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
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 protein 2B (NS2B), which is involved in posttranslational protein processing and virus assembly. Serial deletion mutation of the JEV NS2B protein revealed that two transmembrane domains, NS2B(1-49) and NS2B(84-131), interact with SPCS1. Further mutagenesis analysis of conserved flavivirus residues in two SPCS1 interaction domains of NS2B demonstrated that G12A, G37A, and G47A in NS2B(1-49) and P112A in NS2B(84-131) weakened the interaction with SPCS1. Deletion mutation of SPCS1 revealed that SPCS1(91-169), which contains two transmembrane domains, was involved in interactions with both NS2B(1-49) and NS2B(84-131). Taken together, these results demonstrate that SPCS1 affects viral replication by interacting with NS2B, thereby influencing the posttranslational processing of JEV proteins and the assembly of virions. Understanding virus-host interactions is important for elucidating the molecular mechanisms of virus propagation and identifying potential antiviral targets. Previous reports demonstrated that SPCS1 is involved in the flavivirus life cycle, but the mechanism remains unknown. In this study, we confirmed that SPCS1 participates in the posttranslational protein processing and viral assembly stages of the JEV life cycle but not in the cell entry, genome RNA replication, or translation stages. Furthermore, we found that SPCS1 interacts with two independent transmembrane domains of the flavivirus NS2B protein. NS2B also interacts with NS2A, which is proposed to mediate virus assembly. Therefore, we propose a protein-protein interaction model showing how SPCS1 participates in the assembly of JEV particles. These findings expand our understanding of how host factors participate in the flavivirus replication life cycle and identify potential antiviral targets for combating flavivirus infection.