N-linked glycosylation of GP5 of porcine reproductive and respiratory syndrome virus is critically important for virus replication in vivo

Abstract

It has been proposed that the N-linked glycan addition at certain sites in GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) is important for production of infectious viruses and viral infectivity. However, such specific N-linked glycosylation sites do not exist in some field PRRSV isolates. This implies that the existence of GP5-associated glycan per se is not vital to the virus life cycle. In this study, we found that mutation of individual glycosylation sites at N30, N35, N44, and N51 in GP5 did not affect virus infectivity in cultured cells. However, the mutants carrying multiple mutations at N-linked glycosylation sites in GP5 had significantly reduced virus yields compared with the wild-type (wt) virus. As a result, no viremia and antibody response were detected in piglets that were injected with a mutant without all N-linked glycans in GP5. These results suggest that the N-linked glycosylation of GP5 is critically important for virus replication in vivo. The study also showed that removal of N44-linked glycan from GP5 increased the sensitivity of mutant virus to convalescent-phase serum samples but did not elicit a high-level neutralizing antibody response to wt PRRSV. The results obtained from the present study have made significant contributions to better understanding the importance of glycosylation of GP5 in the biology of PRRSV.

Fig 1

Site-directed mutagenesis of N-linked glycosylation sites of PRRSV GP5. (A) Schematic representation of the 3′ part of the PRRSV genome. Boxed numbers represent the open reading frames encoding structural proteins, including the recently reported ORF5a. (B) Alignment of amino acid sequences of the N-terminal portions of GP5 of different PRRSV strains. The GenBank accession numbers for GP5 of the eight PRRSV strains from top to bottom are ACV95344, EF112445, U87392, ADN42874, AAD37076, ABY68567, AF184212, and JF422072, respectively. The amino acids identical to those in GP5 of JXM100 are represented by dots. Four potential N-glycosylation sites (N-X-T/S) at positions N30, N35, N44, and N51 in PRRSV strain JXM100 and the corresponding amino acids in other strains are boxed. (C) Knockout of the glycosylation signal by site-directed mutagenesis. Alignment of amino acid sequences of GP5 ectodomains of the pAJXM backbone and mutants is shown. In the N-glycosylation consensus sequence of all mutants, N-X-T/S, the first amino acid of the motif N codon is replaced with an S, Y, or K, or the third codon of the motif S is replaced with N or R. To avoid the regeneration of the N33 or N34 glycosylation site, when the N35 glycosylation site mutant was constructed the overlapping glycosylation site NNNSS was mutated to YSSSS. The rationale of the mutagenesis is based on the mutated amino acids that are present in the natural isolates as shown in panel B. (D) The amino acid mutations in the small ORF5a protein. The amino acids identical to those of the ORF5a protein of pAJXM are represented by dots.

Fig 2

Single mutations at N-linked glycosylation sites in GP5 do not affect the infectious virus recovery in MARC-145 cells. The mutants with single amino acid substitutions at N-glycosylation sites, along with the parental pAJXM full-length plasmids, were transfected into BHK-21 cells. The transfectant supernatants were harvested at 48 hpt and inoculated into young MARC-145 cells. (A) Expression of PRRSV N protein was visualized in IFA. The infected MARC-145 cells were fixed and stained at 24 hpi with the anti-N protein monoclonal antibody (D5-4) and anti-mouse secondary antibody labeled with Alexa Fluor. Images were taken at a magnification of ×200. (B) Multistep growth kinetics of wt and mutant viruses in MARC-145 cells. Cells in six-well plates were infected with PRRSV at an MOI of 0.01. The culture supernatants were collected at the indicated time points and titrated. The geometric mean titers with standard deviations (error bars) from three independent experiments are shown. (C) Viral plaque size. Transfectant supernatants were 10-fold serially diluted and inoculated into young MARC-145 cells in six-well-plates. The MARC-145 cell monolayers were cultured in EMEM containing a 1% agarose overlay, fixed at 4 dpi, and stained with 1% crystal violet.

Fig 3

N-linked glycosylation of GP5 is not essential for virus viability in MARC-145 cells. (A) Examination of MARC-145 cells at 24 hpi with wt and mutants bearing double, triple, or quadruple glycosylation site mutations in GP5. The IFA analysis was performed at 24 hpi using the anti-N protein monoclonal antibody (D5-4). Images were taken at a magnification of ×200. (B) Multistep growth kinetics of wt and mutants bearing double and triple glycosylation site mutations in GP5 in MARC-145 cells. Cells in six-well plates were infected with PRRSV at an MOI of 0.01. Culture supernatants were collected at the indicated time points and titrated. The geometric mean titers with standard deviations (error bars) from three independent experiments are shown. (C) Plaque size in MARC-145 cells infected with wt and mutant viruses.

Fig 4

Confirmation of glycosylation status of GP5 in MARC-145 cells infected with the mutants. (A) Expression profiles of GP5 of PRRSV wt and mutants carrying single glycosylation site mutations in the infected MARC-145 cells. The infected MARC-145 cells were harvested at 24 hpi and solubilized. The lysates were treated with (+) or without (−) PNGase F and then separated by 12% SDS-PAGE followed by blotting against anti-GP5 monoclonal antibody (HP-GP5) and staining with HRP-labeled antibody. Molecular mass markers are shown (kDa) to the left of each panel. (B) Expression profiles of GP5 of PRRSV wt and mutants carrying multiple glycosylation site mutations in the infected MARC-145 cells. The experiments were performed as described for panel A.

Fig 5

Infectivity of wt virus and mutants bearing multiple glycosylation site mutations in GP5 in PAMs. (A) Examination of infected PAMs in IFA. Equal numbers of PRRSV particles were used to inoculate PAMs. The IFA was performed at 24 hpi using the anti-N protein monoclonal antibody (D5-4). (B) Titers of wt and mutants bearing glycosylation site mutations in GP5. Cells in six-well plates were infected with PRRSV at equal numbers of viral particles. Culture supernatants were collected at 24 hpi and titrated. The geometric mean titers (TCID₅₀/ml) with standard deviations (error bars) from three independent experiments are shown. Means with different symbols (#, ★, and §) indicate significant differences (P < 0.05).

Fig 6

Antibody responses in piglets infected with wt and mutants. (A) Mean anti-N antibody levels. PRRSV-specific N antibody development was monitored throughout the experimental period and represented as S/P ratios. The S/P ratios greater than 0.4 were considered positive. Anti-N-PRRSV antibodies were quantitated in a HerdChek ELISA. (B) Kinetics of neutralization antibody response to homologous viruses. Three 4-month-old piglets were inoculated with the indicated viruses as described in Materials and Methods. The neutralizing activities of serum samples were tested against homologous viruses. Neutralization titers are expressed as means ± standard errors of the means.