Effects of variation in N-linked glycosylation site on antibody mediated neutralization in PRRSV

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is a 15Kb enveloped positive-sense, single stranded RNA virus. More than two decades after the emergence of PRRSV, much is known about the viral genome, spread, and pathogenesis of the disease but our knowledge of the protective immune response of the host to the PRRS virus remains incomplete. Neutralizing antibodies that are produced late during the course of the infection play a significant role in clearing virus infection. One of the most effective ways for the virus to combat this immune response is to have mutants that can escape the neutralization antibody response. Various studies have been done to indicate that the envelope glycoprotein 5 bears a major neutralizing epitope. The ectodomain of glycoprotein 5 in PRRSV has a neutralization epitope which is flanked by three N-linked glycosylation sites, N34, N44 and N51. It is believed that these glycosylation sites play a role in glycan shielding which is a primary mechanism by which the virus evades neutralizing immune responses. Analysis of GP5 sequences of a subset of field strains showed that the N34 glycosylation site can shift to N33 or N35. Comparing these variations to the virus neutralization titers, we observed that the closer the glycosylation site (N35) was to the epitope, the virus neutralizing titer was significantly lower than when the glycosylation site was further away (N33). The purpose of this study was to study the association between the variation in glycosylation site and its effects on virus neutralization. We hypothesized that the position of the glycosylation site is under selection, it alters the virus neutralization titer and is involved in virus neutralization escape. Mutations carrying the change in glycosylation site at N33, N34 and N35 were introduced into a full length cDNA clone. Western Blots confirmed that predicted glycosylated sites were in fact glycosylated. Serum neutralization assays were performed on glycosylation variant FL-12 constructs using antiserum raised against N34 FL-12. Variation in location of the glycosylation site on the amino terminal side of the putative GP5 neutralization epitope explains some of the variation in neutralization of field strains and could be a novel mechanism by which viruses are adapted to serologic selection. According to our findings, virus neutralization titers for N35 were higher than those for N33 or N34