Abstract
Duck Tembusu virus (DTMUV), a mosquito-borne Flavivirus, has caused serious economic losses for the Chinese poultry industry. The genome is translated into a polyprotein that is cleaved to mature protein by host and viral proteases in the host cell, and this proteolytic process is important for the viral life cycle. However, the cleavage mechanism of DTMUV polyprotein is still unclear. In this study, we identified that several amino acids (P1-R, P1′-G, P2-R, P3-T, and P4-V) were vital for NS2A/2B cleavage. Meanwhile, both NS2A and NS2B were essential in cis for polyprotein NS2A/2B intramolecular cleavage. Subsequently, a DTMUV replicon and an infectious clone showed that the P1 site is essential to viral replication, while a mutation in P1′ could boost viral RNA replication. Furthermore, a recombinant virus with P1 and P1′ site mutations named rDTMUV-NS2A/2B-P1P1′(AA) was rescued from transfected BHK21 cells. The maximum viral titers and viral genome copies of rDTMUV-NS2A/2B-P1P1′(AA) were much lower than those of rDTMUV-WT both in the intracellular and extracellular samples of transfected and infected BHK21 cells. Taken together, the NS2A/2B cleavage sites processed by the NS2B3 protease are vital for DTMUV proliferation and virulence.
Highlights
Duck Tembusu virus (DTMUV), a mosquito-borne Flavivirus, has caused serious economic losses for the Chinese poultry industry
The viral genome is first translated into a polyprotein, the polyprotein is cleaved by host protease and a viral protease (NS2B3) in endoplasmic reticulum (ER) to form ten functional proteins (Fig. 1A) including three structural proteins (C, prM and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).The NS3 protease can cleave the polyprotein with its cofactor N S2B9–11
NS2B is a transmembrane protein, and the junction between NS2B and NS3 is self-cleaved by the viral protease, whereby NS2B binding to NS3 causes conformational changes in NS3, which converts NS3 into the more active and stable NS2B3 proteinase[19,20]
Summary
Duck Tembusu virus (DTMUV), a mosquito-borne Flavivirus, has caused serious economic losses for the Chinese poultry industry. The genome is translated into a polyprotein that is cleaved to mature protein by host and viral proteases in the host cell, and this proteolytic process is important for the viral life cycle. The N-terminal 180 amino acids possess protease activity, and as previously escribed, the flavivirus NS2B3 protease can cleave 6 sites in the viral polyproteins C/prM, NS2A/2B, NS2B/3, NS3/4A, NS4A/2K, and NS4B/5 (Fig. 1A)[12,13]. All of these cleavages are necessary for the virus life cycle, for example, NS2A/2B (39 kDa) can be Scientific Reports | (2020) 10:12423. The cleavage of the DTMUV polyprotein by NS2B3 has not been described
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