Recoding of viral mRNAs by –1 programmed ribosome frameshifting

Recoding of viral mRNAs by –1 programmed ribosome frameshifting

Natalia Korniy

Dissertation

Datum der mündl. Prüfung: 2019-05-17

Erschienen: 2019-06-11

Betreuer: Rodnina, Marina Prof. Dr.

Gutachter: Rodnina, Marina Prof. Dr.

Gutachter: Stark, Holger Prof. Dr.

Zum Verlinken/Zitieren: http://hdl.handle.net/21.11130/00-1735-0000-0003-C124-A

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Zusammenfassung

Englisch

–1 programmed ribosome frameshifting (–1PRF) is commonly used in many viruses and cellular genes. The current study focuses on the –1PRF in two human viruses, human immunodeficiency virus type 1 (HIV-1) and Semliki Forest virus (SFV), which share the same slippery site (SS) sequence, but have different mRNA secondary structures. –1PRF on the gag-pol mRNA of HIV-1 results in the production of structural proteins of the capsid (Gag, 0-frame) and viral enzymes (Gag-Pol, –1-frame), and its dysregulation is detrimental for the structural integrity of the virion and its infectivity. In SFV –1PRF is responsible for the synthesis of the structural proteins 6K and TransFrame (TF), which contribute to the virus pathogenicity. Here we show that –1PRF in both viruses operates via two conserved kinetic pathways: tRNA dual-slippage during translocation under saturated translation conditions and single P-site tRNA slippage when the A site is vacant due to the limited supply of tRNAs. –1PRF is modulated by the abundance of Leu-tRNALeu that reads the rare UUA codon at the mRNA SS. In HIV-1 the Gag to Gag-Pol ratio is maintained by switching between two frameshifting mechanisms depending on the tRNALeu(UUA) availability. Leu-tRNALeu(UUA) is rare in human cell lines derived from CD4+ T-lymphocytes – a natural target of HIV-1 infection. –1PRF in HIV-1 is additionally stimulated by a downstream enhancer sequence located within the first 12 nt after the SS. Finally, a second potential SS downstream of the first one is normally inefficient but can also support –1-frameshifting when altered by a compensatory resistance mutation in response to current anti-HIV drug therapy. With regard to SFV, we determined that its frameshifting site contains an extended stem-loop (SL) following the SS, which could act as a modulator of frameshifting under the limited supply of Leu-tRNALeu(UUA). The previously predicted enhancer sequence does not seem to play a role in –1PRF in SFV. Described different frameshifting regimes and stimulators allow the viruses to maintain a constant –1PRF efficiency regardless of environmental or therapeutical influence to ensure high viral load and successful virus propagation.

Keywords: ribosome; translation; programmed ribosome frameshifting; HIV-1; SFV; tRNA abundance; mRNA secondary structure; enhancer