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Host Factor SPCS1 Regulates the Replication of Japanese Encephalitis Virus through Interactions with Transmembrane Domains of NS2B.

J. Virol.. 2018; 
MaLe,LiFang,ZhangJing-Wei,LiWei,ZhaoDong-Ming,WangHan,HuaRong-Hong,BuZhi
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Rabbit polyclonal antibodies (GenScript 369 A01388-40) and mouse monoclonal antibodies (GenScript A00185-40) againse GFP 370 were obtained from GenScript. Alexa Fluor 680-conjugated donkey anti-mouse IgG 371 antibodies were obtained from Life (A10038). IRDye 800CW-conjugated donkey 372 anti-rabbit IgG antibodies were obtained from LI-COR (926-32213). To visualise proteins by immunoblotting, the following were used as 483 primary antibodies: anti-myc-tag mouse monoclonal (ThermoFisher MA1-980), 484 anti-FLAG mouse monoclonal (ThermoFisher MA1-91878), anti-GAPDH mouse 485 monoclonal (Proteintech 10494-1-AP), anti-GFP rabbit polyclonal (GenScript 486 A01388-40), anti-GFP mouse monoclonal (GenScript A00185-40), and anti-E, 487 anti-NS1, anti-PrM and anti-NS2B mouse monoclonal antibodies. Primary antibodies 488 were detected using Alexa Fluor 680 Donkey Anti-Mouse IgG secondary antibodies 489 (Life A10038) by incubating membranes at a 1:15,000 dilution, and using IRDye 490 800CW Donkey Anti-Rabbit IgG (LI-COR 926-32213) by incubating membranes at a 491 1:20,000 dilution, both for 1 h at room temperature.Mouse monoclonal antibodies against HA (SIGMA 368 H9658) were obtained from Sigma-Aldrich. Rabbit polyclonal antibodies (GenScript 369 A01388-40) and mouse monoclonal antibodies (GenScript A00185-40) againse GFP 370 were obtained from GenScript. Alexa Fluor 680-conjugated donkey anti-mouse IgG 371 antibodies were obtained from Life (A10038). IRDye 800CW-conjugated donkey 372 anti-rabbit IgG antibodies were obtained from LI-COR (926-32213).

摘要

Signal peptidase complex subunit 1 (SPCS1) is a newly identified host factor that regulates flavivirus replication, but the molecular mechanism is not fully understood. Here, using Japanese encephalitis virus (JEV) as a model, we investigated the mechanism through which the host factor SPCS1 regulates the replication of flaviviruses. We first validated the regulatory function of SPCS1 in JEV propagation by knocking down and knocking out endogenous SPCS1. The loss of SPCS1 function markedly reduced intracellular virion assembly and the production of infectious JEV particles but did not affect cell entry, RNA replication, or translation of the virus. SPCS1 was found to interact with nonstructural protei... More

关键词

assembly,flavivirus,host factor,protein-protein interactions,viral replica