ABSTRACT
The global pandemic prompted by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has already caused more than 6 million deaths worldwide, calling for urgent effective therapeutic measures. A deep understanding of the mechanisms involved in viral replication is required. Among the nonstructural proteins (nsps) encoded by SARS-CoV-2 genome, there is the nsp14 ribonuclease, the main object of study in this work. Ribonucleases are key factors in the control of all biological processes, ensuring maturation, degradation, and quality control of all types of RNAs. Nsp14 is a bifunctional protein, holding a 3'- 5' exoribonucleolytic activity (ExoN) in the N-terminal domain, stimulated through the interaction with nsp10, and a C-terminal N7-methyltransferase activity (MTase). Both are critical for the coronavirus life cycle. In this work, we provide a complete biochemical characterization of SARS-CoV-2 nsp14-nsp10, addressing several aspects of the complex for the first time. Moreover, using a homology model, we have identified residues involved in the nsp14-nsp10 interaction that were extensively studied. We have confirmed the SARS-CoV-2 nsp14 dual function and we have shown that both ExoN and MTase activities are functionally independent. We demonstrate that the nsp14 MTase activity is independent of nsp10, contrarily to nsp14 ExoN that is upregulated in the presence of the cofactor. Additionally, our results show that the ExoN motif I has a prominent role on the ribonucleolytic activity of SARS-CoV-2 nsp14, contrasting to what was previously observed in other coronaviruses, which can be related to the pathogenesis of SARS-CoV-2. The knowledge provided in this work can serve as a basis to design effective drugs that target the pinpointed residues in order to disturb the complex assembly and affect the viral replication, ultimately, treating COVID-19 and other CoV infections.
ABSTRACT
Objective. COVID-19 has spread worldwide and Portugal decreed the State of Emergency on March 18th, 2020. During this period, the population was encouraged to stay at home. Still, there were no restrictions on access to health care. Therefore, we aimed to compare the major causes for attending the Obstetrics and Gynaecology Emergency Department (ED) from a referral centre (Maternidade Dr. Alfredo da Costa, in Lisbon). Materials and Methods. Several variables were collected and compared between two periods of time: from 19th March to 2nd April 2020 and the same period of 2019. Results. During the COVID-19 pandemic period, 49.4% fewer patients visited the ED. We observed a higher number of urgent patients and hospitalization rate than previous year. Conclusions. We experienced a reduction number of admissions to the Obstetrics and Gynaecology ED, but apparently the severity of cases that visited the ED increased. © 2022, EDRA S.p.A. All rights reserved.