title: Efficient -2 frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein.
abstract: Programmed -1 ribosomal frameshifting (-1 PRF) is a gene-expression mechanism used to express many viral and some cellular genes. In contrast, efficient natural utilization of -2 PRF has not been demonstrated previously in eukaryotic systems. Like all nidoviruses, members of the Arteriviridae (a family of positive-stranded RNA viruses) express their replicase polyproteins pp1a and pp1ab from two long ORFs (1a and 1b), where synthesis of pp1ab depends on -1 PRF. These polyproteins are posttranslationally cleaved into at least 13 functional nonstructural proteins. Here we report that porcine reproductive and respiratory syndrome virus (PRRSV), and apparently most other arteriviruses, use an additional PRF mechanism to access a conserved alternative ORF that overlaps the nsp2-encoding region of ORF1a in the +1 frame. We show here that this ORF is translated via -2 PRF at a conserved G_GUU_UUU sequence (underscores separate ORF1a codons) at an estimated efficiency of around 20%, yielding a transframe fusion (nsp2TF) with the N-terminal two thirds of nsp2. Expression of nsp2TF in PRRSV-infected cells was verified using specific Abs, and the site and direction of frameshifting were determined via mass spectrometric analysis of nsp2TF. Further, mutagenesis showed that the frameshift site and an unusual frameshift-stimulatory element (a conserved CCCANCUCC motif 11 nucleotides downstream) are required to direct efficient -2 PRF. Mutations preventing nsp2TF expression impair PRRSV replication and produce a small-plaque phenotype. Our findings demonstrate that -2 PRF is a functional gene-expression mechanism in eukaryotes and add another layer to the complexity of arterivirus genome expression.
authors.affiliation: Departments of Veterinary and Biomedical Science and Biology/Microbiology, South Dakota State University, Brookings, SD 57007, USA. ying.fang@sdstate.edu
authors.forename: Ying
authors.initials: Y
authors.lastname: Fang
authors.forename: Emmely E
authors.initials: EE
authors.lastname: Treffers
authors.forename: Yanhua
authors.initials: Y
authors.lastname: Li
authors.forename: Ali
authors.initials: A
authors.lastname: Tas
authors.forename: Zhi
authors.initials: Z
authors.lastname: Sun
authors.forename: Yvonne
authors.initials: Y
authors.lastname: van der Meer
authors.forename: Arnoud H
authors.initials: AH
authors.lastname: de Ru
authors.forename: Peter A
authors.initials: PA
authors.lastname: van Veelen
authors.forename: John F
authors.initials: JF
authors.lastname: Atkins
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Snijder
authors.forename: Andrew E
authors.initials: AE
authors.lastname: Firth
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018018:Arterivirus / Q000378:metabolism*; D001483:Base Sequence; D002451:Cell Compartmentation; D003062:Codon; D016368:Frameshift Mutation; D008322:Mammals; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016366:Open Reading Frames; D012270:Ribosomes / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D003062:Codon; D014764:Viral Proteins