen interactions between infectious bursal disease virus (IBDV) and target B-lymphocytic cells, a cDNA T7 phage display library from the chicken bursa of Fabricius was constructed and screened for virus binding. Surface immunoglobulin M (sIgM) was isolated as a putative candidate binding site and its interactions with IBDV were further investigated using a chicken bursal lymphoma-derived cell line DT40. The results showed that the λ light chain of sIgM specifically interacted with IBDV in a virulence-independent manner in vitro, and most of the binding of IBDV to DT40 cells was inhibited by sIgM-specific monoclonal antibodies. Further, the infectivity of IBDV in vitro was reduced by sIgM-specific monoclonal antibodies. Our data provided evidence that sIgM may participate as one of the putative membrane binding sites responsible for IBDV infection.

Nonmuscle Myosin Heavy Chain IIA Recognizes Sialic Acids on Sialylated RNA Viruses To Suppress Proinflammatory Responses via the DAP12-Syk Pathway.

Viral infections induce proinflammatory signaling cascades and inflammatory cytokine production, which is precisely regulated for host benefits. In the current study, we unravel a previously unappreciated role of nonmuscle myosin heavy chain IIA (NMHC-IIA) as a negative regulator in inflammatory responses. We identified that cell surface NMHC-IIA recognized sialic acids on sialylated RNA viruses during early infections and interacted with an immune adaptor DNAX activation protein of 12 kDa (DAP12) to recruit downstream spleen tyrosine kinase (Syk), leading to suppressed virus-triggered proinflammatory responses. More importantly, recognition of sialylated RNA viruses or sialic acid mimics by NMHC-IIA was shown to inhibit lipopolysaccharide (LPS)-induced proinflammatory responses via the DAP12-Syk pathway. These findings uncover a novel negative regulation mechanism of proinflammatory responses and provide a molecular basis to design anti-inflammatory drugs.IMPORTANCE NMHC-IIA, a subunit of nonmuscle myosin IIA (NM-IIA), takes part in diverse physiological processes, including cell movement, cell shape maintenance, and signal transduction. Recently, NMHC-IIA has been demonstrated to be a receptor or factor contributing to viral infections. Here, we identified that NMHC-IIA recognizes sialic acids on sialylated RNA viruses, vesicular stomatitis virus (VSV) and porcine reproductive and respiratory syndrome virus (PRRSV). Upon recognition, NMHC-IIA associates with the transmembrane region of DAP12 to recruit Syk. Activation of the DAP12-Syk pathway impairs the host antiviral proinflammatory cytokine production and signaling cascades. More importantly, sialic acid mimics and sialylated RNA viruses enable the antagonism of LPS-triggered proinflammatory responses through engaging the NMHC-IIA-DAP12-Syk pathway. These results actually support that NMHC-IIA is involved in negative modulation of the host innate immune system, which provides a molecular basis for prevention and control of the sialylated RNA viruses and treatment of inflammatory diseases.

The CD163 long-range scavenger receptor cysteine-rich repeat: expression, purification and X-ray crystallographic characterization.

Scavenger receptors (SRs) play critical roles in various physiological and pathological pathways. One of them, CD163, is a multifunctional endocytic receptor and is characterized by a long-range scavenger receptor cysteine-rich (SRCR) repeat. However, the structural and functional details of this long-range SRCR repeat have not yet been elucidated. In this study, the CD163 long-range SRCR repeat was expressed in Drosophila Schneider 2 cells. The recombinant protein was homogeneous after purification by metal-affinity, cation-exchange and size-exclusion chromatography. Single crystals were obtained using 20% PEG 4000, 0.15 M potassium sodium tartrate tetrahydrate pH 8.5 and diffracted to 3.30 Šresolution. As the first view of a long-range SRCR repeat, this work lays the structural basis for a deep understanding of SRs and their multiple functions.

Characterization of the interaction between recombinant porcine aminopeptidase N and spike glycoprotein of porcine epidemic diarrhea virus.

Porcine epidemic diarrhea (PED) has caused huge economic losses to the global pork industry. Infection by its causative agent PED virus (PEDV), an Alpha-coronavirus, was previously proven to be mediated by its spike (S) glycoprotein and a cellular receptor porcine aminopeptidase N (pAPN). Interestingly, some recent studies have indicated that pAPN is not a functional receptor for PEDV. To date, there is a lack of a direct evidence for the interaction between pAPN and PEDV S protein in vitro. Here, we prepared pAPN ectodomain and the truncated variants of PEDV S protein in Drosophila S2 cells. These recombinant proteins were homogeneous after purification by metal-affinity and size-exclusion chromatography. We then assayed the purified target proteins through immunogenicity tests, PEDV binding interference assays, circular dichroism (CD) measurements, pAPN activity assay and structural determination, demonstrating that they were biologically functional. Finally, we characterized their interactions by gel filtration chromatography, native-polyacrylamide gel electrophoresis (PAGE) and surface plasmon resonance (SPR) analyses. The results showed that their affinities were too low to form complexes, which suggest that pAPN may be controversial as the genuine receptor for PEDV. Therefore, further research needs to be carried out to elucidate the interaction between PEDV and its genuine receptor.

Development of an immunochromatographic strip for detection of antibodies against porcine reproductive and respiratory syndrome virus.

A simple and rapid immunochromatographic test strip incorporating a colloidal gold-labeled recombinant Nsp7 antigen probe was successfully developed for the detection of anti-porcine reproductive and respiratory syndrome virus (PRRSV) antibodies in swine. Recombinant Nsp7 protein of PRRSV labeled with colloidal gold was dispensed on a conjugate pad for use as the detector. Staphylococcal protein A and purified porcine anti-Nsp7 antibodies were blotted on a nitrocellulose membrane to form test and control lines, respectively. A comparison of the strip with standard diagnostic tests, enzyme-linked immunosorbent assays and immunoperoxidase monolayer assay, was also performed. The immunochromatographic test strip was shown to be of high specificity and sensitivity. Furthermore, the strip assay is rapid and easy to perform with no requirement for professional-level skills or equipment. It is suggested that the immunochromatographic test strip can be used to quickly and accurately detect PRRSV antibody and to be suitable for diagnostic purposes in the field.

Development of an immunochromatographic strip for the detection of antibodies against Porcine circovirus-2.

A rapid (<5 min) immunochromatographic strip using a colloidal gold-labeled antigen probe was successfully developed and applied for the detection of Porcine circovirus-2 (PCV-2) antibodies in swine. Recombinant Cap protein truncated nuclear localization signal of PCV-2, was expressed and labeled with colloidal gold. This conjugate was dispensed on a conjugate pad as the detector. Staphylococcal protein A and purified porcine anti-PCV-2 antibodies were blotted on a nitrocellulose membrane for the test and control lines, respectively. Sensitivity and specificity of this strip test was evaluated using PCV-2 antisera as well as other sera from pigs infected with a variety of swine viruses. For the validation of this strip test, 500 clinical swine serum samples were assessed both by the strip and a commercial enzyme-linked immunosorbent assay (ELISA) kit. The agreement between the immunochromatographic strip and ELISA kit was 94.00%. This strip possesses high sensitivity and specificity and may be useful as a candidate for rapid diagnosis of PCV-2 antibodies in the field.

The nonstructural protein 1 papain-like cysteine protease was necessary for porcine reproductive and respiratory syndrome virus nonstructural protein 1 to inhibit interferon-ß induction.

Porcine reproductive and respiratory syndrome virus nonstructural protein 1 (nsp1) could be auto-cleaved into nsp1α and nsp1ß, both of which had the papain-like cysteine protease activities. Previous studies have shown that porcine reproductive and respiratory syndrome virus nsp1 was an interferon (IFN) antagonist. However, the mechanism by which nsp1 inhibited IFN-ß production was unclear. Here, we used site-directed mutagenesis that inactivated the papain-like cysteine protease activities of nsp1 to explore whether the papain-like cysteine protease activities were required for nsp1 to disrupt IFN-ß production. The results showed that mutations that inactivated papain-like cysteine protease activity of nsp1α made nsp1 lose its IFN antagonism activity, whereas mutations that inactivated papain-like cysteine protease activity of nsp1ß did not influence the IFN antagonism activity of nsp1. In conclusion, our present work indicated that the papain-like cysteine protease activity of nsp1α was necessary for nsp1 to inhibit IFN-ß induction.

Development of a lateral flow colloidal gold immunoassay strip for the rapid detection of olaquindox residues.

A rapid immunochromatographic lateral flow test strip of competitive format has been developed for the specific determination of olaquindox (OLA) residues in pig urine and muscle tissues. The sensitivity of the test strip was found to be 1.58 ± 0.27 µg/kg and 1.70 ± 0.26 µg/kg of OLA in pig urine and muscle tissues, and the lower detection limit was 0.27 ± 0.08 µg/kg and 0.31 ± 0.07 µg/kg respectively. For negative pig urine and muscle samples spiked with 4, 12, and 36 µg/kg, the recovery range was 83.0-94.0% and 78.8-87.4% and the coefficient of variation scope [CV (%)] was 3.17-7.41% and 4.66-7.64% respectively. Parallel analysis of OLA samples from pig urine and muscle tissue showed comparable results from the test strip and HPLC. Each test requires 5-8 min, and the test strip can provide a useful screening method for quantitative, semiquantitative, or qualitative detection of OLA residues.

Porcine reproductive and respiratory syndrome virus (PRRSV) could be sensed by professional beta interferon-producing system and had mechanisms to inhibit this action in MARC-145 cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes an economically important disease in swine-producing area, and interferon beta (IFN-beta) is the first responder against the animal virus infection. However, whether PRRSV could induce the production of IFN-beta is controversial. In this paper, we first time found that PRRSV could phosphorylate IFN-regulatory factor 3 (IRF-3) and weakly activate the IFN-beta promoter in MARC-145 cells in early infection, but the activations of IRF-3 and IFN-beta promoter were rapidly inhibited in the following infection. Furthermore, which components or products of the invading PRRSV cause PRRSV to inhibit IFN-beta promoter activity attracted our attentions. The obtained results showed that PRRSV nsp1 could inhibit Poly(I:C)-induced IFN-beta promoter activity in MARC-145 cells by down-regulating the protein level of IRF-3 and inhibiting the phosphorylation of IRF-3. In conclusion, our results suggested that PRRSV could be sensed by professional IFN-beta-producing system and had mechanisms to inhibit this action in MARC-145 cells.

Development of an immunochromatographic strip for the detection of antibodies against foot-and-mouth disease virus serotype O.

An immunochromatographic strip was developed for the serological detection of type O foot-and-mouth disease (FMD) in swine. In the strip, the expressed protein of VP1, the main protective antigen of FMDV, labeled with colloidal gold was used as the detector, the staphylococcal protein A (SPA) and swine anti-foot-and-mouth disease virus (FMDV) antibody were blotted on the nitrocellulose membrane for the test and control lines, respectively. 296 swine serum samples were collected to evaluate the characteristics of the strip in comparison with existing commercial liquid-phage blocking ELISA (LPB ELISA) kit and peptide ELISA kit. The strip was shown to be of high specificity and sensitivity. Furthermore, the dipstick assay based on the strip is rapid (5 min) and easy to perform with no requirement of professional skills, reagents nor equipment. This suggests that the immunochromatographic strip is an acceptable alternative for use in clinical laboratories lacking specialized equipment and for field diagnosis.