Analysis of the Antiviral Restriction Factor Shiftless in the context of HIV-1 Programmed –1 Ribosomal Frameshifting
by Niklas Jäger
Date of Examination:2024-05-29
Date of issue:2024-07-05
Advisor:Prof. Dr. Stefan Pöhlmann
Referee:Prof. Dr. Stefan Pöhlmann
Referee:Prof. Dr. Lutz Walter
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Abstract
English
The human immunodeficiency virus 1 (HIV 1) is responsible for a major global health crisis, the acquired immunodeficiency syndrome (AIDS) pandemic. Retroviruses integrate their genetic material into the genome of host cells, where the virus can persist in its latent form for large time periods. The interferon system, a component of innate immunity, represents the first line of defense against viral infection. Pathogen-induced activation of the interferon system leads to the expression of roughly 400 interferon-stimulated genes (ISGs), many of which encode antiviral proteins, termed restriction factors. As a consequence, cells establish an antiviral state. Restriction factors can inhibit viral infection by targeting one or multiple steps of the viral replication cycle. The recently discovered restriction factor Shiftless (SFL) blocks HIV 1 replication by suppressing programmed –1 ribosomal frameshifting (–1PRF), an RNA recoding event required for the expression of the HIV 1 Gag Pol polyprotein. However, the determinants of suppression of –1PRF and SFL antiviral activity are poorly understood. SFL interacts with RNAs, ribosomes and other SFL proteins and the goal of this thesis was to determine the contribution of these interactions to SFL-mediated inhibition of –1PRF and antiviral activity. A comparative study of SFL and its inactive splice variant, Shiftless short (SFLS), demonstrated that association with the ribosome, binding to the –1PRF RNA and SFL SFL interactions are insufficient for the inhibition of –1PRF and antiviral activity. Further, the SFL core domain was found to drive SFL multimerization and was required for suppression of Gag Pol expression and antiviral activity. Mutation of two zinc ribbon motifs (ZRM) modulated SFL SFL interactions, binding to the ribosome and –1PRF RNA. The mutants ZRMA+ and ZRMAXXA exerted antiviral activity against authentic virus but failed to suppress Gag Pol expression, indicating that SFL inhibits HIV 1 by more than one mechanism. The newly discovered –1PRF-independent mechanism did not rely on SFL multimerization and interactions with ribosomes or the –1PRF RNA. Finally, SFL not only suppressed –1PRF but also programmed stop codon readthrough, which is utilized by murine leukemia virus (MLV) for Gag Pol expression. In summary, SFL serves as a multifunctional restriction factor that inhibits –1PRF, programmed stop codon readthrough, and exerts an as yet undefined antiviral activity.
Keywords: HIV-1; Gag-Pol; Frameshifting; Shiftless; C19orf66; Restriction factor; -1PRF