Next Article in Journal
Unveiling Infection Strategies across Diverse Marine Phage–Host Systems
Previous Article in Journal
Yellow Fever Virus Vaccine Reduces T Cell Receptor Signaling and the Levels of Phosphatase PTPRE In Vivo
Open AccessAbstract

Usutu Virus NS5: Characterization of Polymerase Activity, Protein–Protein Interaction and Cellular Localization %MCEPASTEBIN%

Centro Regional de Investigaciones Biomédicas (CRIB-UCLM), 02008 Albacete, Spain
*
Author to whom correspondence should be addressed.
Presented at Viruses 2020—Novel Concepts in Virology, Barcelona, Spain, 5–7 February 2020.
Proceedings 2020, 50(1), 98; https://doi.org/10.3390/proceedings2020050098
Published: 24 June 2020
Usutu virus (USUV) is a mosquito-borne arbovirus that has rapidly propagated in birds across several European countries over the last two decades, leading to substantial avian mortalities. USUV infection in humans has been associated with a growing number of cases of neurological disease in the last years, underlining the need for increased awareness and suitable treatments. Our group is working on the characterization of the NS5 protein of USUV. This protein is responsible for the replication activity of the viral genome and can be a suitable viral target to treat the infection. NS5 contains a RNA-dependent RNA polymerase (RdRpD) and a methyltransferase domains. Recombinant NS5 and RdRpD proteins expressed in bacteria were purified and biochemically characterized to determine the best conditions for their polymerase activities. Both proteins showed de novo and primer extension activities. Optimal RNA–polymerase reaction conditions included low NaCl (less than 20 mM), 2.5 mM MgCl2 and 5 mM MnCl2, 30 °C, and pH 7.25. Polymerase activity was cooperative for the polymerase domain (Hill coefficient = 5.8) but not for the complete NS5 (Hill coefficient = 1.2). To study their subcellular location, suitable sequences were cloned into a pcDNA3 vector and expressed in Huh7.5 and HEK293T cells. Both proteins were preferentially located in the cytoplasmic region, although a significant amount was found in the nucleus. Preliminary results showed that the concentration of sofosbuvir (SOFTP) necessary to achieve its incorporation by NS5 in 50% of the nascent RNA is higher than 100 µM, as already observed for dengue virus DENV. In this work, we describe the main features of the full-length USUV NS5, including the polymerase activity as well as the effect of protein–protein interactions and subcellular localization. Our results will be very useful for the study of this viral enzyme as a suitable target against the infection and the effect of antiviral drugs.
Keywords: USUV; NS5; RdRp; MTase USUV; NS5; RdRp; MTase
MDPI and ACS Style

Albentosa-González, L.; Clemente-Casares, P.; Sabariegos, R.; Mas, A. Usutu Virus NS5: Characterization of Polymerase Activity, Protein–Protein Interaction and Cellular Localization %MCEPASTEBIN%. Proceedings 2020, 50, 98.

Show more citation formats Show less citations formats
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Access Map by Country/Region

1
Back to TopTop