Dataset Information

Expression, Purification and Characterization of a GII.4 Norovirus Protease from Minerva Virus.

ABSTRACT: BACKGROUND:Noroviruses are the leading cause of acute gastroenteritis worldwide. Norovirus proteases, which are responsible for cleavage of the viral polyprotein, have become an attractive drug target to treat norovirus infections. Genogroup II (GII) noroviruses are responsible for a majority of outbreaks; however, limited data exists regarding GII norovirus proteases. METHODS:We report here successful expression, purification, characterization, and inhibition of the Minerva virus protease (MVpro), a genogroup II genotype 4 (GII.4) norovirus protease. We observed MVpro as both a monomer and dimer in solution through sizeexclusion chromatography. In addition, MVpro cleaves the synthetic substrate mimicking the MVpro NS2/NS3 cleavage site more efficiently than other norovirus proteases such as the Norwalk virus protease (GI.1) and the MD145 protease (GII.4). RESULTS AND CONCLUSION:Compound A, a potent inhibitor of MVpro, is a good starting point for the design of inhibitors to target GII.4 noroviruses. Furthermore, the results presented here will allow for future characterization of MVpro inhibitors as they are synthesized.

SUBMITTER: Kuiper BD

PROVIDER: S-EPMC7734621 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Expression, Purification and Characterization of a GII.4 Norovirus Protease from Minerva Virus.

Kuiper Benjamin D BD Muzzarelli Kendall M KM Keusch Bradley J BJ Holcomb Joshua J Amblard Franck F Liu Peng P Zhou Shaoman S Kovari Iulia A IA Yang Zhe Z Schinazi Raymond F RF Kovari Ladislau C LC

Infectious disorders drug targets 20180101 3

<h4>Background</h4>Noroviruses are the leading cause of acute gastroenteritis worldwide. Norovirus proteases, which are responsible for cleavage of the viral polyprotein, have become an attractive drug target to treat norovirus infections. Genogroup II (GII) noroviruses are responsible for a majority of outbreaks; however, limited data exists regarding GII norovirus proteases.<h4>Methods</h4>We report here successful expression, purification, characterization, and inhibition of the Minerva virus ...[more]

PMID: 29779487

Similar Datasets

Project description:UnlabelledHuman noroviruses (NoVs) are the main etiological agents of acute gastroenteritis worldwide. While NoVs are highly diverse (more than 30 genotypes have been detected in humans), during the last 40 years most outbreaks and epidemics have been caused by GII.4 genotype strains, raising questions about their persistence in the population. Among other potential explanations, immune evasion is considered to be a main driver of their success. In order to study antibody recognition and evasion in detail, we analyzed a conformational epitope recognized by a monoclonal antibody (3C3G3) by phage display, site-directed mutagenesis, and surface plasmon resonance. Our results show that the predicted epitope is composed of 11 amino acids within the P domain: P245, E247, I389, Q390, R397, R435, G443, Y444, P445, N446, and D448. Only two of them, R397 and D448, differ from the homologous variant (GII.4 Den-Haag_2006b) and from a previous variant (GII.4 VA387_1996) that is not recognized by the antibody. A double mutant derived from the VA387_1996 variant containing both changes, Q396R and N447D, is recognized by the 3C3G3 monoclonal antibody, confirming the participation of the two sites in the epitope recognized by the antibody. Furthermore, a single change, Q396R, is able to modify the histo-blood group antigen (HBGA) recognition pattern. These results provide evidence that the epitope recognized by the 3C3G3 antibody is involved in the virus-host interactions, both at the immunological and at the receptor levels.ImportanceHuman noroviruses are the main cause of viral diarrhea worldwide in people of all ages. Noroviruses can infect individuals who had been previously exposed to the same or different norovirus genotypes. Norovirus genotype GII.4 has been reported to be most prevalent during the last 40 years. In the present study, we describe a novel viral epitope identified by a monoclonal antibody and located within the highly diverse P domain of the capsid protein. The evolution of this epitope along with sequential GII.4 variants has allowed noroviruses to evade previously elicited antibodies, thus explaining how the GII.4 genotype can persist over long periods, reinfecting the population. Our results also show that the epitope participates in the recognition of host receptors that have evolved over time, as well.

Project description:The genotype II.4 (GII.4) variants of human noroviruses (HuNVs) are recognized as the major agent of global gastroenteritis outbreaks. Due to the lack of an efficient cell culture system for HuNV propagation, the exact roles of HuNV-encoded nonstructural proteins (including Nterm, NTPase, P22, VPg, Pro, and RdRp) in viral replication or pathogenesis have not yet been fully understood. Here, we report the molecular characterization of the GII.4 HuNV-encoded NTPase (designated GII-NTPase). Results from our studies showed that GII-NTPase forms vesicular or nonvesicular textures in the cell cytoplasm, and the nonvesicular fraction of GII-NTPase significantly localizes to the endoplasmic reticulum (ER) or mitochondria. Deletion analysis revealed that the N-terminal 179-amino-acid (aa) region of GII-NTPase is required for vesicle formation and for ER colocalization, whereas the C-terminal region is involved in mitochondrial colocalization. In particular, two mitochondrion-targeting domains were identified in the C-terminal region of GII-NTPase which perfectly colocalized with mitochondria when the N-terminal region of GII-NTPase was deleted. However, the corresponding C-terminal portions of NTPase derived from the GI HuNV did not show mitochondrial colocalization. We also found that GII-NTPase physically interacts with itself as well as with Nterm and P22, but not VPg, Pro, and RdRp, in cells. The Nterm- and P22-interacting region was mapped to the N-terminal 179-aa region of GII-NTPase, whereas the self-assembly of GII-NTPase could be achieved via a head-to-head, tail-to-tail, or head-to-tail configuration. More importantly, we demonstrate that GII-NTPase possesses a proapoptotic activity, which can be further enhanced by coexpression with Nterm or P22.IMPORTANCE Despite the importance of human norovirus GII.4 variants in global gastroenteritis outbreaks, the basic biological functions of the viral nonstructural proteins in cells remain rarely investigated. In this report, we focus our studies on characteristics of the GII.4 norovirus-encoded NTPase (GII-NTPase). We unexpectedly find that GII-NTPase can perfectly colocalize with mitochondria after its N-terminal region is deleted. However, such a phenomenon is not observed for NTPase encoded by a GI strain. We further reveal that the N-terminal 179-aa region of GII-NTPase is sufficient to mediate (i) vesicle formation, (ii) ER colocalization, (iii) the interaction with two other nonstructural proteins, including Nterm and P22, (iv) the formation of homodimers or homo-oligomers, and (v) the induction of cell apoptosis. Taken together, our findings emphasize that the virus-encoded NTPase must have multiple activities during viral replication or pathogenesis; however, these activities may vary somewhat among different genogroups.

Dataset Information

Expression, Purification and Characterization of a GII.4 Norovirus Protease from Minerva Virus.

Publications

Expression, Purification and Characterization of a GII.4 Norovirus Protease from Minerva Virus.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets