Identification of Cleavage Sites Proteolytically Processed by NS2B-NS3 Protease in Polyprotein of Japanese Encephalitis Virus

Abstract

Understanding the proteolytic processing of polyprotein mediated by NS2B-NS3 protease contributes to the exploration of the mechanisms underlying infection of Japanese encephalitis virus (JEV), a zoonotic flavivirus. In this study, eukaryotic and prokaryotic cell models were employed to identify the cleavage sites mediated by viral NS2B-NS3 protease in JEV polyprotein. Artificial green fluorescent protein (GFP) substrates that contained the predicted cleavage site sequences of JEV polyprotein were expressed in swine testicle (ST) cells in the presence and absence of JEV infection, or co-expressed in E. coli with the recombinant NS2B-NS3 protease that was generated by fusing the N-terminal protease domain of NS3 to the central hydrophilic domain of NS2B. The cleavage of GFP substrates was examined by western blot. Among twelve artificial GFP substrates containing the cleavage site sequences predictively processed by host cell and/or NS2B-NS3 proteases, all sites were found to be cleaved by host cell proteases with different efficiencies. The sites at internal C, NS2A/NS2B, NS2B/NS3 and NS3/NS4A junctions, but not the sites at internal NS3, internal NS4A and NS4B/NS5 junctions were identified to be cleaved by JEV NS2B-NS3 protease. These data provide insight into the proteolytic processing of polyprotein, which is useful for understanding JEV replication and pathogenesis.

NS2B-D55E and NS2B-E65D Variations are Responsible for Differences in NS2B-NS3 Protease Activities Between Japanese Encephalitis Virus Genotype I and III in Fluorogenic Peptide Model

“…Cleavage of artificial GFP substrates was determined by western blot using antibodies specific to GFP and expression of viral proteases were determined by anti-NS3. Here our results demonstrated that GI-SH7 was able to cleave the sites at internal C, NS2A/NS2B, NS2B/NS3, and NS3/NS4A junctions and exhibited the same cleavage pattern as previously reported for SH-GIII [51] , suggesting that viral protease substitution and protein conformation do not interfere with selection of cleavage sites for proteolytic processing and possibly play no role in genotype displacement.…”

Potential Role of Flavivirus NS2B-NS3 Proteases in Viral Pathogenesis and Anti-flavivirus Drug Discovery Employing Animal Cells and Models: A Review

“…NS2B (H) (hydrophilic domain of NS2B) essentially acts as a cofactor for the protease activity of the NS3 protein. The initial characterization of the cofactor requirement for various flaviviruses has revealed that the minimal essential region for protease activity is positioned in a 40-50 residue central hydrophilic segment of NS2B (amino acid 45 to 95) [32,58,66,67]. NS2B contains a hydrophilic region, the central region of which contains a β-barrel, which folds around the β-barrel of the NS3 protease for its stability [55].…”

“…NS2B-NS3 proteases have been involved in carrying out a variety of important phases, e.g., RNA replication (viral), polypeptide cleavage, and the processing and assembly of viral particles [104,105]. The protease activity of the JEV NS2B/NS3 leads to the viral polyprotein cleavage of the capsid (internal), NS2A/NS2B, NS2B/NS3, and NS3/NS4A sites [32]. Moreover, NS2B-NS3 proteases may also play an important role in the immune evasion by the virus [105].…”

“…NS3 encodes the RNA helicase [25,26] serine protease [24,27], RNA triphosphatase (RTPase) [27,28], and nucleocapsid triphosphatase (NTPase) activities [29][30][31]. In particular, NS2B and NS3 are mainly responsible for performing the proteolytic cleavage in the virus (the remainder of the cleavage is performed by the proteases from the cellular origin [23,[32][33][34]), in addition to encoding guanyl and methyltransferase (GTase and MTase activities) [35][36][37]. Flaviviruses replicate inside macrophages, monocytes, and dendritic cells, and they tend to replicate in the cytoplasm of the host cell in order to induce a variety of cytopathic alternations in the cells [38,39].…”

Flavivirus Persistence in Wildlife Populations

“…These particles that contain RNA, 12 kb of positive-sense genome that encodes a single polyprotein that is cleaved into 10 proteins predominately by the NS2B-3 protease, except for the maturation digestion of prM into pr and a fully mature M. Once the virus particle is uncoated following infection, flavivirus RNA genomes are replicated. Following replication of the RNA genome, the polyprotein is translated, consisting of three structural proteins (capsid (C), precursor membrane/membrane (prM), and envelope (E)) and seven non-structural proteins, designated as: NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 [ 25 , 26 , 27 ] ( Figure 3 ).…”