In the present study, we examined the influence of glycosylation of GP5 of PRRSV on recovery of infectious virus and its role in the ability of the mutant viruses to be neutralized by antibodies and in inducing neutralizing antibodies in vivo. We have found that all three potential glycosylation sites (N34, N44, and N51) in GP5 are used for the addition of glycan moieties. Our results reveal that glycan addition at the N44 site is most critical for the recovery of infectious virus. Furthermore, our results show that PRRSVs containing hypoglycosylated forms of GP5 are exquisitely sensitive to neutralization by antibodies and that the mutant viruses induce significantly higher levels of neutralizing antibodies not only to the homologous mutant viruses but also to wt PRRSV.
Confirmation that all three potential N-linked glycosylation sites are used for glycan addition in GP5 was provided by using mutants with alterations at single or multiple sites (Fig. ). Biochemical studies showed that the PRRSV GP5 protein, when coexpressed with M protein in transfected cells, contains Endo H-sensitive high-mannose-type glycans. The observation that the majority of GP5 incorporated into virions is resistant to Endo H (Fig. ) whereas GP5 expressed in the presence of M protein in transfected cells is fully Endo H sensitive is intriguing. It is possible that GP5 expressed in the presence of M protein in transfected cells accumulates mostly in the endoplasmic reticulum or in the cis-Golgi region and therefore remains Endo H sensitive. However, in PRRSV-infected cells, GP5 may interact with additional viral proteins and the transport of GP5 beyond the endoplasmic reticulum or cis-Golgi apparatus is facilitated through the formation of complexes with other viral proteins. Consistent with this interpretation, we have observed that in wt or mutant PRRSV-infected cells, GP5 protein is also resistant to Endo H.
We suggest that GP5, which is synthesized in the endoplasmic reticulum in infected cells, is transported to the medial and/or trans
-Golgi region where the majority of GP5 molecules acquire Endo H resistance prior to being incorporated into PRRSV virions. Several studies with arteriviruses, including PRRSV, suggest that GP5 and M protein form a heterodimer, which may play a key role in viral infectivity (13
). In EAV and LDV, direct interaction of GP5 and M protein through the formation of disulfide bridges has been demonstrated (13
). Such interactions may occur prior to further processing of N-linked oligosaccharide side chains, presumably before GP5 is transported out of the endoplasmic reticulum or the cis
It is interesting that the pattern of Endo H resistance of GP5 incorporated into wt and mutant virions is different. While the majority of GP5 molecules in wt PRRSV were Endo H resistant, most of the GP5 molecules in the single-site mutant virions (FL-N34A and FL-N51A) were Endo H sensitive (Fig. ). Furthermore, of the two glycan moieties in these mutants, only one was sensitive, while the other was resistant. The double mutant (FL-N34/51A) virion also incorporated GP5 that contained glycans, some of which were also sensitive to Endo H. These data are consistent with the interpretation that wt as well as mutant PRRSV virions incorporate a mixed population of GP5 molecules that contain different glycan moieties at different sites. Previous studies demonstrating incorporation of differentially glycosylated forms of GP5 into wt PRRSV virions (28
) further strengthen our interpretation.
From the pattern of Endo H sensitivity of GP5 incorporated into the virions, it is tempting to speculate that the N44 site may contain the Endo H-resistant glycans, although some GP5 molecules with Endo H-sensitive glycans at this site were incorporated into virions. Whether this unusual pattern of glycans at various sites in GP5 and incorporation of various forms of GP5 into virions has any relevance to the pattern of immune response seen in PRRSV-infected animals remains to be investigated.
In a recent study, it was shown that of the two N-linked glycosylation sites (N46 and N53) in GP5 of Lelystad PRRSV, glycosylation of N46 residue was strongly required for virus particle production. Infectious virus yield was reduced by approximately 100-fold by a mutation at N46 (53
). Our results suggest that glycan addition at N44 (for North American PRRSV) is absolutely essential for recovery of infectious PRRSV. It is possible that the European and North American isolates of PRRSV differ somewhat in their requirements for N-linked glycosylation for production of infectious viruses. In this regard, it is of note that the Lelystad virus contains only two N-linked glycosylation sites, whereas the North American isolates we have used in this study contain three such sites.
GP5 is the most important glycoprotein of PRRSV involved in the generation of PRRSV-neutralizing antibodies and protective immunity. Our results reveal that the absence of glycans at residues 34 and 51 in the GP5 ectodomain, while generating viable PRRSV mutants, enhances both the sensitivity of these mutants to neutralization by antibodies and the immunogenicity of the nearby neutralization epitope. The immediate effect of the absence of glycans in GP5 of mutant PRRSVs has been increased sensitivity of the viruses to neutralization by convalescent-phase sera from pigs infected with wt PRRSV (Table ).
Studies with human immunodeficiency virus type 1 and simian immunodeficiency virus have shown that acquisition or removal of glycans in the variable loops of gp160 modifies their sensitivity to neutralization (5
). Therefore, it has been postulated (41
) that glycans play at least two types of essential roles during biosynthesis of viral envelope glycoproteins. In one case, lack of glycans entails defects of the glycoprotein and thus in the overall viability of the viral strain. We postulate that the glycans at N44 of PRRSV GP5 serve a similar role. In the second case, the glycans potentially serve to shield viral proteins against neutralization by antibodies (41
). For PRRSV GP5, glycans at N34 and N51 may have a similar role. In the case of human immunodeficiency virus, “glycan shielding” is postulated to be a primary mechanism to explain evasion from neutralizing immune response, ensuring in vivo persistence of human immunodeficiency virus (50
This invites us to draw some parallels with PRRSV. Infection with PRRSV, which is known to persist for several months in individual animals (3
), presents an unusual behavior in terms of induction of virus-specific neutralizing immune response (31
). It is well documented that animals infected with PRRSV usually take longer than normal to establish a detectable PRRSV-neutralizing antibody response. Once established, this PRRSV-neutralizing response is weak, and varies significantly from animal to animal (31
). The delay in neutralizing antibody response has been postulated to be due to the presence of a nearby immunodominant decoy epitope (amino acid positions 27 to 30), which evokes a robust, early, nonprotective immune response that masks and/or slows the response to the neutralizing epitope (amino acid positions 37 to 45) (26
). While this is a plausible explanation for the atypical character of the PRRSV-neutralizing antibody response, it remains to be tested. In our laboratory, deletion of the decoy epitope has consistently proven lethal to the recovery of infectious PRRSV (I. H. Ansari et al., unpublished results), making it difficult to test this hypothesis.
It is possible that an alternative or complementary mechanism to explain the peculiar nature of the PRRSV-neutralizing response could be envisioned by the glycan-shielding phenomenon proposed for the human and simian immunodeficiency viruses (22
). The use of mutant PRRSVs lacking one or two glycan moieties in our studies provides evidence for the first time of the presence of large amounts of PRRSV-neutralizing antibodies in the sera of wt PRRSV-infected animals that were otherwise undetectable because of the use of wt PRRSV in the serum neutralization assays. The PRRSV-neutralizing antibodies, while present in the host's response, are unable to react with the infecting wt PRRSV virions due to blocking or shielding of the neutralizing epitope by the glycan moieties on GP5.
One important precedent for neutralization escape by glycosylation of glycoproteins in arteriviruses has been described for LDV. It is highly resistant to antibody neutralization due to the heavy glycan shielding of its major glycoprotein, VP-3 (8
). However, certain naturally occurring strains of LDV are highly susceptible to neutralization due to loss of two glycosylation sites on the ectodomain of VP-3. Interestingly, this neutralization-sensitive phenotype correlates with a high degree of neurotropism in the host acquired by these easily neutralizable LDV strains. Such enhancement of neuropathogenicity probably reflects the facilitation of interaction of the viral glycoproteins with receptors in neural cells, possibly due to the absence of glycan shielding (8
In the young-pig model that we used for inoculation with PRRSV, we were not able to detect pathogenic differences between any of the mutant PRRSVs and wt PRRSV, although we limited our observations to temperature and viremia measurements. It is possible that under different experimental conditions (i.e., in a pregnant-sow model), some alterations in the pathogenicity of these mutant PRRSVs might be observed. It is not known whether naturally occurring hypoglycosylated PRRSV strains are common, although previous reports have suggested their presence (27
A remarkable observation in our experiments has been that the GP5 mutants, when infecting pigs in vivo, can outperform wt PRRSV in their ability to mount a sizable wt PRRSV-neutralizing response at late phases of infection (Table ). Our results seem to mimic one of the most dramatic effects of viral glycoprotein carbohydrate removal so far reported in the literature by Reitter et al. (40
). These authors observed that rhesus monkeys infected with simian immunodeficiency virus mutants lacking selected glycans moieties mount higher neutralization responses not only against the mutant virus but also against wt simian immunodeficiency virus than those caused by the wt virus itself. In a parallel scenario, we have observed not only higher neutralizing titers against homologous PRRSV mutants but also sizable titers against wt PRRSV (Table ). In addition, the response occurred earlier, with neutralizing titers detectable at 14 days postinfection, an observation not typically noted with wt PRRSV infection (Table ).
The increased neutralization of wt PRRSV by sera from pigs infected with the PRRSV mutants suggests that glycans were masking a neutralizing epitope(s) that does not induce neutralizing antibodies when glycans are present. This observation has great significance for the design of better, more efficacious PRRSV vaccines, suggesting that new, rationally designed vaccines should carry modifications in the glycosylation pattern of GP5 in order to enhance the production of neutralizing antibodies. In addition, it will be important to study the effects that this removal of carbohydrates from immunologically prominent glycoproteins of PRRSV may have on increasing serum neutralization titers not only to the homologous immunizing strain, but also to diverse unrelated PRRSV strains.