Virol. 68:5755C5764 [PMC free article] [PubMed] [Google Scholar] 46. PRRSV and EAV are only distantly related and utilize diversified transcription-regulating sequences (TRSs), a viable chimeric progeny computer virus was rescued. Strikingly, this chimeric computer virus (vAPRRS-EAV2ab34) acquired the broad cell tropism of EAV, demonstrating that this 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. INTRODUCTION The and (2). The arterivirus family consists of equine arteritis computer virus (EAV), porcine reproductive and respiratory syndrome computer virus (PRRSV), lactate dehydrogenase-elevating computer virus (LDV), and simian hemorrhagic fever computer virus (SHFV) (42). PRRSV isolates segregate into European (type I) and North American (type II) genotypes, which share only about 60% sequence identity Rabbit polyclonal to AnnexinA1 (32). In general, the host range of arteriviruses is very restricted: EAV is only known to infect horses and donkeys; the tropism of PRRSV is usually specific for LY2922470 swine; LDV infects only mice; and SHFV targets several genera of monkeys, in which it can cause either acute or persistent infections (42). PRRSV contamination, in particular, often leads to high-mortality disease outbreaks and is considered one of the greatest threats to the swine industry worldwide (24, 33, 48, 64). Recently, a large outbreak of highly virulent PPRSV (type II) affected the Asian swine industry, causing considerable economic losses (48, 64). The arterivirus genome is usually a polyadenylated RNA molecule of about 12 to 16 kb (see Fig. 1), comprising short 5- and 3-terminal untranslated regions (UTRs) flanking a large replicase gene (open reading frame 1a [ORF1a] and ORF1b) andin the case of PRRSV, EAV, and LDVeight known structural protein genes (17, 19, 42, 43). The SHFV genome contains four additional ORFs, which appear to be derived from an ancient duplication of ORFs 2a to 4 and may encode additional envelope proteins (18). Like coronaviruses, arteriviruses employ a unique mechanism of discontinuous RNA synthesis to produce an extensive nested set of subgenomic (sg) mRNAs. These transcripts are 3 coterminal but also carry a common 5 leader sequence that is identical to the 5-terminal part of the genome (37). Synthesis of sg mRNA is usually thought to start with the generation of subgenome-length negative-stranded templates, one for each mRNA species, which derive from a process of discontinuous negative-strand synthesis directed by short, conserved transcription-regulating sequences (TRSs). During discontinuous RNA synthesis, the genomic 3-proximal region is usually copied up to a so-called body TRS (TRS-B), after which RNA synthesis is usually interrupted and the 3 end of the nascent unfavorable strand base pairs to a complementary sequence (leader TRS [TRS-L]) in the 5 UTR. Following the base-pairing interaction between the negative-stranded TRS-B and the positive-stranded TRS-L, RNA synthesis is usually resumed to add the complement of the leader sequence to the subgenome-length negative-stranded RNA, which can subsequently serve as a template for mRNA synthesis (7, 26, 37C39, 46). Open in a separate windows Fig 1 Schematic business of parental and chimeric PRRSV and EAV genomes. The genomic businesses of the parental viruses, vEAV030 LY2922470 and vAPRRS, are represented using filled and open rectangles, respectively. The intermediate construct vAPRRSasc was generated by inserting an AscI restriction enzyme recognition sequence ((50). However, another study documented that the minor envelope proteins GP2 and GP4 interact with CD163 (4). Van Breedam and coworkers proposed a model in which LY2922470 pSn and CD163 would together mediate PRRSV entry, with the former serving as the authentic receptor for adsorption and the latter subsequently mediating penetration (49). Nevertheless, it is clear that more genetic and biochemical information is required to clarify the exact roles of the different viral and cellular proteins claimed to be involved. Such information will also be key for the rational design of vaccines and antivirals. In this study, we provide genetic evidence that this minor envelope proteins GP2, GP3, GP4, and E together play a key role in the entry of arteriviruses into cultured cells. Utilizing PRRSV and EAV infectious cDNA clones (52, 62), we constructed a chimera in which PRRSV ORFs 2a to 4 were replaced by the corresponding genes.