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. 2010 Feb;84(4):1731-40.
doi: 10.1128/JVI.01774-09. Epub 2009 Nov 25.

The minor envelope glycoproteins GP2a and GP4 of porcine reproductive and respiratory syndrome virus interact with the receptor CD163

Phani B Das  1 , Phat X Dinh, Israrul H Ansari, Marcelo de Lima, Fernando A Osorio, Asit K Pattnaik
Affiliations

The minor envelope glycoproteins GP2a and GP4 of porcine reproductive and respiratory syndrome virus interact with the receptor CD163

Phani B Das et al. J Virol. 2010 Feb.

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) contains the major glycoprotein, GP5, as well as three other minor glycoproteins, namely, GP2a, GP3, and GP4, on the virion envelope, all of which are required for generation of infectious virions. To study their interactions with each other and with the cellular receptor for PRRSV, we have cloned each of the viral glycoproteins and CD163 receptor in expression vectors and examined their expression and interaction with each other in transfected cells by coimmunoprecipitation (co-IP) assay using monospecific antibodies. Our results show that a strong interaction exists between the GP4 and GP5 proteins, although weak interactions among the other minor envelope glycoproteins and GP5 have been detected. Both GP2a and GP4 proteins were found to interact with all the other GPs, resulting in the formation of multiprotein complex. Our results further show that the GP2a and GP4 proteins also specifically interact with the CD163 molecule. The carboxy-terminal 223 residues of the CD163 molecule are not required for interactions with either the GP2a or the GP4 protein, although these residues are required for conferring susceptibility to PRRSV infection in BHK-21 cells. Overall, we conclude that the GP4 protein is critical for mediating interglycoprotein interactions and, along with GP2a, serves as the viral attachment protein that is responsible for mediating interactions with CD163 for virus entry into susceptible host cell.

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Figures

FIG. 1.
FIG. 1.
Expression of individual minor envelope glycoproteins of PRRSV in transfected cells. (A) Schematic representation of GP2a, GP3, and GP4 proteins, with their sizes in amino acids shown. Potential N-linked glycosylation sites (with residue number and letter Y above of the rectangular boxes), predicted signal sequences (SS), and transmembrane regions (TM) are shown. The amino (N) and carboxy (C) termini are identified. (B) BHK-21 cells were infected with vTF7-3 and subsequently mock transfected or transfected with plasmids encoding individual envelope glycoproteins as shown above the lanes. The cells were radiolabeled with 35S protein labeling mix as described in Materials and Methods, cell extracts were prepared and immunoprecipitated with monospecific polyclonal antibodies as shown below the lanes, and the proteins were detected by SDS-10% PAGE and fluorography. The fully glycosylated proteins are identified by white dots, whereas the partially glycosylated forms of the proteins are identified by black dots, on the left sides of the lanes. Relative mobilities of molecular mass markers in kilodaltons (kDa) are shown on the right. (C) Expression of the GPs in MARC-145 cells. The experiment was done as described for panel B, but the plasmid encoding GP2a with a FLAG tag was used in place of GP2a and the fusion protein was detected with anti-FLAG antibody. The full-length mature GPs are identified with white dots in lanes 2, 4, and 6, whereas the partially glycosylated forms of the proteins are identified by black dots in the lanes. Relative mobilities of molecular mass markers in kDa are shown on the right. (D) Endo H sensitivity of the GPs. The proteins expressed in BHK-21 cells as described above were recovered by immunoprecipitation, treated without (−) or with (+) endo H, and detected by electrophoresis as described above. Mock-transfected cells immunoprecipitated with the antibodies and treated without endo H are shown in lanes 1, 4, and 7. White dots show the fully glycosylated proteins, and black dots show the protein species generated by the enzyme treatment. The protein band identified by an asterisk is most likely the signal-cleaved deglycosylated form of GP2a. Relative mobilities of molecular mass markers in kDa are shown on the right.
FIG. 2.
FIG. 2.
Interaction of PRRSV GPs. (A) Examination of GP interactions using GP2a antibody. BHK-21 cells were infected with vTF7-3 virus and mock transfected (lane 1) or transfected with plasmids encoding the GPs as shown below the panel. − and + indicate transfection without or with the plasmid shown on the left. At 16 h posttransfection, the cells were radiolabeled with 35S protein labeling mix as described in Materials and Methods, cell extracts were prepared and immunoprecipitated with monospecific polyclonal antibodies as shown below the lanes, and the proteins were detected by SDS-12% PAGE and fluorography. The full-length mature GPs are identified with white dots, whereas the partially glycosylated forms of the proteins are identified by black dots, on the left sides of the lanes. Relative mobilities of molecular mass markers in kDa are shown on the right. (B) Examination of GP interactions using anti-GP3 antibody. The experiment was performed as described for panel A but using anti-GP3 antibody. (C) Examination of GP interactions using anti-GP5 antibody. The experiment was performed as described for panel A but using anti-GP5 antibody. Relative mobilities of molecular mass markers in kDa are shown on the right.
FIG. 3.
FIG. 3.
Interaction of GP5 with GP4 is necessary for multiprotein complex formation. BHK-21 cells were infected with vTF7-3 and subsequently mock transfected (lane 1) or transfected with individual or combinations of plasmids encoding various GPs as shown below each lane. Cells were radiolabeled as described in Materials and Methods and immunoprecipitated with antibodies as shown below the lanes. The proteins were detected by SDS-12% PAGE and fluorography. Various GPs are identified on the right. The fully glycosylated GPs are identified with white dots on the left side of the lanes. GP2a (1N), the monoglycosylated form of GP2a, is identified with black dots on the left sides of the lanes. Relative mobilities of molecular mass markers in kDa are shown on the left.
FIG. 4.
FIG. 4.
Expression and functional analysis of CD163. (A) Expression of CD163 in transfected cells. BHK-21 cells were infected with vTF7-3 and transfected with pcDNA3.1 (E. Vector, lane 1) or CD163 cDNA containing clones (no. 1, 2, 3, and 4 [lanes 2 to 5, respectively]). Cells were radiolabeled for 4 h at 16 h posttransfection, and the radiolabeled proteins were analyzed by immunoprecipitation with porcine anti-CD163 monoclonal antibody, resolved by SDS-10% PAGE, and detected by fluorography. Mobilities of full-length CD163 and its truncated form (CD163ΔTM) are shown on the right. Relative mobilities of molecular mass markers in kDa are shown on the left. (B) Susceptibility of BHK-21 cells expressing CD163 encoded in the clones to PRRSV infection. Cells were transfected with the plasmid clones as shown (EV, empty vector control), and expression of CD163 was driven by the CMV promoter in pcDNA3.1 vector. At 48 h after transfection, cells were infected with PRRSV and expression of N protein was examined using the monoclonal antibody SDOW17 and secondary antibody conjugated to Alexa-488. (C) Immunofluorescent staining of BHK-21 cells transfected with empty vector (panels a and b), full-length CD163 receptor encoding clone 2 (panels c and d), or the CD163ΔTM-encoding clone (panels e and f). Cell surface or cytoplasmic staining was performed with CD163 monoclonal antibody and Alexa-488 conjugated secondary antibody.
FIG. 5.
FIG. 5.
Interaction of CD163 with PRRSV GPs. (A) BHK-21 cells were infected with vTF7-3 and transfected with various GPs alone or in combination with CD163 (clone 2). The cells were radiolabeled, and the proteins were immunoprecipitated with antibodies as shown below the lanes. Immunoprecipitated proteins were detected by SDS-10% PAGE and fluorography. Mobilities of molecular mass marker proteins in kDa are shown on the right. CD163 and the fully glycosylated viral GPs are identified on the left. Fully glycosylated GPs are identified with white dots in the fluorograms on the left side of each lane. The monoglycosylated GP2a is identified with black dots on the left side in lanes 3 and 11. (B). Interaction of GP2a and GP4 with CD163ΔTM. The experiment was performed as described for panel A using the clone encoding the CD163ΔTM protein.
FIG. 6.
FIG. 6.
A preliminary model of the PRRSV envelope protein complex and its interaction with CD163 on the host cell plasma membrane. For the sake of simplicity and convenience, the GPs are depicted as globular structures with lines spanning the viral envelope. CD163 is shown as a structure with an extracellular region having repeating units (corresponding to the nine SRCR domains) projecting from the plasma membrane of a host cell. The carboxy-terminal cytoplasmic domain and the transmembrane domains are also shown. The dotted lines in the schematic represent the regions of the proteins that are masked by GP2a.
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