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

Abstract

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.

Cellular Interleukin Enhancer-Binding Factor 2, ILF2, Inhibits Japanese Encephalitis Virus Replication In Vitro

“…The interaction of virus and host factors is a key step for the virus infection process and plays an important role in viral RNA replication, translation, and post-translational modification, aiding in our understanding of the molecular mechanism of viral pathogenesis and the development of antiviral drugs [38]. In recent years, several studies have identified anti-JEV host restriction factors with different mechanisms [17,39,40,41,42,43,44,45]. In this study, host cellular protein ILF2 was identified to specifically interact with JEV NS3.…”

Tick-Borne Encephalitis Virus: A Structural View

“…The particle and NC assembly processes do not solely rely on viral proteins. In JEV-infected cells, transmembrane domains of NS2B interact with the host factor SPCS1 to secure particle production and in DENV infections, the interaction of C protein and nucleolin is essential for formation of virions [ 141 , 142 ]. Additionally, WNV particle production requires the presence of the host helicase DDX56 at the viral assembly sites [ 143 , 144 ].…”

Avian Influenza A Virus Polymerase Recruits Cellular RNA Helicase eIF4A3 to Promote Viral mRNA Splicing and Spliced mRNA Nuclear Export

“…The BiFC assay was performed according to a previous study (Ma et al, 2018). Briefly, HEK-293T cells grown in 24 well plates were transiently co-transfected with combinations of plasmids VC-PB2 (500 ng) and VN-eIF4A3 (500 ng), or VC-PB2 (500 ng) and VN-vector (500 g) and incubated at 37°C for 24 h. Fluorescence emission was detected using a fluorescence microscope.…”