The Experts below are selected from a list of 132 Experts worldwide ranked by ideXlab platform
Hans Nauwynck - One of the best experts on this subject based on the ideXlab platform.
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sialoadhesin and cd163 join forces during entry of the porcine reproductive and respiratory syndrome Virus
Journal of General Virology, 2008Co-Authors: Hanne Van Gorp, Peter Delputte, Wander Van Breedam, Hans NauwynckAbstract:The porcine reproductive and respiratory syndrome Virus (PRRSV) shows a restricted tropism for subsets of porcine macrophages in vivo. To date, two PRRSV receptors have been identified on primary macrophages, heparan sulphate for binding and sialoadhesin for binding and internalization. However, additional factors are needed because the expression of both receptors in non-permissive cells results in Virus internalization but not in Virus Uncoating and productive infection. Recently, CD163 was described as a PRRSV receptor on Marc-145 cells that renders non-permissive cells susceptible to PRRSV. Therefore, the potential role of CD163 in PRRSV entry in macrophages and its potential interplay with sialoadhesin were studied. Incubation of macrophages at 37 °C with either sialoadhesin- or CD163-specific antibodies reduced PRRSV infection by up to 75 %, while infection was completely blocked by a combination of both antibodies. When incubated at 4 °C, only sialoadhesin- and not CD163-specific antibodies reduced PRRSV infection. In addition, confocal analysis of PRRSV entry in non-permissive cells expressing only sialoadhesin showed PRRSV internalization but no Uncoating. In contrast, when both sialoadhesin and CD163 were expressed, PRRSV was uncoated upon internalization, resulting in productive infection. Virus internalization was not observed when only CD163 was expressed; although, cells became productively infected. Thus, sialoadhesin is confirmed as a PRRSV internalization receptor and CD163 is shown to be involved in PRRSV entry, probably during Uncoating. Co-expression of recombinant sialoadhesin and CD163 in non-permissive cells increased Virus production 10–100 times compared with cells expressing only CD163, sustaining the requirement of both for efficient PRRSV infection.
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Involvement of proteases in porcine reproductive and respiratory syndrome Virus Uncoating upon internalization in primary macrophages
Veterinary Research, 2008Co-Authors: Gerald Misinzo, Peter Delputte, Hans NauwynckAbstract:Porcine reproductive and respiratory syndrome Virus (PRRSV) replicates in differentiated macrophages. In macrophages, heparan sulphate glycosaminoglycans mediate the initial PRRSV attachment and the receptor sialoadhesin mediates both PRRSV attachment and internalization into endosomes. Upon a pH drop, PRRSV is uncoated and its genome is released from the endosomes into the cytoplasm, which allows Virus replication. However, expression of heparan sulphate and sialoadhesin in non-susceptible cells only allows Virus internalization, but no Virus Uncoating and infection, indicating that other factors are involved. In the present study, it is shown that treatment of macrophages with serum (mainly the alpha-globulin fraction) inhibited PRRSV infection without affecting attachment and internalization. Because alpha-globulins contain several protease inhibitors, macrophages were treated with different protease inhibitors to investigate the involvement of proteases in PRRSV Uncoating. Treatment of macrophages with broadly active inhibitors of serine or aspartic proteases, but not cysteine- or metallo-proteases, inhibited PRRSV Uncoating and infection. Further investigation using specific inhibitors indicated that the aspartic protease cathepsin E is involved during PRRSV Uncoating, but did not allow identification of the serine protease involved. The involvement of cathepsin E during PRRSV Uncoating was confirmed by partial co-localization of internalized PRRSV with cathepsin E. Furthermore, cathepsin E expression increased with macrophage cultivation, which was positively correlated with an increased susceptibility to PRRSV infection. Together, these data show that, in macrophages, both the aspartic protease cathepsin E and an unidentified trypsin-like serine protease are involved in Uncoating of internalized PRRSV and subsequent infection.
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involvement of sialoadhesin in entry of porcine reproductive and respiratory syndrome Virus into porcine alveolar macrophages
Journal of Virology, 2003Co-Authors: Nathalie Vanderheijden, Peter Delputte, Herman Favoreel, Joel Vandekerckhove, Jozef Van Damme, Petrus Alphonsus Maria Van Woensel, Hans NauwynckAbstract: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.
Peter Delputte - One of the best experts on this subject based on the ideXlab platform.
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sialoadhesin and cd163 join forces during entry of the porcine reproductive and respiratory syndrome Virus
Journal of General Virology, 2008Co-Authors: Hanne Van Gorp, Peter Delputte, Wander Van Breedam, Hans NauwynckAbstract:The porcine reproductive and respiratory syndrome Virus (PRRSV) shows a restricted tropism for subsets of porcine macrophages in vivo. To date, two PRRSV receptors have been identified on primary macrophages, heparan sulphate for binding and sialoadhesin for binding and internalization. However, additional factors are needed because the expression of both receptors in non-permissive cells results in Virus internalization but not in Virus Uncoating and productive infection. Recently, CD163 was described as a PRRSV receptor on Marc-145 cells that renders non-permissive cells susceptible to PRRSV. Therefore, the potential role of CD163 in PRRSV entry in macrophages and its potential interplay with sialoadhesin were studied. Incubation of macrophages at 37 °C with either sialoadhesin- or CD163-specific antibodies reduced PRRSV infection by up to 75 %, while infection was completely blocked by a combination of both antibodies. When incubated at 4 °C, only sialoadhesin- and not CD163-specific antibodies reduced PRRSV infection. In addition, confocal analysis of PRRSV entry in non-permissive cells expressing only sialoadhesin showed PRRSV internalization but no Uncoating. In contrast, when both sialoadhesin and CD163 were expressed, PRRSV was uncoated upon internalization, resulting in productive infection. Virus internalization was not observed when only CD163 was expressed; although, cells became productively infected. Thus, sialoadhesin is confirmed as a PRRSV internalization receptor and CD163 is shown to be involved in PRRSV entry, probably during Uncoating. Co-expression of recombinant sialoadhesin and CD163 in non-permissive cells increased Virus production 10–100 times compared with cells expressing only CD163, sustaining the requirement of both for efficient PRRSV infection.
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Involvement of proteases in porcine reproductive and respiratory syndrome Virus Uncoating upon internalization in primary macrophages
Veterinary Research, 2008Co-Authors: Gerald Misinzo, Peter Delputte, Hans NauwynckAbstract:Porcine reproductive and respiratory syndrome Virus (PRRSV) replicates in differentiated macrophages. In macrophages, heparan sulphate glycosaminoglycans mediate the initial PRRSV attachment and the receptor sialoadhesin mediates both PRRSV attachment and internalization into endosomes. Upon a pH drop, PRRSV is uncoated and its genome is released from the endosomes into the cytoplasm, which allows Virus replication. However, expression of heparan sulphate and sialoadhesin in non-susceptible cells only allows Virus internalization, but no Virus Uncoating and infection, indicating that other factors are involved. In the present study, it is shown that treatment of macrophages with serum (mainly the alpha-globulin fraction) inhibited PRRSV infection without affecting attachment and internalization. Because alpha-globulins contain several protease inhibitors, macrophages were treated with different protease inhibitors to investigate the involvement of proteases in PRRSV Uncoating. Treatment of macrophages with broadly active inhibitors of serine or aspartic proteases, but not cysteine- or metallo-proteases, inhibited PRRSV Uncoating and infection. Further investigation using specific inhibitors indicated that the aspartic protease cathepsin E is involved during PRRSV Uncoating, but did not allow identification of the serine protease involved. The involvement of cathepsin E during PRRSV Uncoating was confirmed by partial co-localization of internalized PRRSV with cathepsin E. Furthermore, cathepsin E expression increased with macrophage cultivation, which was positively correlated with an increased susceptibility to PRRSV infection. Together, these data show that, in macrophages, both the aspartic protease cathepsin E and an unidentified trypsin-like serine protease are involved in Uncoating of internalized PRRSV and subsequent infection.
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involvement of sialoadhesin in entry of porcine reproductive and respiratory syndrome Virus into porcine alveolar macrophages
Journal of Virology, 2003Co-Authors: Nathalie Vanderheijden, Peter Delputte, Herman Favoreel, Joel Vandekerckhove, Jozef Van Damme, Petrus Alphonsus Maria Van Woensel, Hans NauwynckAbstract: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.
Hanne Van Gorp - One of the best experts on this subject based on the ideXlab platform.
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sialoadhesin and cd163 join forces during entry of the porcine reproductive and respiratory syndrome Virus
Journal of General Virology, 2008Co-Authors: Hanne Van Gorp, Peter Delputte, Wander Van Breedam, Hans NauwynckAbstract:The porcine reproductive and respiratory syndrome Virus (PRRSV) shows a restricted tropism for subsets of porcine macrophages in vivo. To date, two PRRSV receptors have been identified on primary macrophages, heparan sulphate for binding and sialoadhesin for binding and internalization. However, additional factors are needed because the expression of both receptors in non-permissive cells results in Virus internalization but not in Virus Uncoating and productive infection. Recently, CD163 was described as a PRRSV receptor on Marc-145 cells that renders non-permissive cells susceptible to PRRSV. Therefore, the potential role of CD163 in PRRSV entry in macrophages and its potential interplay with sialoadhesin were studied. Incubation of macrophages at 37 °C with either sialoadhesin- or CD163-specific antibodies reduced PRRSV infection by up to 75 %, while infection was completely blocked by a combination of both antibodies. When incubated at 4 °C, only sialoadhesin- and not CD163-specific antibodies reduced PRRSV infection. In addition, confocal analysis of PRRSV entry in non-permissive cells expressing only sialoadhesin showed PRRSV internalization but no Uncoating. In contrast, when both sialoadhesin and CD163 were expressed, PRRSV was uncoated upon internalization, resulting in productive infection. Virus internalization was not observed when only CD163 was expressed; although, cells became productively infected. Thus, sialoadhesin is confirmed as a PRRSV internalization receptor and CD163 is shown to be involved in PRRSV entry, probably during Uncoating. Co-expression of recombinant sialoadhesin and CD163 in non-permissive cells increased Virus production 10–100 times compared with cells expressing only CD163, sustaining the requirement of both for efficient PRRSV infection.
Nathalie Vanderheijden - One of the best experts on this subject based on the ideXlab platform.
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involvement of sialoadhesin in entry of porcine reproductive and respiratory syndrome Virus into porcine alveolar macrophages
Journal of Virology, 2003Co-Authors: Nathalie Vanderheijden, Peter Delputte, Herman Favoreel, Joel Vandekerckhove, Jozef Van Damme, Petrus Alphonsus Maria Van Woensel, Hans NauwynckAbstract: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.
Gerald Misinzo - One of the best experts on this subject based on the ideXlab platform.
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Involvement of proteases in porcine reproductive and respiratory syndrome Virus Uncoating upon internalization in primary macrophages
Veterinary Research, 2008Co-Authors: Gerald Misinzo, Peter Delputte, Hans NauwynckAbstract:Porcine reproductive and respiratory syndrome Virus (PRRSV) replicates in differentiated macrophages. In macrophages, heparan sulphate glycosaminoglycans mediate the initial PRRSV attachment and the receptor sialoadhesin mediates both PRRSV attachment and internalization into endosomes. Upon a pH drop, PRRSV is uncoated and its genome is released from the endosomes into the cytoplasm, which allows Virus replication. However, expression of heparan sulphate and sialoadhesin in non-susceptible cells only allows Virus internalization, but no Virus Uncoating and infection, indicating that other factors are involved. In the present study, it is shown that treatment of macrophages with serum (mainly the alpha-globulin fraction) inhibited PRRSV infection without affecting attachment and internalization. Because alpha-globulins contain several protease inhibitors, macrophages were treated with different protease inhibitors to investigate the involvement of proteases in PRRSV Uncoating. Treatment of macrophages with broadly active inhibitors of serine or aspartic proteases, but not cysteine- or metallo-proteases, inhibited PRRSV Uncoating and infection. Further investigation using specific inhibitors indicated that the aspartic protease cathepsin E is involved during PRRSV Uncoating, but did not allow identification of the serine protease involved. The involvement of cathepsin E during PRRSV Uncoating was confirmed by partial co-localization of internalized PRRSV with cathepsin E. Furthermore, cathepsin E expression increased with macrophage cultivation, which was positively correlated with an increased susceptibility to PRRSV infection. Together, these data show that, in macrophages, both the aspartic protease cathepsin E and an unidentified trypsin-like serine protease are involved in Uncoating of internalized PRRSV and subsequent infection.
