EXPLORING THE INTERPLAY OF La-RELATED PROTEINS AND PABPC1 IN SARS-COV-2 REPLICATION

Few events in recent history have influenced science, medicine, and society as deeply as the Coronavirus Disease 2019 (COVID-19) pandemic. It started in late December 2019 as a cluster of pneumonia cases of unknown aetiology reported in Wuhan (China), and was shortly followed by the identification of a new coronavirus, later named Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), as the causative agent. Official data on the first three years of SARS-CoV-2 circulation reflect its terrifying magnitude, which is depicted in more than 700 million infections and almost 7 million deaths worldwide. These devastating effects were mitigated largely due to the unprecedented effort of the scientific community, aimed at the rapid identification of effective strategies to reduce the spread of the infection and the impact of severe outbreaks. Among these strategies, novel platform vaccines and therapeutic treatments - including antiviral agents and neutralizing antibody therapies - proved to be crucial weapons against SARS‑CoV‑2, limiting disease progression and improving patient outcomes. Nonetheless, the pandemic has emphasized critical gaps in our understanding of the molecular mechanisms of the coronavirus. Indeed, although scientific knowledge advanced significantly during this period, many host systems that the virus hijacks or manipulates to enhance its fitness remain poorly understood, and an effective strategy to manage future coronavirus outbreaks is still lacking. In this context, drawing on proteomic evidence identifying host interactors of SARS‑CoV‑2, this PhD project investigated the role of La-Related Proteins (LaRPs), a subset of host RNA-binding proteins, in the SARS-CoV-2 life cycle. Specifically, it explored their potential involvement in viral replication through interactions with key viral proteins and the host factor PABPC1, aiming to unravel novel molecular mechanisms relevant for viral RNA infection. Indeed, proteomic studies reported direct interactions among some LaRPs and members of the SARS-CoV-2 Replication and Transcription complex (RTC), specifically LaRP4B-nsp12, and LaRP7-nsp8. Additionally, LaRPs are reported to associate with SARS-CoV-2 RNA, similarly to PABPC1, a known physiological binding partner of some LaRPs. Thus, following in-cell studies to validate LaRP4s in viral lifecycle, direct interactions were assessed using biophysical techniques, which included Bio-layer Interferometry and Isothermal Titration Calorimetry, and cross-linking assays. This investigation provides insight into the regions of LaRPs that are not involved in the recognition.Moreover, this project also addressed LaRP4s/PABPC1 interaction to define the molecular basis of their association and the synergic role in viral infection. Research findings suggested that this molecular crosstalk is crucial for the cellular functioning of LaRP4s. Both LaRP4A and LaRP4B recognise PABPC1 through two interacting regions known as the PABP-interacting motif 2w (PAM2w) and the PABP-binding motif (PBM). While recognition via the PAM2w was extensively studied, the exact boundaries of the PBM and its binding region within PABPC1 remain elusive. Here, in vitro assays and biophysical methodologies such as Isothermal Titration Calorimetry, Nuclear Magnetic Resonance, Dynamic Light Scattering, and Nano Differential Scanning Fluorimetry were employed to isolate and characterize the LaRP4s/PABPC1 complex. These analyses identified the PBM region in LaRP4A and its interacting site on PABPC1, also providing insights into the role of RNA in the interplay.