bArteriviruses are enveloped positive-strand RNA viruses for which the attachment proteins and cellular receptors have remained largely controversial. Arterivirus particles contain at least eight envelope proteins, an unusually large number among RNA viruses. These appear to segregate into three groups: major structural components (major glycoprotein GP5 and membrane protein [M]), minor glycoproteins (GP2a, GP3, and GP4), and small hydrophobic proteins (E and the recently discovered ORF5a protein). Biochemical studies previously suggested that the GP5-M heterodimer of porcine reproductive and respiratory syndrome virus (PRRSV) interacts with porcine sialoadhesin (pSn) in porcine alveolar macrophages (PAM). However, another study proposed that minor protein GP4, along with GP2a, interacts with CD163, another reported cellular receptor for PRRSV. In this study, we provide genetic evidence that the minor envelope proteins are the major determinant of arterivirus entry into cultured cells. A PRRSV infectious cDNA clone was equipped with open reading frames (ORFs) encoding minor envelope and E proteins of equine arteritis virus (EAV), the only known arterivirus displaying a broad tropism in cultured cells. Although PRRSV and EAV are only distantly related and utilize diversified transcription-regulating sequences (TRSs), a viable chimeric progeny virus was rescued. Strikingly, this chimeric virus (vAPRRS-EAV2ab34) acquired the broad in vitro cell tropism of EAV, demonstrating that the minor envelope proteins play a critical role as viral attachment proteins. We believe that chimeric arteriviruses of this kind will be a powerful tool for further dissection of the arterivirus replicative cycle, including virus entry, subgenomic RNA synthesis, and virion assembly.
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 glycanper seis 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 replicationin 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.
It has been proposed that the N-linked glycan of the minor proteins of porcine reproductive and respiratory syndrome virus (PRRSV) is important for the production of infectious virus. In this study, we showed that N-linked glycosylation of GP2 is not essential for virus viability and none of the individual glycosylation sites in GP3 has a vital effect on the production of infectious virus. Moreover, mutations of single and double glycosylation sites in GP4 are not critically important for infectious virus recovery, triple and quadruple mutations are lethal. The bimolecular fluorescence complementation (BiFC) analysis also showed that GP4, but might be not GP2, is involved in interaction with cellular receptor CD163 and that glycosylation of GP4 might not play a vital role in the interaction with CD163. The study further revealed that none of the N-glycosylation sites in the minor proteins is critical for the susceptibility of mutants to neutralizing antibody.
Nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) is the most abundant viral structural protein with high immunogenicity. Previously, the nonessential sequences for virus infectivity were identified at both N and C terminal ends of N protein. Here, by means of reverse genetics, a marker virus (v7APMa) was generated with a mutant N protein that differs from the wild-type strains (vAPRRS, type 2 PRRSV). v7APMa shows stable inheritance in cell culture and the virologic characteristics of the marker virus in vitro showed that v7APMa replicates well as its parental strain. In the pig model, the v7APMa marker virus induced a similar level of anti-N protein antibodies and robust antibodies against the marker peptide, from 14 days post infection. In addition, a peptide-based ELISA was developed to detect the specific antibodies for the introduced 7APMa peptide. This approach, using a rationally designed marker virus, provides a new potential mutant basis for further development of PRRSV novel vaccines.
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