Interfering with critical steps of the viral replication cycle to diminish SARS-CoV-2 propagation
by Kim Maren Stegmann
Date of Examination:2022-09-22
Date of issue:2022-10-07
Advisor:Prof. Dr. Matthias Dobbelstein
Referee:Prof. Dr. Matthias Dobbelstein
Referee:Prof. Dr. Bernd Wollnik
Referee:Prof. Dr. Stefan Pöhlmann
Referee:Prof. Dr. Uwe Groß
Referee:Prof. Dr. Kai Tittmann
Referee:Prof. Dr. Heidi Hahn
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EnglishThe ongoing COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, has so far infected more than 0.5 billion people worldwide, with at least 6.3 million deaths, and constitutes one of the largest global health crises in the recent past. The fast progression of the COVID-19 pandemic and the rapid emergence of SARS-CoV-2 variants raised urgent need for effective therapies. In order to contribute to combating the disease, we isolated SARS-CoV-2 from a local COVID-19 patient. Using this virus strain for cell culture experiments, we conducted and contributed to several projects, aiming at interfering with different steps of the viral replication cycle. The results presented in this thesis demonstrate that newly developed and variant-adapted single-domain (VHH) antibodies, also known as nanobodies, neutralize SARS-CoV-2 at low picomolar concentrations, thereby preventing viral entry. Moreover, we show that redox-independent crosslinkers inhibit the main protease of SARS-CoV-2, which is essential for the cleavage of large viral precursor polyproteins into functional proteins. Besides viral entry and proteolytic processing of precursor proteins, viral RNA replication constitutes an important vulnerability of the viral replication cycle. RNA replication can be diminished through inhibition of the cellular nucleotide biosynthesis and/or by administration of non-natural nucleoside analogues. We found that the folate antagonist methotrexate (MTX) significantly reduces SARS-CoV-2 RNA progeny, indicating that purine synthesis is critical for viral replication. Furthermore, the combination of MTX with the purine nucleoside analogue remdesivir further reduces the propagation of the virus. We then expanded the principle of limiting nucleotide biosynthesis in combination with the incorporation of nucleoside analogues to interfere with SARS-CoV-2 replication. Specifically, we combined dihydroorotate dehydrogenase (DHODH) inhibitors, which inhibit the synthesis of pyrimidines, with the pyrimidine nucleoside analogue N4-hydroxycytidine (NHC). Strikingly, the combination of both diminished SARS-CoV-2 RNA yields far more efficiently compared to single drugs. However, since NHC suppresses virus replication by inducing mutations within the viral genome, we modelled NHC-induced virus mutagenesis in vitro, in the presence of a neutralizing nanobody. Our findings disclose the possibility that NHC-treatment might give rise to replication-competent immune-escape variants of the virus. In summary, several treatment strategies were evaluated for antagonizing SARS-CoV-2 replication, ultimately to combat COVID-19.
Keywords: SARS-CoV-2; Coronavirus; COVID-19; Nanobody; Main protease; Methotrexate; Remdesivir; DHODH; Molnupiravir; N4-hydroxycytidine; RNA replication; Nucleotide synthesis; Nucleoside analogues