Molecular characterization of the RNA-protein complex directing -2/-1 programmed ribosomal frameshifting during arterivirus replicase expression
| Autor: | Trushar R. Patel, Markus Meier, Eric J. Snijder, Emmely E. Treffers, Brian L. Mark, Ankoor Patel, Jörg Stetefeld |
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| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Swine viruses Reading frame translation nsp1 Viral Nonstructural Proteins Virus Replication Biochemistry Ribosome poly(C)-binding protein structural biology Translational frameshift biology Chemistry virus diseases RNA-Binding Proteins 3. Good health Cell biology virology DNA-Binding Proteins PCPB2 ribosome Host-Pathogen Interactions RNA Viral βnsp1 ribosome function β RNA virus Porcine Reproductive and Respiratory Syndrome RNA-dependent RNA polymerase 03 medical and health sciences Animals Humans Porcine respiratory and reproductive syndrome virus Editors' Picks nonstructural protein 1 Molecular Biology Immune Evasion nsp1beta 030102 biochemistry & molecular biology βsmall-angle X-ray scattering RNA Frameshifting Ribosomal Cell Biology biology.organism_classification nidovirus 030104 developmental biology Viral replication Structural biology PRRSV small-angle X-ray scattering viral replication PCBP sedimentation velocity |
| Zdroj: | The Journal of Biological Chemistry Journal of Biological Chemistry, 295(52), 17904-17921. AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC |
| ISSN: | 1083-351X |
| Popis: | Programmed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading frames within its RNA genome. PRRSV employs -1 PRF directed by RNA secondary and tertiary structures within its viral genome (canonical PRF), as well as a noncanonical -1 and -2 PRF that are stimulated by the interactions of PRRSV nonstructural protein 1 beta (nsp1 beta) and host protein poly(C)-binding protein (PCBP) 1 or 2 with the viral genome. Together, nsp1 beta and one of the PCBPs act as transactivators that bind a C-rich motif near the shift site to stimulate -1 and -2 PRF, thereby enabling the ribosome to generate two frameshift products that are implicated in viral immune evasion. How nsp1 beta and PCBP associate with the viral RNA genome remains unclear. Here, we describe the purification of the nsp1 beta:PCBP2:viral RNA complex on a scale sufficient for structural analysis using small-angle X-ray scattering and stochiometric analysis by analytical ultracentrifugation. The proteins associate with the RNA C-rich motif as a 1:1:1 complex. The monomeric form of nsp1 beta within the complex differs from previously reported homodimer identified by X-ray crystallography. Functional analysis of the complex via mutational analysis combined with RNA-binding assays and cell-based frameshifting reporter assays reveal a number of key residues within nsp1 beta and PCBP2 that are involved in complex formation and function. Our results suggest that nsp1 beta and PCBP2 both interact directly with viral RNA during formation of the complex to coordinate this unusual PRF mechanism. |
| Databáze: | OpenAIRE |
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