Host RNA-binding protein LARP6 controls SARS-CoV-2 programmed −1 ribosomal frameshifting

Programmed -1 ribosomal frameshifting (-1PRF) is essential for the balanced expression of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins and efficient viral replication, yet how host factors modulate this process remains incompletely understood. Here, we identify the host RNA-binding protein La-related protein 6 (LARP6) as a host regulator of this mechanism. Structural analysis and mutational dissection of the SARS-CoV-2 frameshifting element reveal that a three-stem-loop pseudoknot downstream of the slippery sequence is critical for efficient -1PRF. Using RNA pulldown and mass spectrometry, we identify LARP6 as a host factor that associates with the -1PRF pseudoknot. LARP6 associates with the viral RNA in a pseudoknot-dependent manner through multiple RNA-binding domains, and both loss- and restore-of-function analyses demonstrate that LARP6 promotes -1PRF efficiency. Mechanistically, LARP6 associates with ribosomal components in an mRNA-dependent manner, and changes in LARP6 expression are accompanied by corresponding changes in ribosome-associated RNA occupancy at the frameshifting region, consistent with a potential role in modulating ribosomal pausing. In a SARS-CoV-2 luciferase replicon, LARP6 overexpression increased RdRp protein levels, providing additional support for LARP6-mediated promotion of -1PRF in a viral genomic context. Together, our findings identify LARP6 as a host regulator of SARS-CoV-2 -1PRF and suggest that LARP6 may regulate frameshifting through its association with the viral pseudoknot and ribosomes.IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) relies on programmed -1 ribosomal frameshifting (-1PRF) to regulate the production of viral replicase proteins, making this process an important point of control in the viral life cycle. Although the viral RNA structures that promote -1PRF have been extensively studied, how host factors contribute to this process remains less well understood. Our study identifies LARP6 as a host protein associated with the SARS-CoV-2 -1PRF pseudoknot and reveals a potential link between LARP6-viral RNA interaction and ribosome behavior at the frameshifting site. These findings expand the current understanding of -1PRF regulation beyond viral RNA structures and suggest that host RNA-binding proteins may contribute to the control of viral gene expression by interacting with structured RNA elements. This work highlights the interaction of host RNA-binding proteins with viral RNA structures as a potentially important layer of translational regulation in coronaviruses.