Influence of N-linked glycosylation of porcine reproductive and respiratory syndrome virus GP5 on virus infectivity, antigenicity, and ability to induce neutralizing antibodies

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 5 (GP5) is the most abundant envelope glycoprotein and a major inducer of neutralizing antibodies in vivo. Three putative N-linked glycosylation sites (N34, N44, and N51) are located on the GP5 ectodomain, where a major neutralization epitope also exists. To determine which of these putative sites are used for glycosylation and the role of the glycan moieties in the neutralizing antibody response, we generated a panel of GP5 mutants containing amino acid substitutions at these sites. Biochemical studies with expressed wild-type (wt) and mutant proteins revealed that the mature GP5 contains high-mannose-type sugar moieties at all three sites. These mutations were subsequently incorporated into a full-length cDNA clone. Our data demonstrate that mutations involving residue N44 did not result in infectious progeny production, indicating that N44 is the most critical amino acid residue for infectivity. Viruses carrying mutations at N34, N51, and N34/51 grew to lower titers than the wt PRRSV. In serum neutralization assays, the mutant viruses exhibited enhanced sensitivity to neutralization by wt PRRSV-specific antibodies. Furthermore, inoculation of pigs with the mutant viruses induced significantly higher levels of neutralizing antibodies against the mutant as well as the wt PRRSV, suggesting that the loss of glycan residues in the ectodomain of GP5 enhances both the sensitivity of these viruses to in vitro neutralization and the immunogenicity of the nearby neutralization epitope. These results should have great significance for development of PRRSV vaccines of enhanced protective efficacy.

FIG. 1.

Transient expression of PRRSV GP5 and M protein. (A) Schematic of the bicistronic construct showing the GP5 and M coding regions flanking the IRES from EMCV (I_E). The coding regions are under the control of the T7 RNA polymerase promoter (black rectangle) present immediately upstream of the GP5 coding region. The bent arrow shows the position and direction of transcription by T7 RNA polymerase from the vector. (B) Expression of GP5 and M proteins in cells transfected with the bicistronic vector. Mock-transfected (lane 1) or plasmid-transfected cells (lanes 2 to 7) were radiolabeled as described in Materials and Methods and immunoprecipitated with anti-GP5 antibody (lanes 1 to 5) or anti-M antibody (lanes 6 and 7). Immunoprecipitated proteins were left untreated (−) (lanes 1, 2, 6, and 7) or treated (+) with Endo H (lane 3) or PNGase F (lane 4) and analyzed by electrophoresis. Lane 5 contains immunoprecipitated proteins from transfected cells treated or not with tunicamycin. The mobility of proteins is shown in kilodaltons. The asterisk identifies a cellular protein that coimmunoprecipitates with anti-M antibody.

FIG. 2.

Glycosylation analysis of wt GP5 and its mutants in transfected cells. (A) Schematic of the bicistronic vector and PRRSV GP5 with the three putative glycosylation sites at amino acid positions 34, 44, and 51 shown. (B) Various mutants used in the present study. (C) Expression of wt and mutant GP5 and their sensitivity to Endo H. The experiment was performed as described in the legend to Fig. 1; proteins were immunoprecipitated with anti-GP5 antibody, digested with Endo H (+) or left undigested (−), and analyzed by electrophoresis. Mutant GP5 proteins are shown by open and shaded arrows. The protein band (∼17 kDa) identified by the asterisk is generated by Endo H digestion of the wt and mutant GP5 proteins. (D) Expression of the triple mutant GP5 N34/44/51A from plasmids with and without M. Cells were mock transfected (lane 1) or transfected with plasmids encoding the triple mutant-containing M coding region (GP5-N34/44/51A, lanes 2 and 3) or without the M coding region (GP5-N34/44/51A*, lanes 4 and 5). Proteins were radiolabeled and detected with anti-GP5 antibody as described above. The mobility of proteins is shown in kilodaltons on the right.

FIG. 3.

Characterization of mutant viruses encoding mutant GP5. (A) Single-step growth kinetics of wt (FL-12) and various mutant PRRSVs in MARC-145 cells. Cells in six-well plates were infected with PRRSV at an MOI of 3, culture supernatants were collected at the indicated times after infection, and virus titers were determined. Average titers with standard deviations (error bars) from three independent experiments are shown. (B) Plaque morphology of mutant viruses. Arrows and arrowheads show plaques that are less clear. Boxes show representative plaques that have been magnified in succeeding panels. (C) Magnified images of representative plaques from wt PRRSV (FL-WT) and one mutant virus (FL-N34/51A). (D) trans-Complementation to recover mutant PRRSVs; quantitative analysis of mutant virus recovery from cells expressing wt GP5 protein. The average yields of viruses from three independent experiments with standard deviations (represented by error bars) are shown.

FIG. 4.

Examination of GP5 incorporated into mutant virions and synthesized in mutant virus-infected cells or in transfected cells. (A) Radiolabeled virions from culture supernatants of infected cells were pelleted, GP5 protein was immunoprecipitated, treated with Endo H (+) or not (−), and analyzed by electrophoresis. The positions of wt GP5 without and with Endo H digestion (lanes 1 and 2, respectively) are shown by white brackets. (B) Cells infected with various mutant viruses were radiolabeled, GP5 was immunoprecipitated, treated with Endo H (+) or not (−), and analyzed by electrophoresis. The positions of wt GP5 without and with Endo H digestion (lanes 2 and 3, respectively) are shown by white brackets. (C) Pulse-chase analysis and Endo H sensitivity of GP5 expressed in transfected cells. Cells were transfected with GP5-I_E-M, pulse-labeled for 2 h (2hrP), and subsequently chased for 2 h (2hrC) or 4 h (4hrC). Proteins were immunoprecipitated with anti-GP5 antibody, digested with Endo H (+) or not (−), and analyzed by electrophoresis as described in Materials and Methods. (D) Pulse-chase analysis and Endo H sensitivity of GP5 in cells infected with wt PRRSV. The experiment was performed as in panel C. The mobility of proteins is shown in kilodaltons on the right side of each panel.