Efficient - 2 Frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein

Ying Fang, Emmely E. Treffers, Yanhua Li, Ali Tas, Zhi Sun, Yvonne Van Der Meer, Arnoud H. De Ru, Peter A. Van Veelen, John F. Atkins, Eric J. Snijder, Andrew E. Firth

Research output: Contribution to journalArticle

Abstract

Programmed -1 ribosomal frameshifting ( -1 PRF) is a geneexpression 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 PRRSVinfected cells was verifi ed using speci fi c 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.

Original languageEnglish (US)
Pages (from-to)E2920-E2928
JournalProceedings of the National Academy of Sciences of the United States of America
Volume109
Issue number43
DOIs
StatePublished - Oct 23 2012
Externally publishedYes

Keywords

  • Genetic recoding
  • Nidovirales
  • Overlapping gene
  • Translation
  • Virology

ASJC Scopus subject areas

  • General

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  • Cite this

    Fang, Y., Treffers, E. E., Li, Y., Tas, A., Sun, Z., Van Der Meer, Y., De Ru, A. H., Van Veelen, P. A., Atkins, J. F., Snijder, E. J., & Firth, A. E. (2012). Efficient - 2 Frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein. Proceedings of the National Academy of Sciences of the United States of America, 109(43), E2920-E2928. https://doi.org/10.1073/pnas.1211145109