An intra-family conserved high-order RNA structure within the M ORF is important for arterivirus subgenomic RNA accumulation and infectious virus production.

Synthesis of subgenomic RNAs is a strategy commonly used by polycistronic positive-sense single-stranded RNA viruses to express 3'-proximal genes. Members of the order , including coronaviruses and arteriviruses, use a unique discontinuous transcription strategy to synthesize subgenomic RNAs. In this study, synonymous site conservation analysis and RNA structure folding predicted the existence of intra-family conserved high-order RNA structure within the M ORF of arteriviral genomes, which was further confirmed by RNA secondary structure probing.

Structural and molecular basis for Cardiovirus 2A protein as a viral gene expression switch.

Programmed -1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein, a multi-functional virulence factor that also inhibits cap-dependent translational initiation. Here we present the X-ray crystal structure of 2A and show that it selectively binds to a pseudoknot-like conformation of the PRF stimulatory RNA element in the viral genome. Using optical tweezers, we demonstrate that 2A stabilises this RNA element, likely explaining the increase in PRF efficiency in the presence of 2A.

Investigating molecular mechanisms of 2A-stimulated ribosomal pausing and frameshifting in Theilovirus.

The 2A protein of Theiler's murine encephalomyelitis virus (TMEV) acts as a switch to stimulate programmed -1 ribosomal frameshifting (PRF) during infection. Here, we present the X-ray crystal structure of TMEV 2A and define how it recognises the stimulatory RNA element. We demonstrate a critical role for bases upstream of the originally predicted stem-loop, providing evidence for a pseudoknot-like conformation and suggesting that the recognition of this pseudoknot by beta-shell proteins is a conserved feature in cardioviruses.

Modulation of Viral Programmed Ribosomal Frameshifting and Stop Codon Readthrough by the Host Restriction Factor Shiftless.

The product of the interferon-stimulated gene , Shiftless (SHFL) restricts human immunodeficiency virus replication through downregulation of the efficiency of the viral frameshifting signal. In this study, we demonstrate that bacterially expressed, purified SHFL can decrease the efficiency of programmed ribosomal frameshifting in vitro at a variety of sites, including the RNA pseudoknot-dependent signals of the coronaviruses IBV, SARS-CoV and SARS-CoV-2, and the protein-dependent stimulators of the cardioviruses EMCV and TMEV. SHFL also reduced the efficiency of stop-codon readthrough at the murine leukemia virus signal.

Characterization of the stimulators of protein-directed ribosomal frameshifting in Theiler's murine encephalomyelitis virus.

Many viruses utilize programmed -1 ribosomal frameshifting (-1 PRF) to express additional proteins or to produce frameshift and non-frameshift protein products at a fixed stoichiometric ratio. PRF is also utilized in the expression of a small number of cellular genes. Frameshifting is typically stimulated by signals contained within the mRNA: a 'slippery' sequence and a 3'-adjacent RNA structure.

Programmed -2/-1 Ribosomal Frameshifting in Simarteriviruses: an Evolutionarily Conserved Mechanism.

The -2/-1 programmed ribosomal frameshifting (-2/-1 PRF) mechanism in porcine reproductive and respiratory syndrome virus (PRRSV) leads to the translation of two additional viral proteins, nonstructural protein 2TF (nsp2TF) and nsp2N. This -2/-1 PRF mechanism is transactivated by a viral protein, nsp1β, and cellular poly(rC) binding proteins (PCBPs). Critical elements for -2/-1 PRF, including a slippery sequence and a downstream C-rich motif, were also identified in 11 simarteriviruses.

Protein-directed ribosomal frameshifting temporally regulates gene expression.

Programmed -1 ribosomal frameshifting is a mechanism of gene expression, whereby specific signals within messenger RNAs direct a proportion of translating ribosomes to shift -1 nt and continue translating in the new reading frame. Such frameshifting normally occurs at a set ratio and is utilized in the expression of many viral genes and a number of cellular genes. An open question is whether proteins might function as trans-acting switches to turn frameshifting on or off in response to cellular conditions.

A novel role for poly(C) binding proteins in programmed ribosomal frameshifting.

Translational control through programmed ribosomal frameshifting (PRF) is exploited widely by viruses and increasingly documented in cellular genes. Frameshifting is induced by mRNA secondary structures that compromise ribosome fidelity during decoding of a heptanucleotide 'slippery' sequence. The nsp2 PRF signal of porcine reproductive and respiratory syndrome virus is distinctive in directing both -2 and -1 PRF and in its requirement for a trans-acting protein factor, the viral replicase subunit nsp1β.

Characterization of Ribosomal Frameshifting in Theiler's Murine Encephalomyelitis Virus.

Theiler's murine encephalomyelitis virus (TMEV) is a member of the genus Cardiovirus in the Picornaviridae, a family of positive-sense single-stranded RNA viruses. Previously, we demonstrated that in the related cardiovirus, Encephalomyocarditis virus, a programmed-1 ribosomal frameshift (1 PRF) occurs at a conserved G_GUU_UUU sequence within the 2B-encoding region of the polyprotein open reading frame (ORF). Here we show that-1 PRF occurs at a similar site during translation of the TMEV genome.

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.

Identification of immune and viral correlates of norovirus protective immunity through comparative study of intra-cluster norovirus strains.

Whether or not primary norovirus infections induce protective immunity has become a controversial issue, potentially confounded by the comparison of data from genetically distinct norovirus strains. Early human volunteer studies performed with a norovirus-positive inoculum initially led to the conclusion that primary infection does not generate long-term, protective immunity. More recently though, the epidemiological pattern of norovirus pandemics has led to the extrapolation that primary norovirus infection induces herd immunity.

Characterization of the stop codon readthrough signal of Colorado tick fever virus segment 9 RNA.

Termination codon readthrough is utilized as a mechanism of expression of a growing number of viral and cellular proteins, but in many cases the mRNA signals that promote readthrough are poorly characterized. Here, we investigated the readthrough signal of Colorado tick fever virus (CTFV) segment 9 RNA (Seg-9). CTFV is the type-species of the genus Coltivirus within the family Reoviridae and is a tick-borne, double-stranded, segmented RNA virus.

Expression of the VP2 protein of murine norovirus by a translation termination-reinitiation strategy.

Background: Expression of the minor virion structural protein VP2 of the calicivirus murine norovirus (MNV) is believed to occur by the unusual mechanism of termination codon-dependent reinitiation of translation. In this process, following translation of an upstream open reading frame (ORF) and termination at the stop codon, a proportion of 40S subunits remain associated with the mRNA and reinitiate at the AUG of a downstream ORF, which is typically in close proximity. Consistent with this, the VP2 start codon (AUG) of MNV overlaps the stop codon of the upstream VP1 ORF (UAA) in the pentanucleotide UAAUG.

Characterization of the termination-reinitiation strategy employed in the expression of influenza B virus BM2 protein.

Coupled expression of the M1 and BM2 open-reading frames (ORFs) of influenza B from the dicistronic segment 7 mRNA occurs by a process of termination-dependent reinitiation. The AUG start codon of the BM2 ORF overlaps the stop codon of the upstream M1 ORF in the pentanucleotide UAAUG, and BM2 synthesis is dependent upon translation of the M1 ORF and termination at the stop codon. Here, we have investigated the mRNA sequence requirements for BM2 expression.

Reprogrammed genetic decoding in cellular gene expression.

Reprogrammed genetic decoding signals in mRNAs productively overwrite the normal decoding rules of translation. These "recoding" signals are associated with sites of programmed ribosomal frameshifting, hopping, termination codon suppression, and the incorporation of the unusual amino acids selenocysteine and pyrrolysine. This review summarizes current knowledge of the structure and function of recoding signals in cellular genes, the biological importance of recoding in gene regulation, and ways to identify new recoded genes.

Prokaryotic-style frameshifting in a plant translation system: conservation of an unusual single-tRNA slippage event.

Ribosomal frameshifting signals are found in mobile genetic elements, viruses and cellular genes of prokaryotes and eukaryotes. Typically they comprise a slippery sequence, X XXY YYZ, where the frameshift occurs, and a stimulatory mRNA element. Here we studied the influence of host translational environment and the identity of slippery sequence-decoding tRNAs on the frameshift mechanism.