Transactivation of programmed ribosomal frameshifting by a viral protein

Programmed −1 ribosomal frameshifting (−1 PRF) is a widely used translational mechanism facilitating the expression of two polypeptides from a single mRNA. Commonly, the ribosome interacts with an mRNA secondary structure that promotes −1 frameshifting on a homopolymeric slippery sequence. Recently, we described an unusual −2 frameshifting (−2 PRF) signal directing efficient expression of a transframe protein [nonstructural protein 2TF (nsp2TF)] of porcine reproductive and respiratory syndrome virus (PRRSV) from an alternative reading frame overlapping the viral replicase gene. Unusually, this arterivirus PRF signal lacks an obvious stimulatory RNA secondary structure, but as confirmed here, can also direct the occurrence of −1 PRF, yielding a third, truncated nsp2 variant named “nsp2N.” Remarkably, we now show that both −2 and −1 PRF are transactivated by a protein factor, specifically a PRRSV replicase subunit (nsp1β). Embedded in nsp1β’s papain-like autoproteinase domain, we identified a highly conserved, putative RNA-binding motif that is critical for PRF transactivation. The minimal RNA sequence required for PRF was mapped within a 34-nt region that includes the slippery sequence and a downstream conserved CCCANCUCC motif. Interaction of nsp1β with the PRF signal was demonstrated in pull-down assays. These studies demonstrate for the first time, to our knowledge, that a protein can function as a transactivator of ribosomal frameshifting. The newly identified frameshifting determinants provide potential antiviral targets for arterivirus disease control and prevention. Moreover, protein-induced transactivation of frameshifting may be a widely used mechanism, potentially including previously undiscovered viral strategies to regulate viral gene expression and/or modulate host cell translation upon infection.Among the repertoire of mechanisms that viruses use to control or regulate their gene expression, noncanonical translation plays an important role, in particular for positive-strand RNA viruses whose genomic RNA serves a dual function as mRNA and genome (reviewed in ref. 1). A commonly used strategy is −1 programmed ribosomal frameshifting (−1 PRF), in which mRNA signals induce a significant proportion of translating ribosomes to change reading frame, with ribosomes slipping back (in the 5′ direction) by 1 nt into an overlapping ORF before continuing translation, generating a fusion protein composed of the products of both upstream and downstream ORFs (reviewed in refs. 1–4). PRF was first described as the mechanism by which the Gag-Pol polyprotein of the retrovirus Rous sarcoma virus is expressed from overlapping gag and pol ORFs (5, 6) and related signals have since been documented in many other viruses of medical, veterinary, and agricultural importance (7–11). PRF has also been increasingly recognized in cellular genes of both prokaryotes and eukaryotes as well as in other replicating elements, such as insertion sequences and transposons (12).Recently, we identified an unusual −2 programmed ribosomal frameshifting (−2 PRF) event that operates during the translation of the genome of porcine reproductive and respiratory syndrome virus (PRRSV), a member of the arterivirus family in the order Nidovirales (13). PRRSV can be divided into distinct European (EU, type 1) and North American (NA, type 2) genotypes. The viral genome comprises a positive-sense RNA molecule, ∼15 kb in length (14). As in other nidoviruses, its 5′ proximal region contains two large replicase ORFs (ORF1a and ORF1b) (15), with the ORF1b product being expressed as a fusion with the ORF1a product following −1 PRF in the short ORF1a/ORF1b overlap region (Fig. 1). Four ORF1a-encoded proteinases (residing in nsp1α, nsp1β, nsp2, and nsp4) subsequently cleave the pp1a and pp1ab polyproteins into (at least) 14 different nonstructural proteins (nsps; Fig. 1A). The recently identified −2 PRF signal is located several kilobases upstream of the ORF1a/ORF1b −1 PRF signal, and maps to the part of ORF1a that encodes nsp2. This large, multifunctional replicase subunit is involved in diverse steps of the arterivirus replicative cycle, including replicase polyprotein processing (16), the formation of replication structures (17, 18), and innate immune evasion (19–22). At the PRRSV −2 PRF signal, a proportion of ribosomes back up 2 nt, to generate a transframe fusion protein (nsp2TF) comprising the N-terminal two-thirds of nsp2 and the product encoded by a conserved alternative ORF [transframe (TF)] in the −2 reading frame. Compared with full-length nsp2, the nsp2TF product is truncated, equipped with an alternative C-terminal transmembrane domain (Fig. 1A), and targeted to a different subcellular compartment (13). Mutations preventing nsp2TF expression reduce PRRSV replication efficiency in cell culture 50- to 100-fold, highlighting the biological importance of the frameshifting event and nsp2TF expression. The −2 PRF takes place at a highly conserved RG_GUU_UUU slippery sequence (R = G or A), and frameshifting is remarkably efficient (around 20% in virus-infected cells and up to 50% in expression systems) (13).Open in a separate windowFig. 1.PRRSV genome organization and location of ribosomal frameshifting signals. (A) Overview of the ∼15-kb PRRSV genome. The long 5′ ORFs 1a and 1b encode nonstructural polyproteins, and at least eight shorter 3′ ORFs (2a-7) encode structural proteins. The 3′ ORFs are translated from a nested set of subgenomic mRNAs, two of which are bicistronic. ORF1a and ORF1b are translated from the genomic RNA, with translation of ORF1b depending on −1 PRF at the end of ORF1a. The TF ORF overlaps the central ORF1a region in the −2 reading frame and is accessed via −2 PRF (13). A −1 frameshift at the same site generates the nsp2N product (see details under the section “Alternative −2 and −1 PRF at the Same PRRSV Slippery Sequence”). The vertical red line indicates the location of the RG_GUU_UUU shift site (R = A or G, in different arteriviruses). Domains in nsp2/nsp2TF: C, Cys-rich domain HVR, hypervariable region; PLP2, papain-like proteinase;TM/TM′, (putative) transmembrane domains. (B) Sequence of the SD01-08 RNA in the region of the −2/−1 PRF signal, with the slippery sequence (red) and C-rich motif (blue) highlighted. The −1 reading frame stop codon is underlined and codons for each of the reading frames are indicated. (C) Features of the canonical −1 PRF signal present in the PRRSV ORF1a/ORF1b overlap region. The stimulatory RNA pseudoknot is composed of two stems connected by single-stranded loops.As depicted in Fig. 1 B and C, the elements that promote PRF in PRRSV are quite distinct. The −1 PRF signal at the ORF1a/1b junction comprises a slippery sequence (generally U_UUA_AAC) where the ribosome changes frame, and a stimulatory RNA pseudoknot structure immediately downstream, an organization that is conserved throughout the Nidovirales order (23, 24) and widely used in other viral −1 PRF mechanisms. It is thought that interaction of the translating ribosome with the pseudoknot confounds its RNA-unwinding activity (25, 26) and may induce tension in the mRNA that assists in the uncoupling of codon–anticodon interactions at the shift site (27–29). In contrast, only a few cases of −2 PRF in mammalian cells have been documented thus far (13, 29) and the elements involved are poorly understood. Our previous computer-based RNA-folding analysis suggested that the RNA downstream of the slippery sequence (RG_GUU_UUU) used for −2 PRF in PRRSV is rather unstructured and does not fold into a structure compatible with canonical RNA-structure-stimulated PRF. However, mutations within a conserved CCCANCUCC motif located 11 nt downstream of the shift site can reduce or inhibit frameshifting, consistent with the presence of a 3′ stimulatory element of some form (13). Remarkably, our previous study also provided indications for the occurrence of efficient −1 frameshifting at (or near) the same slippery sequence. Due to the presence of a translation termination codon in the −1 reading frame immediately following the slippery sequence, this would yield a truncated form of nsp2, termed “nsp2N” (Fig. 1A).In this report, we identify PRRSV replicase subunit nsp1β as a transactivator of efficient −2 and −1 PRF at the same slippery sequence and provide evidence that its frameshift-stimulatory activity requires interaction with the viral mRNA. In support of this, a highly conserved putative RNA-binding motif (GKYLQRRLQ), integrated into the structure of nsp1β’s papain-like autoproteinase domain, was found to be critical for the stimulation of frameshifting and for interacting with the RNA sequence of the PRRSV PRF signal. The minimal RNA sequence required to direct efficient PRF was mapped within a 34-nt region of the PRRSV nsp2-coding sequence that includes the shift site and the conserved CCCANCUCC motif. Our findings reveal an unusual noncanonical translation mechanism in which a viral protein functions as a transactivator of efficient −2 and −1 PRF. This study advances our understanding of noncanonical translation, suggests that viruses may use additional strategies to modulate viral and potentially host cell translation during infection, and has practical implications in biotechnology and the design of antiviral strategies.