Konformacijske preureditve proteina S SARS-CoV-2, potrebne za vstop v gostiteljsko celico, in od receptorja ACE2 neodvisna tvorba sincicijev

Govednik Hropot, Tea (avtor), Manček Keber, Mateja (mentor)

Pojav novega patogenega koronavirusa SARS-CoV-2 v letu 2020 je svet hitro postavil v krizo mednarodnih razsežnosti. Kljub koncu pandemije mnoga vprašanja glede samega virusa in podrobnega mehanizma okužbe gostiteljskih celic ostajajo neodgovorjena, vendar so ključna za razumevanje bolezni in obvladovanje morebitnih prihodnjih pandemij. Zaradi hitrega prenosa virusa, porajajočih se različic SARS-CoV-2 ter dolgotrajnih posledic COVID-19, se iskanje ustreznih zdravil, ki bi presegla omejitve cepiv, nadaljuje. V sklopu doktorske naloge smo pokazali potencial tioredoksin mimetičnih (TXM) peptidov in s tem potrdili smiselnost strategije redukcije disulfidnih mostičkov v boju proti redoks občutljivim virusom. Peptidi TXM, ki delujejo tudi kot antioksidativna in protivnetna sredstva, so preprečili vezavo proteina S SARS-CoV-2 na receptor ACE2 in fuzijo celic. Neodvisno od različice virusa so peptidi TXM zavrli virusni vstop v celice ter poleg inhibicije vstopa psevdovirusov SARS-CoV-2 negativno vplivali tudi na okužbo s psevdovirusi HIV ter s tem pokazali univerzalnost njihove uporabe. Preko uvedbe substitucij in insercij fleksibilnih peptidnih povezovalcev v različnih domenah proteina S SARS-CoV-2 smo želeli prispevati k razumevanju potrebnih strukturnih sprememb, do katerih pride med preureditvijo iz prefuzijske v postfuzijsko konformacijo, ki je nujna za uspešno okužbo gostiteljske celice. S stabilizacijo oziroma destabilizacijo specifičnih regij proteina smo pokazali funkcionalno pomembnost ustrezno stabilnega trimera in neokrnjenost regije SD1 ter v povezavi s tem potrdili vpliv regije S1. Naši rezultati kažejo na občutljivost zmožnosti cepljenja proteina S s furinom in uspešnost fuzije povežejo s pomembnostjo cepitve s TMPRSS2, ki je v nekaterih primerih zmožna povrniti fuzijsko aktivnost proteina S. Raziskavo smo razširili s preučevanjem sestavljanja in dinamike trimernega proteina S. Pokazali smo, da mešani trimeri, ki vsebujejo protomere različnih mutant, lahko nastanejo ter da je za uspešno fuzijo dovolj že en sam polno funkcionalen protomer. S pomočjo daljših inkubacijskih časov smo pokazali, da ob prisotni proteazi TMPRSS2 sinciciji med protein S izražajočimi in receptorskimi celicami lahko nastanejo neodvisno od prisotnosti receptorja ACE2. Glede na profil izražanja ACE2 in TMPRSS2 ter obsežne pljučne poškdobe, ki jih opazimo ob težjem poteku bolezni COVID-19, naše ugotovitve poudarijo pomembnost TMPRSS2 v procesu fuzije, ne glede na prisotnost ACE2.

• Jezik: Slovenski jezik
• Ključne besede: SARS-CoV-2, protein S, konformacijske spremembe, ACE2, TMPRSS2, furin
• Vrsta gradiva: Doktorsko delo/naloga
• Organizacija: MF - Medicinska fakulteta
• Leto izida: 2025
• PID: 20.500.12556/RUL-170534
• Datum objave v RUL: 09.07.2025

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Conformational changes in S protein of SARS-CoV-2 necessary for host cell entry and ACE2 receptor-independent syncytia formation

Izvleček

The emergence of the novel pathogenic coronavirus SARS-CoV-2 in 2020 rapidly escalated into a global crisis. Despite the official end of the pandemic, many questions regarding the virus itself and the intricate mechanisms of host cell infection remain unanswered. However, addressing these gaps is crucial for understanding the disease and mitigating the impact of potential future pandemics. Due to the rapid transmission of SARS-CoV-2, the continuous emergence of viral variants, and the long-term consequences of COVID-19, the search for effective therapeutics beyond the limitations of vaccines remains ongoing. In this doctoral research, we demonstrated the potential of thioredoxin mimetic (TXM) peptides, validating the efficacy of disulfide bond reduction strategies in combating redox-sensitive viruses. TXM peptides, which also exhibit antioxidant and anti-inflammatory properties, successfully inhibited the binding of the SARS-CoV-2 spike (S) protein to the ACE2 receptor and prevented cell fusion. Importantly, these peptides blocked viral entry into host cells across different SARS-CoV-2 variants and, beyond inhibiting SARS-CoV-2 pseudovirus entry, also impaired HIV pseudovirus infection, highlighting their broad-spectrum antiviral potential. To further investigate the structural rearrangements required for the transition of the SARS-CoV-2 S protein from its prefusion to postfusion conformation, we introduced substitutions and flexible peptide linkers in various domains of the S protein. By stabilizing or destabilizing specific regions, we demonstrated the functional significance of maintaining a properly assembled trimer and preserving the integrity of the SD1 region while confirming the role of the S1 subunit in this process. Our findings highlight the sensitivity of S protein cleavage by furin and link successful fusion to the importance of cleavage by TMPRSS2, which, in certain cases, can restore the fusogenic activity of the S protein. Furthermore, we expanded our research by studying the assembly and dynamics of the trimeric S protein. We demonstrated that mixed trimers composed of protomers with different mutations can form and that a single fully functional protomer is sufficient to drive successful cell fusion. Using extended incubation times, we further showed that in the presence of the TMPRSS2 protease, syncytia can form between S-expressing and receptor cells independently of the ACE2 receptor. Given the expression profiles of ACE2 and TMPRSS2, along with the extensive lung damage observed in severe cases of COVID-19, our findings emphasize the critical role of TMPRSS2 in viral fusion, regardless of ACE2 presence.

• Ključne besede: SARS-CoV-2, protein S, conformational changes, ACE2, TMPRSS2, furin