Molecular mechanisms of protein-mediated ribosomal frameshifting in SARS-CoV-2 and HIV-1

As obligate parasites, viruses rely on the host machinery for translating their genome and, consequently, for replication. Many viruses have developed a translational recoding mechanism known as –1 programmed ribosomal frameshifting (–1PRF), which allows for expansion of the genomes’ repertoire by accessing an alternative reading frame. It has long been considered that intrinsic features of the viral RNA regulate –1PRF. However, more recently, host- and viral-encoded regulatory proteins and molecules have been discovered to also regulate –1PRF. This project’s aim was to identify novel host-encoded factors that affect –1PRF and alter the stoichiometry of the viral proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human immunodeficiency virus 1 (HIV-1). To address this, an unbiased genome-wide CRISPR interference (CRISPRi) screen was established and combined with a dual-fluorescence reporter assay containing the frameshifting element of SARS-CoV-2 or HIV-1, respectively. Several ribosomal proteins, RNA-binding proteins, as well as components of the ubiquitin-proteasome system were identified to be putative –1PRF modulators of both viral genomes and were further investigated by different ex vivo and in vitro experiments. One leading candidate of the SARS-CoV-2 screen, RBM8A (also known as Y14), upon knockdown showed a decrease in the mCherry/EGFP ratio of the dual-fluorescence reporter used for the calculation of the frameshifting efficiency, while the EGFP levels were increased. In contrast, the transient overexpression had no effect on frameshifting. Finally, in SARS-CoV-2 infection assays in collaboration with Dr. Ulfert Rand, a stable overexpression of RBM8A in A549-ACE2 cells showed increased viral levels suggesting a proviral activity of RBM8A. Nevertheless, more investigations are required to further prove a frameshifting-related role of RBM8A. In contrast, the HIV-1 CRISPRi data suggests more putative –1PRF modulators that could be investigated for their role on frameshifting and viral replication.