Characterization of #Usutu virus NS5 protein. #Polymerase activity, protein-protein interaction and cellular localization (Antimicrob Agents Chemother., abstract)

[Source: Antimicrobial Agents and Chemotherapy, full page: (LINK). Abstract, edited.]

Characterization of Usutu virus NS5 protein. Polymerase activity, protein-protein interaction and cellular localization.

L. Albentosa-González, P. Clemente-Casares, R. Sabariegos, A. Mas

DOI: 10.1128/AAC.01573-19

 

ABSTRACT

Usutu virus (USUV) has become increasingly relevant in recent years with large outbreaks that sporadically have affected humans, being reported in wildlife. Similarly to the rest of flaviviruses, USUV contains a positive single-stranded RNA genome which is replicated by the activity of the non-structural protein 5 (NS5). USUV NS5 shows high sequence identity with the remaining viruses in this genus. This permitted us to identify the predicted methyl-transferase domain and the RNA-dependent RNA polymerase domain (RdRpD). Owing to their high degree of conservation, viral polymerases are considered priority targets for the development of antiviral compounds. In the present study, we have cloned and expressed the entire NS5 and the RdRpD in a heterologous system and have used purified preparations for protein characterizations. We have determined the optimal reaction conditions by investigating how variations in different physicochemical parameters, such as buffer concentration, temperature, and pH, affect RNA polymerization activity. We also found that USUV polymerase, but not the full-length NS5, exhibits cooperative activity in the synthesis of RNA, and that the RdRp activity is not inhibited by Sofosbuvir. To further examine the characteristics of USUV polymerase in a more biological context, we have expressed NS5 and the RdRpD in eukaryotic cells and analyzed its subcellular location. NS5 is predominantly found in the cytoplasm, a significant proportion is directed to the nucleus and this translocation involves nuclear location signals (NLS) located, at least, between the MTase and RdRpD domains.

Copyright © 2019 American Society for Microbiology. All Rights Reserved.

Keywords: Flavivirus; Usutu Virus; Sofosbuvir.

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Giuseppe Michieli

I am an Italian blogger, active since 2005 with main focus on emerging infectious diseases such as avian influenza, SARS, antibiotics resistance, and many other global Health issues. Other fields of interest are: climate change, global warming, geological and biological sciences. My activity consists mainly in collection and analysis of news, public services updates, confronting sources and making decision about what are the 'signals' of an impending crisis (an outbreak, for example). When a signal is detected, I follow traces during the entire course of an event. I started in 2005 my blog ''A TIME'S MEMORY'', now with more than 40,000 posts and 3 millions of web interactions. Subsequently I added an Italian Language blog, then discontinued because of very low traffic and interest. I contributed for seven years to a public forum (FluTrackers.com) in the midst of the Ebola epidemic in West Africa in 2014, I left the site to continue alone my data tracking job.