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.
Department of Veterinary and Biomedical Sciences and Department of Biology/Microbiology, South Dakota State University, Brookings, SD 57007;Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506;
Yanhua
Y
Li
Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands;Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands;
Emmely E
EE
Treffers
Division of Virology, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom; and.
Sawsan
S
Napthine
Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands;
Ali
A
Tas
Department of Veterinary and Biomedical Sciences and Department of Biology/Microbiology, South Dakota State University, Brookings, SD 57007;Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506;
Longchao
L
Zhu
Department of Veterinary and Biomedical Sciences and Department of Biology/Microbiology, South Dakota State University, Brookings, SD 57007;
Zhi
Z
Sun
Division of Virology, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom; and.
Susanne
S
Bell
Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada R3T 2N2.
Brian L
BL
Mark
Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands;
Peter A
PA
van Veelen
Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands;
Martijn J
MJ
van Hemert
Division of Virology, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom; and.
Andrew E
AE
Firth
Division of Virology, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom; and yfang@vet.k-state.edu ib103@mole.bio.cam.ac.uk e.j.snijder@lumc.nl.
Ian
I
Brierley
Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands; yfang@vet.k-state.edu ib103@mole.bio.cam.ac.uk e.j.snijder@lumc.nl.
Eric J
EJ
Snijder
Department of Veterinary and Biomedical Sciences and Department of Biology/Microbiology, South Dakota State University, Brookings, SD 57007;Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506; yfang@vet.k-state.edu ib103@mole.bio.cam.ac.uk e.j.snijder@lumc.nl.
Ying
Y
Fang
D000818:Animals; D002460:Cell Line; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D018965:Frameshifting, Ribosomal / Q000502:physiology*; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D057809:HEK293 Cells; D000882:Haplorhini; D006801:Humans; D007118:Immunoassay; D008156:Luciferases; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics*; D012394:Rosaniline Dyes; D053719:Tandem Mass Spectrometry; D015533:Transcriptional Activation / Q000502:physiology*; D017361:Viral Nonstructural Proteins / Q000502:physiology*
D012394:Rosaniline Dyes; D017361:Viral Nonstructural Proteins; D008156:Luciferases; C004692:coomassie Brilliant Blue
genetic recoding; nsp1beta; translational control