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

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 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.Nidovirales | virology | genetic recoding | overlapping gene | translation

Asc1, homolog of human RACK1, prevents frameshifting in yeast by ribosomes stalled at CGA codon repeats

“…We have provided evidence that the Asc1/RACK1 protein plays a role in reading frame maintenance, a critical feature of translation, and infer that Asc1-mediated cessation of translation is important to avoid synthesis of aberrant proteins through frameshifting, although the precise nature of the frameshifting event (+1 or −2) (Fang et al 2012) that occurs in the asc1-Δ mutant is unknown. Moreover, the effects of the asc1-Δ mutant may be physiologically relevant, since Asc1 protein levels are decreased substantially during hypoxic stress (Bruckmann et al 2009), and since frameshifting is detectable (albeit minimally) at CGA-CGA codon pairs, which are found in 26 yeast genes, with two occurrences of CGA-CGA pairs in one gene (BUD8).…”

Immunological and biochemical characterisation of 7ap, a short protein translated from an alternative frame of ORF7 of PRRSV

“…However, more conservative estimations put the mutation rate somewhere between 1.8 and 7 × 10 −3 /nucleotide/year (Chang et al, 2002;Forsberg, 2005), which still places PRRSV among the most rapidly evolving viruses known. Despite the rapidly changing codon sequences of the main ORFs, recently several positionally conserved alternative ORFs have been identified in the PRRSV genome and all of them proved to be protein coding: ORFs partially coding the nsp2TF and nsp2N proteins in the NSP2 region (Fang et al, 2012), ORF5a and ORF2b overlapping with the ORF5 and ORF2a genes (Wu et al, 2001;Firth et al, 2011), respectively. Genome comparison of 46 divergent PRRSV genomes revealed another short ORF in a conserved position, named ORF7a overlapping with the ORF7 gene.…”

“…In addition, three accessory proteins were identified translating from fully overlapping ORFs by noncanonical translation mechanisms (Wu et al, 2001;Firth et al, 2011;Fang et al, 2012).…”

Expression of peptide nanoparticles containing a porcine reproductive and respiratory syndrome (PRRS) virus epitope in plants

“…Recently, the nsp2 protein, which is coded for by the most variable region of the genome, was demonstrated to be incorporated into virions of several PRRSV strains as a set of differently sized protein isomers (Kappes et al, 2013(Kappes et al, , 2015b. This unexpected result increases the number of viral proteins to at least 10 (full-length nsp2 and its isomers, nsp2TF, E,M,N,ORF5a), that are exposed to the porcine immune system on entry of PRRSV into swine alveolar macrophages (Fang et al, 2012;Veit et al, 2014). The enormous genetic and protein variation of all of these structural proteins, from the least conserved nsp2 to the most conserved M protein shows the complexity and plasticity of the PRRSV genome and virion structure (Rascón-Castelo et al, 2015;Kappes and Faaberg, 2015a).…”