INVESTIGATION OF NEW THERAPEUTIC TARGETS OF RESPIRATORY VIRUSES: A NOVEL METHOD EXPLOITING UV RADIATION ¿ PROOF OF CONCEPT BASED ON SARS-COV-2 ¿

The COVID-19 pandemic has highlighted the urgent need for rapid and effective methods to investigate pathogens, particularly those with high mutation rates like RNA respiratory viruses. Understanding the virus's seasonal dynamics and identifying new potential therapeutic targets are key factors in controlling outbreaks. In this context, our study has investigated the inhibitory effects of ultraviolet (UV) radiation that reaches the Earth's surface (UV-B, and UV-A) on SARS-CoV-2 to better characterize seasonal viral spread. Subsequently, UV-C radiation was exploited in an in vitro infection assay to develop an unbiased/multidisciplinary roadmap coupling Surface Plasmon Resonance (SPR), Mass Spectrometry (MS) and Computational Analysis (CA) to thoroughly dissect and identify microbe molecular domains that could be new potential therapeutic targets. We first characterized the UV light’s ability to inactivate SARS-CoV-2, finding that irradiation with LED light at 278 nm (UV-C), 308 nm (UV-B), and 366 nm (UV-A) significantly decreased the infectivity of the virus. Specifically, UV-C (278 nm) doses as low as 4 mJ/cm², UV-B (308 nm) at 200 mJ/cm², and UV-A (366 nm) at 4000 mJ/cm² were sufficient to completely inhibit viral replication. FISH analysis confirmed that UV irradiation of these wavelengths could effectively prevent SARS-CoV-2 infection of target cells. The study further revealed that even UV-A light showed notable efficiency in viral inactivation, indicating its potential role in the seasonal behavior of airborne viruses. Interestingly, the UV-inhibiting effect occurred in a very upstream step of infection since the virus was no longer able to infect cells. Thus, an alteration of spike (S) protein following UV light exposure was assumed. Based on this, we developed and applied the aforementioned workflow (in vitro infection assay – SPR – MS – CA) in order to identify S protein domains more susceptible to alteration. As a result, Mass spectrometry and computational analyses revealed UV-induced modifications, including the disruption of a disulfide bond (Cys1032-Cys1043) in the S2 subunit. This modification reduced the protein's binding affinity to the ACE2 receptor, impairing the virus’s ability to infect cells. These results pinpoint the S2 subunit of the spike protein as a new potential therapeutic target. This workflow could be used to screen a wide variety of pathogens, resulting in a precise molecular fingerprint and providing new insights to address future epidemics by defining the molecular and structural alterations of microbial domains involved in pathogen-host cell interaction, which is a prerequisite for specific drug design.