Transactivation of programmed ribosomal frameshifting by a viral protein

Abstract

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.genetic recoding | translational control | nsp1beta

Protein-protein interactions between A. aegypti midgut and dengue virus 2: two-hybrid screens using the midgut cDNA library

“…Despite its crucial role in protein translation, ribosomal protein's involvement in RNA virus replication and dissemination has been well studied [38,39]. A few recently published reports demonstrated the versatility of ribosomal proteins in either facilitating or inhibiting viral growth during infection [40][41][42]. In addition, although ribosome has traditionally been thought to function as the catalytic machinery for translational elongation, Lee et al also showed that ribosomal subunit protein rpL40 acts as a requisite for vesicular stomatitis virus (VSV) capdependent translational regulation [43].…”

The non-structural protein Nsp2TF of porcine reproductive and respiratory syndrome virus down-regulates the expression of Swine Leukocyte Antigen class I

“…The sequence GGUUUUU at the exact frame-shifting site and the downstream conserved motif CCCANCUCC were shown to be required for generating this À 2 PRF product. A follow-up study (Li et al, 2014) further demonstrated that PRRSV Nsp1β is also required for the efficient ribosome frameshifting as a trans-activator protein in Nsp2TF expression. In addition to À 2 PRF, À 1 PRF can also occur at the same site with a much lower efficiency, generating À 1 PRF product Nsp2N.…”

Proteolytic processing of the porcine reproductive and respiratory syndrome virus replicase

“…GP, glycoprotein; M, membrane; E, envelope; N, nucleocapsid. Unlike EAV, the genomes of PRRSV and other arteriviruses contain an alternative transframe (TF) ORF underlying the non-structural protein 2 (nsp2)-coding region, which is expressed by −2 ribosomal frameshifting (yielding nsp2TF; Fang et al, 2012), whereas a −1 ribosomal frameshift at the same position yields a third, truncated nsp2 variant (nsp2N; Li et al, 2014). The positions corresponding to (known or predicted) polyprotein cleavage sites are depicted above the replicase ORFs; red arrowheads, sites cleaved by the nsp4 serine protease (S); blue arrowheads, sites cleaved by papain-like proteases (P) in the nsp1-nsp2 region.…”