ORF5a Protein of Porcine Reproductive and Respiratory Syndrome Virus is Indispensable for Virus Replication

Abstract

Porcine reproductive and respiratory syndrome (PRRS) was first recognized in 1987 in the United States and shortly thereafter in Europe [12, 35]. The disease has since continued to plague nearly all pig-producing countries, causing severe economic losses in the global swine industry [1, 26]. The etiological agent of PRRS, the PRRS virus (PRRSV), was isolated almost simultaneously in Europe and North America in the early 1990s [3, 6, 36]. PRRSV is a member of the family Arteriviridae including equine arteritis virus (EAV), lactate dehydrogenase-elevating virus of mice, and simian hemorrhagic fever virus, which forms the order Nidovirales along with the Coronaviridae family [5, 19, 31]. Since the emergence, PRRSV has evolved divergently on the two continents and consequently, consists of two major genotypes, European (type 1) and North American (type 2) [9, 10, 23, 28]. The two genotypes exhibits antigenic and genetic variations, sharing only about 60% sequence identity at the genome level [24, 18]. PRRSV is a small enveloped virus with a singlestranded, positive-sense RNA genome of ~15 kb in size. The PRRSV genome possesses the 5' cap structure and 3' polyadenylated tail and constitutes the 5' untranslated region (UTR), ten open reading frames (ORF1a, ORF1b, ORF2a, ORF2b, and ORFs 3 through 7 including ORF5a), and the 3' UTR [8, 11, 22, 30, 36]. The two large ORF1a and 1b occupying the 5' two-third of the genome encode the ORF1a and ORF1ab polyproteins by a ribosome frameshifting mechanism that are translated directly from the genomic RNA. The polyproteins are then autocleaved into 14 protease and replicase-associated nonstructural proteins In this study, a DNA-launched reverse genetics system was developed from a type 2 porcine reproductive and respiratory syndrome virus (PRRSV) strain, KNU-12. The complete genome of 15,412 nucleotides was assembled as a single cDNA clone and placed under the eukaryotic CMV promoter. Upon transfection of BHK-tailless pCD163 cells with a full-length cDNA clone, viable and infectious type 2 progeny PRRSV were rescued. The reconstituted virus was found to maintain growth properties similar to those of the parental virus in porcine alveolar macrophage (PAM) cells. With the availability of this type 2 PRRSV infectious clone, we first explored the biological relevance of ORF5a in the PRRSV replication cycle. Therefore, we used a PRRSV reverse genetics system to generate an ORF5a knockout mutant clone by changing the ORF5a translation start codon and introducing a stop codon at the 7 codon of ORF5a. The ORF5a knockout mutant was found to exhibit a lack of infectivity in both BHK-tailless pCD163 and PAM-pCD163 cells, suggesting that inactivation of ORF5a expression is lethal for infectious virus production. In order to restore the ORF5a gene-deleted PRRSV, complementing cell lines were established to stably express the ORF5a protein of PRRSV. ORF5a-expressing cells were capable of supporting the production of the replicationdefective virus, indicating complementation of the impaired ORF5a gene function of PRRSV in trans.