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A study in understanding and inhibiting stop-go translation in the dicistrovirus cricket paralysis virus

University of British Columbia

A subset of RNA viruses can cause significant harm to human health and agriculture. As such, it is important to understand how these RNA viruses replicate and interact with the host machinery in order to identify potential antiviral targets. Some of these RNA viruses contain a Stop-Go sequence, consisting of a conserved C-terminal DxExNPG^P sequence, that mediates co-translational processing in the viral polyproteins. In Stop-Go translation, host ribosomes skip synthesis of a glycine-proline peptide bond, causing release of the translation product. Ribosomes do not dissociate, rather, they continue translating a downstream product. Stop-Go processing occurs without stop codons, and re-initiation occurs without initiation factors. The precise Stop-Go translation mechanism remains unclear. This study aims to better understand the Stop-Go mechanism, its sequence requirements in a dicistrovirus model of infection, and identify inhibitors. I performed a mutational analysis of the Dicistrovirus Cricket Paralysis Virus (CrPV) Stop-Go sequence to investigate effects of mutations on viral infection, yield, and processing. I used various biochemical methods including CrPV RNA transfections, infections, and viral titers to examine effects of these mutants on CrPV infection. To evaluate effects of Stop-Go processing in viral polyprotein processing, I used [₃₅S]-Cysteine-Methionine to conduct in-vitro-translation reactions of mutants and pulse-chase labelling in infected S2 cells. Results of the mutational analysis confirm that wildtype Stop-Go sequences are required for viable CrPV protein production. Certain mutations diminish infectivity, while others ablate viral infection. Viral titers reveal requirements for Stop-Go sequence amino acids under CrPV infection. In- iv vitro-translation and immunoblotting data reveal that Stop-Go processing releases the multifunctional 1A protein to promote infection, suggesting that timing of 1A release is critical in infection. I next aimed to identify the first inhibitors of Stop-Go translation using high-throughput screening methods. I developed a yeast one-hybrid-based high-throughput chemical screen for Stop-Go inhibitors measuring yeast growth as a readout. Using this system, I screened 3346 compounds and identified but did not validate multiple potential inhibitors of Stop-Go translation. This chapter also describes new tools developed to perform secondary validation of hits in yeast, drosophila, mammalian cells, and in sf-21 insect translation extract.

Text

2022-08-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/75797

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/