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

2018 ◽  
Vol 92 (12) ◽  
Author(s):  
Le Ma ◽  
Fang Li ◽  
Jing-Wei Zhang ◽  
Wei Li ◽  
Dong-Ming Zhao ◽  
...  
Keyword(s):  
Life Cycle ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Virus Assembly ◽  
Rna Replication ◽  
Protein Processing ◽  
Host Factor ◽  
Transmembrane Domains ◽  
Cell Entry ◽  
Antiviral Targets

ABSTRACTSignal 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.IMPORTANCEUnderstanding 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.

scholarly journals Transmembrane Domains of NS2B Contribute to both Viral RNA Replication and Particle Formation in Japanese Encephalitis Virus

2016 ◽  
Vol 90 (12) ◽  
pp. 5735-5749 ◽  
Author(s):  
Xiao-Dan Li ◽  
Cheng-Lin Deng ◽  
Han-Qing Ye ◽  
Hong-Lei Zhang ◽  
Qiu-Yan Zhang ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Protease Activity ◽  
Rna Synthesis ◽  
Rna Replication ◽  
Viral Assembly ◽  
Transmembrane Domains ◽  
Viral Rna ◽  
Virion Assembly ◽  
Ns3 Protease

ABSTRACTFlavivirus nonstructural protein 2B (NS2B) is a transmembrane protein that functions as a cofactor for viral NS3 protease. The cytoplasmic region (amino acids 51 to 95) alone of NS2B is sufficient for NS3 protease activity, whereas the role of transmembrane domains (TMDs) remains obscure. Here, we demonstrate for the first time that flavivirus NS2B plays a critical role in virion assembly. Using Japanese encephalitis virus (JEV) as a model, we performed a systematic mutagenesis at the flavivirus conserved residues within the TMDs of NS2B. As expected, some mutations severely attenuated (L38A and R101A) or completely destroyed (G12L) viral RNA synthesis. Interestingly, two mutations (G37L and P112A) reduced viral RNA synthesis and blocked virion assembly. None of the mutations affected NS2B-NS3 protease activity. Because mutations G37L and P112A affected virion assembly, we selected revertant viruses for these two mutants. For mutant G37L, replacement with G37F, G37H, G37T, or G37S restored virion assembly. For mutant P112A, insertion of K at position K127 (leading to K127KK) of NS2B rescued virion assembly. A biomolecular fluorescent complementation (BiFC) analysis demonstrated that (i) mutation P112A selectively weakened NS2B-NS2A interaction and (ii) the adaptive mutation K127KK restored NS2B-NS2A interaction. Collectively, our results demonstrate that, in addition to being a cofactor for NS3 protease, flavivirus NS2B also functions in viral RNA replication, as well as virion assembly.IMPORTANCEMany flaviviruses are important human pathogens. Understanding the molecular mechanisms of the viral infection cycle is essential for vaccine and antiviral development. In this study, we demonstrate that the TMDs of JEV NS2B participate in both viral RNA replication and virion assembly. A viral genetic study and a BiFC assay demonstrated that interaction between NS2B and NS2A may participate in modulating viral assembly in the flavivirus life cycle. Compensatory-mutation analysis confirmed that there was a correlation between viral assembly and NS2B-NS2A interaction. TMDs of NS2B may serve as novel antiviral targets to prevent flavivirus infection, and the structure determination of NS2B will help us to understand the functional mechanism of NS2B in viral RNA replication and assembly. The results have uncovered a new function of flavivirus NS2B in virion assembly, possibly through interaction with the NS2A protein.

scholarly journals Valosin-containing protein/p97 plays critical roles in the Japanese encephalitis virus life cycle

2021 ◽  
Author(s):  
Sapna Sehrawat ◽  
Renu Khasa ◽  
Arundhati Deb ◽  
Surendra Kumar Prajapat ◽  
Suvadip Mallick ◽  
...  
Keyword(s):  
Life Cycle ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Host Factor ◽  
Encephalitis Virus ◽  
Host Factors ◽  
Viral Rna ◽  
High Morbidity ◽  
Replication Complex ◽  
Virus Life Cycle

Host factors provide critical support for every aspect of the virus life cycle. We recently identified the valosin-containing protein (VCP)/p97, an abundant cellular ATPase with diverse cellular functions, as a host factor important for Japanese encephalitis virus (JEV) replication. In cultured cells, using siRNA-mediated protein depletion and pharmacological inhibitors, we show that VCP is crucial for replication of three flaviviruses: JEV, Dengue, and West Nile viruses. An FDA-approved VCP inhibitor, CB-5083, extended survival of mice in the animal model of JEV infection. While VCP depletion did not inhibit JEV attachment on cells, it delayed capsid degradation, potentially through the entrapment of the endocytosed virus in clathrin-coated vesicles (CCVs). Early during infection, VCP-depleted cells showed an increased colocalization of JEV capsid with clathrin, and also higher viral RNA levels in purified CCVs. We show that VCP interacts with the JEV nonstructural protein NS5 and is an essential component of the virus replication complex. The depletion of the major VCP cofactor UFD-1 also significantly inhibited JEV replication. Mechanistically, thus, VCP affected two crucial steps of the JEV life cycle – nucleocapsid release and RNA replication. Our study establishes VCP as a common host factor with a broad antiviral potential against flaviviruses. Importance JEV is the leading cause of viral encephalitis epidemics in South-east Asia, affecting majorly children with high morbidity and mortality. Identification of host factors is thus essential for the rational design of anti-virals that are urgently need as therapeutics. Here we have identified the VCP protein as one such host-factor. This protein is highly abundant in cells and engages in diverse functions and cellular pathways by its ability to interact with different co-factors. Using siRNA mediated protein knockdown, we show that this protein is essential for release of the viral RNA into the cell so that it can initiate replication. The protein plays a second crucial role for the formation of the JEV replication complex. FDA-approved drugs targeting VCP show enhanced mouse survival in JE model of disease, suggesting that this could be a druggable target for flavivirus infections.

scholarly journals Identification of heat-shock protein 90 beta in Japanese encephalitis virus-induced secretion proteins

2011 ◽  
Vol 92 (12) ◽  
pp. 2803-2809 ◽  
Author(s):  
Chun-Yu Hung ◽  
Meng-Chieh Tsai ◽  
Yi-Ping Wu ◽  
Robert Y. L. Wang
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Molecular Chaperones ◽  
Heat Shock Protein 90 ◽  
Rna Replication ◽  
Hsp90 Inhibitor ◽  
Encephalitis Virus ◽  
Virus Infectivity ◽  
Density Fractionation ◽  
Virus Particles

Five host cellular proteins were identified in the secretion medium from Japanese encephalitis virus (JEV)-infected baby hamster kidney-21 (BHK-21) cells, including three molecular chaperones: Hsp70, GRP78 and Hsp90. Hsp90 isoforms were characterized further. Hsp90α was observed to be retained inside the nuclei, whereas Hsp90β associated with virus particles during assembly and was released into the secretion medium upon JEV infection. The association of Hsp90β and viral E protein was demonstrated by using sucrose-density fractionation and Western blot analysis. Moreover, JEV viral RNA replication was not affected by treatment with geldanamycin, an Hsp90 inhibitor, but impaired virus infectivity that was determined by a plaque-forming assay. Our results show that Hsp90β, not Hsp90α, is present in the JEV-induced secretion medium and is required for JEV infectivity in BHK-21 cells.

scholarly journals Human MxB Inhibits the Replication of Hepatitis C Virus

2018 ◽  
Vol 93 (1) ◽  
Author(s):  
Dong-Rong Yi ◽  
Ni An ◽  
Zhen-Long Liu ◽  
Feng-Wen Xu ◽  
Kavita Raniga ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Dengue Virus ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Rna Replication ◽  
Encephalitis Virus ◽  
Common Mechanism ◽  
Type I ◽  
Dependent Manner

ABSTRACTType I interferon (IFN) inhibits viruses by inducing the expression of antiviral proteins. The IFN-induced myxovirus resistance B (MxB) protein has been reported to inhibit a limited number of viruses, including HIV-1 and herpesviruses, but its antiviral coverage remains to be explored further. Here we show that MxB interferes with RNA replication of hepatitis C virus (HCV) and significantly inhibits viral replication in a cyclophilin A (CypA)-dependent manner. Our data further show that MxB interacts with the HCV protein NS5A, thereby impairing NS5A interaction with CypA and NS5A localization to the endoplasmic reticulum, two events essential for HCV RNA replication. Interestingly, we found that MxB significantly inhibits two additional CypA-dependent viruses of theFlaviviridaefamily, namely, Japanese encephalitis virus and dengue virus, suggesting a potential link between virus dependence on CypA and virus susceptibility to MxB inhibition. Collectively, these data have identified MxB as a key factor behind IFN-mediated suppression of HCV infection, and they suggest that other CypA-dependent viruses may also be subjected to MxB restriction.IMPORTANCEViruses of theFlaviviridaefamily cause major illness and death around the world and thus pose a great threat to human health. Here we show that IFN-inducible MxB restricts several members of theFlaviviridae, including HCV, Japanese encephalitis virus, and dengue virus. This finding not only suggests an active role of MxB in combating these major pathogenic human viruses but also significantly expands the antiviral spectrum of MxB. Our study further strengthens the link between virus dependence on CypA and susceptibility to MxB restriction and also suggests that MxB may employ a common mechanism to inhibit different viruses. Elucidating the antiviral functions of MxB advances our understanding of IFN-mediated host antiviral defense and may open new avenues to the development of novel antiviral therapeutics.

scholarly journals Scanning Mutagenesis Studies Reveal a Potential Intramolecular Interaction within the C-Terminal Half of Dengue Virus NS2A Involved in Viral RNA Replication and Virus Assembly and Secretion

2015 ◽  
Vol 89 (8) ◽  
pp. 4281-4295 ◽  
Author(s):  
Ren-Huang Wu ◽  
Ming-Han Tsai ◽  
Day-Yu Chao ◽  
Andrew Yueh
Keyword(s):  
Life Cycle ◽  
Dengue Virus ◽  
Virus Assembly ◽  
Intramolecular Interaction ◽  
Rna Replication ◽  
The Other ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Transmembrane Segments ◽  
Virus Life Cycle

ABSTRACTThe NS2A protein of dengue virus (DENV) has eight predicted transmembrane segments (pTMSs; pTMS1 to pTMS8). NS2A has been shown to participate in RNA replication, virion assembly, and the host antiviral response. However, the role of the amino acid residues within the pTMS regions of NS2A during the virus life cycle is poorly understood. In the study described here, we explored the function of DENV NS2A by introducing a series of double or triple alanine substitutions into the C-terminal half (pTMS4 to pTMS8) of NS2A in the context of a DENV infectious clone or subgenomic replicon. Fourteen (8 within pTMS8) of 35 NS2A mutants displayed a lethal phenotype due to impairment of RNA replication by a replicon assay. Three NS2A mutants with mutations within pTMS7, the CM20, CM25, and CM27 mutants, displayed similar phenotypes, low virus yields (>100-fold reduction), wild-type-like replicon activity, and low infectious virus-like particle yields by transienttrans-packaging experiments, suggesting a defect in virus assembly and secretion. The sequencing of revertant viruses derived from CM20, CM25, and CM27 mutant viruses revealed a consensus reversion mutation, leucine (L) to phenylalanine (F), at codon 181 within pTMS7. The introduction of an L181F mutation into a full-length NS2A mutant, i.e., the CM20, CM25, and CM27 constructs, completely restored wild-type infectivity. Notably, L181F also substantially rescued the other severely RNA replication-defective mutants with mutations within pTMS4, pTMS6, and pTMS8, i.e., the CM2, CM3, CM13, CM31, and CM32 mutants. In conclusion, the results revealed the essential roles of pTMS4 to pTMS8 of NS2A in RNA replication and/or virus assembly and secretion. The intramolecular interaction between pTMS7 and pTMS4, pTMS6, or pTMS8 of the NS2A protein was also implicated.IMPORTANCEThe reported characterization of the C-terminal half of dengue virus NS2A is the first comprehensive mutagenesis study to investigate the function of flavivirus NS2A involved in the steps of the virus life cycle. In particular, detailed mapping of the amino acid residues within the predicted transmembrane segments (pTMSs) of NS2A involved in RNA replication and/or virus assembly and secretion was performed. A revertant genetics study also revealed that L181F within pTMS7 is a consensus reversion mutation that rescues both RNA replication-defective and virus assembly- and secretion-defective mutants with mutations within the other three pTMSs of NS2A. Collectively, these findings elucidate the role played by NS2A during the virus life cycle, possibly through the intricate intramolecular interaction between pTMS7 and other pTMSs within the NS2A protein.

scholarly journals Effect of brefelidin A and monensin on Japanese encephalitis virus maturation and virus release from cells

2011 ◽  
Vol 2 (1) ◽  
pp. 9
Author(s):  
Vaibhavi Jawahar Lad ◽  
Ashok Kumar Gupta
Keyword(s):  
Cell Surface ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Golgi Complex ◽  
Protein Transport ◽  
Virus Assembly ◽  
Brefeldin A ◽  
Antigen Expression ◽  
Encephalitis Virus ◽  
The Individual

Japanese encephalitis virus (JEV) replicates in a variety of cells, the exact intracellular site of virus assembly is somewhat obscure. The aims of this study were to investigate the role Golgi apparatus in JEV maturation by utilizing two Golgi-disrupting agents- brefeldin A (BFA) and monensin (MN) that inhibit virus assembly at specific cellular sites. JEV-infected porcine kidney stable (PS) cells were treated with BFA (2 ug/ mL) or MN (10 uM/ mL) at different h post-infection (p. i.) and the virus contents were assayed after 48 h p. i. The treated cells were further subjected to immuno-fluorescence (IF) using antibodies directed against JEV envelope glycoprotein (gpE) for localization of intracellular viral antigen as well as the antigen expression on the cell surface. Addition of BFA or MN to cells immediately after virus adsorption or at 4 h and 12 h postinfection (p. i.), resulted in 4- or 8- fold reduction in infectious virus contents along with inhibition of its transport to the cell surface, indicating an essential role of the Golgi-associated membranes in JEV replication. Interestingly, the antigenicity of the virus, in contrast, remained unaffected as no difference in epitope presentation/ expression was observed in BFA/MN-treated and control (untreated) infected cells even though in the former cells a loss of hemagglutinating (HA) activity was observed. Further, BFA addition at 18 h or 24 h p. i. showed only a negligible effect on virus suggesting that once the viral-associated membranes are formed, these membranes appear to be stable. In contrast, the inhibition with MN persisted even after its addition to cells at 18 h and 24 h p. i., indicating its sustained effect on JEV. Although BFA inhibits protein transport from endoplasmic reticulum (ER) to the Golgi complex while MN inhibits transport from medial to trans cisternae of the Golgi complex, none of the two agents however affected the gpE synthesis and folding essentially required for the epitope presentation/expression within the cells. As flaviviruses are known to encode three glycoproteins (gps) within their genomes i. e., prM, E, and NS, it will be worthwhile in future to determine whether vesicular transport occurs within or between the virus-induced membranes and how the individual JEV-encoded proteins are transported to discrete compartments further remain to be seen.

scholarly journals A Conserved Proline between Domains II and III of Hepatitis C Virus NS5A Influences both RNA Replication and Virus Assembly

2009 ◽  
Vol 83 (20) ◽  
pp. 10788-10796 ◽  
Author(s):  
Mair Hughes ◽  
Sarah Gretton ◽  
Holly Shelton ◽  
David D. Brown ◽  
Christopher J. McCormick ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Assembly ◽  
Infectious Clone ◽  
Consensus Sequence ◽  
Virus Production ◽  
Rna Replication ◽  
Subgenomic Replicon ◽  
Genotype 1B

ABSTRACT We previously demonstrated that two closely spaced polyproline motifs, with the consensus sequence Pro-X-X-Pro-X-Lys/Arg, located between residues 343 to 356 of NS5A, mediated interactions with cellular SH3 domains. The N-terminal motif (termed PP2.1) is only conserved in genotype 1 isolates, whereas the C-terminal motif (PP2.2) is conserved throughout all hepatitis C virus (HCV) isolates, although this motif was shown to be dispensable for replication of the genotype 1b subgenomic replicon. In order to investigate the potential role of these motifs in the viral life cycle, we have undertaken a detailed mutagenic analysis of these proline residues in the context of both genotype 1b (FK5.1) or 2a subgenomic replicons and the genotype 2a infectious clone, JFH-1. We show that the PP2.2 motif is dispensable for RNA replication of all subgenomic replicons and, furthermore, is not required for virus production in JFH-1. In contrast, the PP2.1 motif is only required for genotype 1b RNA replication. Mutation of proline 346 within PP2.1 to alanine dramatically attenuated genotype 1b replicon replication in three distinct genetic backgrounds, but the corresponding proline 342 was not required for replication of the JFH-1 subgenomic replicon. However, the P342A mutation resulted in both a delay to virus release and a modest (up to 10-fold) reduction in virus production. These data point to critical roles for these proline residues at multiple stages in the HCV life cycle; however, they also caution against extrapolation of data from culture-adapted replicons to infectious virus.

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