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Factorless ribosome assembly on the internal ribosome entry site of cricket paralysis virus
1Department of Microbiology and Immunology, Stanford University School of Medicine, 299 Campus Drive, Stanford, CA 94305, USA.
Abstract:
The cricket paralysis virus (CrPV), a member of the CrPV-like virus family, contains a single positive-stranded RNA genome that encodes two non-overlapping open reading frames separated by a short intergenic region (IGR). The CrPV IGR contains an internal ribosomal entry site (IRES) that directs the expression of structural proteins. Unlike previously described IRESs, the IGR IRES initiates translation by recruiting 80S ribosomes in the absence of initiator Met-tRNA(i) or any canonical initiation factors, from a GCU alanine codon located in the A-site of the ribosome. Here, we have shown that a variety of mutations, designed to disrupt individually three pseudoknot (PK) structures and alter highly conserved nucleotides among the CrPV-like viruses, inhibit IGR IRES-mediated translation. By separating the steps of translational initiation into ribosomal recruitment, ribosomal positioning and ribosomal translocation, we found that the mutated IRES elements could be grouped into two classes. One class, represented by mutations in PKII and PKIII, bound 40S subunits with significantly reduced affinity, suggesting that PKIII and PKII are involved in the initial recruitment of the ribosome. A second class of mutations, exemplified by alterations in PKI, did not affect 40S binding but altered the positioning of the ribosome on the IRES, indicating that PKI is involved in the correct positioning of IRES-associated ribosomes. These results suggest that the IGR IRES has distinct pseudoknot-like structures that make multiple contacts with the ribosome resulting in initiation factor-independent recruitment and correct positioning of the ribosome on the mRNA.
Insights
Cricket paralysis virus (CrPV) internal ribosomal entry site (IRES) uses pseudoknot structures for factor-independent translation initiation. Mutations reveal distinct roles for pseudoknots in ribosome recruitment and positioning.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Cricket paralysis virus (CrPV) possesses a positive-stranded RNA genome.
- The intergenic region (IGR) of CrPV contains an internal ribosomal entry site (IRES).
- CrPV IRES initiates translation independently of canonical initiation factors, using a GCU codon.
Purpose of the Study:
- To investigate the role of pseudoknot (PK) structures within the CrPV IGR IRES.
- To elucidate the mechanism of factor-independent translation initiation mediated by the CrPV IRES.
- To determine how specific mutations affect ribosomal recruitment, positioning, and translocation.
Main Methods:
- Site-directed mutagenesis of three pseudoknot structures (PKI, PKII, PKIII) and conserved nucleotides in the CrPV IRES.
- Analysis of IRES-mediated translation by assessing ribosomal recruitment, positioning, and translocation.
- Categorization of mutations based on their effects on translation initiation steps.
Main Results:
- Mutations disrupting PKII and PKIII significantly reduced 40S ribosomal subunit binding, indicating their role in ribosome recruitment.
- Mutations in PKI did not affect 40S binding but altered ribosome positioning, suggesting its role in correct positioning.
- The CrPV IGR IRES utilizes distinct pseudoknot structures for initiation factor-independent translation.
Conclusions:
- The CrPV IGR IRES employs multiple pseudoknot structures for efficient and accurate translation initiation.
- Pseudoknots PKII and PKIII are crucial for initial ribosomal subunit recruitment.
- Pseudoknot PKI is essential for the correct positioning of ribosomes on the IRES mRNA sequence.