Virions of porcine reproductive and respiratory syndrome virus (PRRSV) contain six membrane proteins: the major proteins GP 5 and M and the minor proteins GP 2a , E, GP 3 , and GP 4 . Here, we studied the envelope protein requirements for PRRSV particle formation and infectivity using full-length cDNA clones in which the genes encoding the membrane proteins were disrupted by site-directed mutagenesis. By transfection of RNAs transcribed from these cDNAs into BHK-21 cells and analysis of the culture medium using ultracentrifugation, radioimmunoprecipitation, and real-time reverse transcription-PCR, we observed that the production of viral particles is dependent on both major envelope proteins; no particles were released when either the GP 5 or the M protein was absent. In contrast, particle production was not dependent on the minor envelope proteins. Remarkably, in the absence of any one of the latter proteins, the incorporation of all other minor envelope proteins was affected, indicating that these proteins interact with each other and are assembled into virions as a multimeric complex. Independent evidence for such complexes was obtained by coexpression of the minor envelope proteins in BHK-21 cells using a Semliki Forest virus expression system. By analyzing the maturation of their N-linked oligosaccharides, we found that the glycoproteins were each retained in the endoplasmic reticulum unless expressed together, in which case they were collectively transported through the Golgi complex to the plasma membrane and were even detected in the extracellular medium. As the PRRSV particles lacking the minor envelope proteins are not infectious, we hypothesize that the virion surface structures formed by these proteins function in viral entry by mediating receptor binding and/or virus-cell fusion.Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the family of Arteriviridae, which also comprises Equine arteritis virus (EAV), Lactate dehydrogenase-elevating virus (LDV), and Simian hemorrhagic fever virus (24,42). This family belongs to the order of Nidovirales, together with the Coronaviridae, Toroviridae, and Roniviridae (2, 3). Arteriviruses are enveloped RNA viruses that contain a positive-strand RNA genome and synthesize a 3Ј nested set of six or eight subgenomic RNAs (sgRNAs) that encode the structural proteins. Analyses of purified virions of PRRSV have indicated that they are composed of seven proteins, i.e., four envelope glycoproteins named GP 2a (encoded by open reading frame 2a [ORF2a]), GP 3 (ORF3), GP 4 (ORF4), and GP 5 (ORF5); a nonglycosylated membrane protein M (ORF6); the nucleocapsid protein N (ORF7); and a nonglycosylated envelope protein, E, that is expressed from a second ORF (ORF2b) entirely contained within ORF2 (22,25,35,48). The 29-to 30-kDa GP 2a and 31-to 35-kDa GP 4 proteins are both putative class I integral membrane proteins with an N-terminal signal sequence and a C-terminal membrane anchor, containing two and four predicted N-glycosylation sites, respectively (22). T...

The two N-terminal cleavage products, nsp1a and nsp1b, of the replicase polyproteins of porcine reproductive and respiratory syndrome virus (PRRSV) each contain a papain-like autoproteinase domain, which have been named PCPa and PCPb, respectively. To assess their role in the PRRSV life cycle, substitutions and deletions of the presumed catalytic cysteine and histidine residues of PCPa and PCPb were introduced into a PRRSV infectious cDNA clone. Mutations that inactivated PCPa activity completely blocked subgenomic mRNA synthesis, but did not affect genome replication. In contrast, mutants in which PCPb activity was blocked proved to be non-viable and no sign of viral RNA synthesis could be detected, indicating that the correct processing of the nsp1b/nsp2 cleavage site is essential for PRRSV genome replication. In conclusion, the data presented here show that a productive PRRSV life cycle depends on the correct processing of both the nsp1a/nsp1b and nsp1b/nsp2 junctions.

A set of neutralizing monoclonal antibodies (mAbs) directed against the GP 5 protein of European type porcine reproductive and respiratory syndrome virus (PRRSV) has been produced previously (Weiland et al., 1999). This set reacted with a plaque-purified virus (PPV) subpopulation of Dutch isolate Intervet-10 (I-10), but not with the European prototype PRRSV LV. In order to map the neutralization epitope in the GP 5 protein of the PPV strain, the ORF5 nucleotide sequence of PPV was determined. When the amino acid sequence derived from this nucleotide sequence was compared with that of PRRSV LV, four amino acid differences were found. Using site-directed mutagenesis, we showed that a proline residue at position 24 of the GP 5 sequence of the PPV strain enabled recognition by the neutralizing mAbs. Pepscan analysis demonstrated that the epitope recognized by the neutralizing mAbs stretched from residues 29 to 35. Surprisingly, the reactivity of the mAbs in the Pepscan system was independent of the presence of a proline in position 24. Moreover, residue 24 is located within the predicted signal peptide, implying that either the signal peptide is not cleaved or is cleaved due to the presence of Pro 24 such that the epitope remains intact. Our results demonstrate the presence of a neutralization epitope in the N-terminal ectodomain of the GP 5 protein of PRRSV and imply a role for the ectodomain of GP 5 in the infection of PRRSV.

The arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) contains four glycoproteins, GP 2a , GP 3 , GP 4 and GP 5 , the functions of which are still largely unresolved. In this study, the significance of the N-glycosylation of the GP 2a and GP 5 proteins of PRRSV strain LV was investigated. Both glycoproteins contain two predicted N-glycosylation sites that are highly conserved between North American-type and European-type PRRSV. Using site-directed mutagenesis, single and double mutant full-length PRRSV cDNA clones were generated. After analysing the expression of the mutant proteins and the actual use of the four putative glycosylation sites in the wild-type proteins, the production of mutant virus particles and their infectivities were investigated. The results showed that the N-linked glycans normally present on the GP 2a protein are not essential for particle formation, as is the oligosaccharide attached to N53 of the GP 5 protein. In contrast, the oligosaccharide linked to N46 of the GP 5 protein is strongly required for virus particle production. The specific infectivities of the mutant viruses were investigated by comparing their infectivity-per-particle ratios with that of wild-type virus. The results showed that the lack of either one or both of the N-linked oligosaccharides on GP 2a or of the oligosaccharide attached to N53 of GP 5 did not significantly affect the infectivities of the viruses. In contrast, the two recombinant viruses lacking the oligosaccharide bound to N46 exhibited a significantly reduced specific infectivity compared with the wild-type virus. The implications of the differential requirements of the modifications of GP 2a and GP 5 for PRRSV assembly and infectivity are discussed.

We used an infectious cDNA clone of Porcine reproductive and respiratory syndrome virus (PRRSV) to investigate the presence of essential replication elements in the region of the genome encoding the structural proteins. Deletion analysis showed that a stretch of 34 nucleotides (14653 to 14686) within ORF7, which encodes the nucleocapsid protein, is essential for RNA replication. Strand-specific reverse transcription-PCR analysis of viral RNA isolated from transfected BHK-21 cells revealed that this region is required for negative-strand genomic RNA synthesis. The 34-nucleotide stretch is highly conserved among PRRSV isolates and folds into a putative hairpin. A 7-base sequence within the loop of this structure was suggested to base-pair with a sequence present in the loop of a hairpin located in the 3 noncoding region, resulting in a kissing interaction. Mutational analyses confirmed that this kissing interaction is required for RNA replication.Positive-strand RNA viruses replicate in infected cells by a process that is mediated by RNA-dependent RNA polymerase (RdRp). In this process, genomic RNA serves as a template for the production of negative-strand antigenomic RNA, which is used in turn as a template for the synthesis of new plus strands. The process of replication requires the recruitment of the RdRp to specific sequences or structures within the templates, also known as cis-acting replication elements. These elements are usually located in the noncoding regions at the termini of the viral RNA, where RdRp complexes initiate the synthesis of plus and minus strands (2). Only a few examples are known of cis-acting replication elements that reside within a coding region (7,10,11,20).Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-stranded RNA virus that belongs to the Arteriviridae family (reviewed in reference 24), together with Equine arteritis virus (EAV), Lactate dehydrogenase-elevating virus (LDV), and Simian hemorrhagic fever virus (SHFV) (17). On the basis of their similar genomic organization and replication strategy, the arteriviruses have been grouped into the order of Nidovirales together with the coronaviruses and the toroviruses (3).The genome of PRRSV is 15.1 kb (17), of which the 5Ј two-thirds is translated into two large polyproteins. These are subsequently cleaved by virus-encoded proteases to yield at least 12 nonstructural proteins, including the viral RdRp (reviewed in reference 24). In addition, a set of subgenomic mRNAs, collectively expressing the viral structural proteins, are produced through a process of discontinuous mRNA transcription (5,8,14,16). This process has not been unraveled completely and may occur during plus-or minus-strand synthesis (reviewed in references 9, 23, and 26). Besides the coding regions, the PRRSV genome contains a 5Ј untranslated region (5ЈUTR) of 221 nucleotides (nt) (24), which carries a cap at its 5Ј end (15, 21), and a 3ЈUTR of 114 nt to which the poly(A) tail is attached (17).Little is known about the requirements for arterivi...