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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.

SUBMITTER: Fang Y

PROVIDER: S-EPMC3491471 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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

Fang Ying Y Treffers Emmely E EE Li Yanhua Y Tas Ali A Sun Zhi Z van der Meer Yvonne Y de Ru Arnoud H AH van Veelen Peter A PA Atkins John F JF Snijder Eric J EJ Firth Andrew E AE

Proceedings of the National Academy of Sciences of the United States of America 20121004 43

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 posttransla ...[more]

PMID: 23043113

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Project description:Proteins involved in mammalian mitochondrial translation, when compared to analogous bacterial proteins, frequently have additional sequence regions whose structural or functional roles are not always clear. For example, an additional short insert sequence in the bovine mitochondrial initiation factor 2 (IF2(mt)) seems sufficient to fulfill the added role of eubacterial initiation factor IF1. Prior to our recent cryo-EM study that showed IF2(mt) to structurally occupy both the IF1 and IF2 binding sites, the spatial separation of these sites, and the short length of the insert sequence, posed ambiguity in whether it could perform the role of IF1 through occupation of the IF1 binding site on the ribosome.The present study probes how well computational structure prediction methods can a priori address hypothesized roles of such additional sequences by creating quasi-atomic models of IF2(mt) using bacterial IF2 cryo-EM densities (that lack the insert sequences). How such initial IF2(mt) predictions differ from the observed IF2(mt) cryo-EM map and how they can be suitably improved using further sequence analysis and flexible fitting are analyzed.By hypothesizing that the insert sequence occupies the IF1 binding site, continuous IF2(mt) models that occupy both the IF2 and IF1 binding sites can be predicted computationally. These models can be improved by flexible fitting into the IF2(mt) cryo-EM map to get reasonable quasi-atomic IF2(mt) models, but the exact orientation of the insert structure may not be reproduced. Specific eukaryotic insert sequence conservation characteristics can be used to predict alternate IF2(mt) models that have minor secondary structure rearrangements but fewer unusually extended linker regions. Computational structure prediction methods can thus be combined with medium-resolution cryo-EM maps to explore structure-function hypotheses for additional sequence regions and to guide further biochemical experiments, especially in mammalian systems where high-resolution structures are difficult to determine.

Dataset Information

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

Publications

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

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