Characterization of the C-terminal sequence of NS5A necessary for the assembly and production of classical swine fever virus infectious particles

Sheng, Chun; Kou, Shumeng; Jiang, Qin; Zhou, Chixing; Xiao, Jing; Li, Jing; Chen, Bing; Zhao, Yu; Wang, Yujing; Xiao, Ming

doi:10.1016/j.rvsc.2014.07.017

Cited by 19 publications

(6 citation statements)

References 26 publications

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“…Consistent with previous studies [26,27], we showed that the intracellular specific infectivity was decreased in shRab18 cells compared to NTshRNA cells, indicating that Rab18 was required for virion assembly. Accumulating studies have indicated that the interactions between cell proteins and NS5A are essential to CSFV RNA replication and virion assembly [12][13][14]. Rab proteins serve as molecular switches, cycling between the inactive GDP-bound form and active GTP-bound form.…”

Section: Discussionmentioning

confidence: 99%

“…The conserved sequence C2717-C2740-C2742-C2767 of NS5A is essential for viral replication [10]. Additionally, another conserved sequence in the C-terminal region (amino acids 478-487) of NS5A protein is required for virion assembly [12]. Although the definitive mechanism of the NS5A protein in the CSFV life cycle and pathogenesis remains unknown, its mechanism in modulating the host cell environment has been confirmed.…”

Section: Introductionmentioning

confidence: 99%

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Rab18 binds to classical swine fever virus NS5A and mediates viral replication and assembly in swine umbilical vein endothelial cells

et al. 2020

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Classical swine fever virus (CSFV), a positive-sense RNA virus, hijacks cell host proteins for its own replication. Rab18, a small Rab GTPase, regulates intracellular membrane-trafficking events between various compartments in cells and is involved in the life cycle of multiple viruses. However, the effect of Rab18 on the production of CSFV remains uncertain. In this study, we showed that knockdown of Rab18 by lentiviruses inhibited CSFV production, while overexpression of Rab18 by lentiviruses enhanced CSFV production. Subsequent experiments revealed that the negative-mutant Rab18-S22 N inhibited CSFV infection, while the positive-mutant Rab18-Q67 L enhanced CSFV infection. Furthermore, we showed that CSFV RNA replication and virion assembly, measured by real-time fluorescence quantitative PCR (RT-qPCR), indirect immunofluorescence assay (IFA), and confocal microscopy, were reduced in cells lacking Rab18 expression. In addition, co-immunoprecipitation, GST-pulldown, and confocal microscopy assays revealed that Rab18 bound to the viral protein NS5A. Further, NS5A was shown to be redistributed in Rab18 knockdown cells. Taken together, these findings demonstrate Rab18 as a novel host factor required for CSFV RNA replication and particle assembly by interaction with the viral protein NS5A.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Rab18 binds to classical swine fever virus NS5A and mediates viral replication and assembly in swine umbilical vein endothelial cells

et al. 2020

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“…However, in our study, the CSFV NS5A protein interacts with GBP1 and antagonizes the antiviral effect of GBP1 by inhibiting its GTPase activity. CSFV NS5A is a component of the viral replicase complex ( 44 ). It has been reported that NS5A can suppress the activity of the IRES located in the 5′ untranslated region (UTR) in the endoplasmic reticulum, induce oxidative stress, interact with the 3′ UTR, and regulate viral replication ( 45 ).…”

Section: Discussionmentioning

confidence: 99%

Guanylate-Binding Protein 1, an Interferon-Induced GTPase, Exerts an Antiviral Activity against Classical Swine Fever Virus Depending on Its GTPase Activity

et al. 2016

J Virol

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Many viruses trigger the type I interferon (IFN) pathway upon infection, resulting in the transcription of hundreds of interferon-stimulated genes (ISGs), which define the antiviral state of the host. Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious viral disease endangering the pig industry in many countries. However, anti-CSFV ISGs are poorly documented. Here we screened 20 ISGs that are commonly induced by type I IFNs against CSFV in lentivirus-delivered cell lines, resulting in the identification of guanylate-binding protein 1 (GBP1) as a potent anti-CSFV ISG. We observed that overexpression of GBP1, an IFN-induced GTPase, remarkably suppressed CSFV replication, whereas knockdown of endogenous GBP1 expression by small interfering RNAs significantly promoted CSFV growth. Furthermore, we demonstrated that GBP1 acted mainly on the early phase of CSFV replication and inhibited the translation efficiency of the internal ribosome entry site of CSFV. In addition, we found that GBP1 was upregulated at the transcriptional level in CSFV-infected PK-15 cells and in various organs of CSFV-infected pigs. Coimmunoprecipitation and glutathione S-transferase (GST) pulldown assays revealed that GBP1 interacted with the NS5A protein of CSFV, and this interaction was mapped in the N-terminal globular GTPase domain of GBP1. Interestingly, the K51 of GBP1, which is crucial for its GTPase activity, was essential for the inhibition of CSFV replication. We showed further that the NS5A-GBP1 interaction inhibited GTPase activity, which was critical for its antiviral effect. Taking our findings together, GBP1 is an anti-CSFV ISG whose action depends on its GTPase activity.IMPORTANCE Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), an economically important viral disease affecting the pig industry in many countries. To date, only a few host restriction factors against CSFV, including interferon-stimulated genes (ISGs), have been characterized. Using a minilibrary of porcine ISGs, we identify porcine guanylate-binding protein 1 (GBP1) as a potent antiviral ISG against CSFV. We further show that the anti-CSFV action of GBP1 depends on its GTPase activity. The K51 of GBP1, critical for its GTPase activity, is essential for the antiviral action of GBP1 against CSFV replication, and the binding of the NS5A protein to GBP1 antagonizes the GTPase activity and thus the antiviral effect. This study will facilitate the development of anti-CSFV therapeutic agents by targeting host factors and may provide a new strategy for the control of CSF.

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“…3a) (Chen et al, 2012). Recently, we have found that the C-terminal sequence from amino acids 478-487 of NS5A is important for the interaction between CSFV NS5A and Core protein, which also might be related to the production of infectious CSFV particles (Sheng et al, 2014). The amino acid fragment responsible for ANXA2 binding is adjacent to the NS5B binding site at the C terminus of CSFV NS5A (Fig.…”

Section: W I T H O U T S I R N a C O N T R O L S I R N A A N T I -A Nmentioning

confidence: 96%

Annexin A2 is involved in the production of classical swine fever virus infectious particles

Sheng

Liu

Jiang

et al. 2015

Journal of General Virology

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Annexin A2 (ANXA2) is an important host factor regulating several key processes in many viruses. To evaluate the potential involvement of ANXA2 in the life cycle of classical swine fever virus (CSFV), an RNA interference (RNAi) approach was utilized. Knockdown of ANXA2 did not impair CSFV RNA replication but significantly reduced CSFV production. A comparable reduction of extracellular and intracellular infectivity levels was detected, indicating that ANXA2 might play a role in CSFV assembly rather than in genome replication and virion release. Furthermore, ANXA2 was found to bind CSFV NS5A, an essential replicase component. Amino acids R338, N359, G378 of NS5A were revealed to be pivotal for the ANXA2-NS5A interaction. Substitutions of these amino acids had no effect on viral RNA replication but substantially reduced CSFV production, which might partly be due to these mutations destroying the ANXA2-NS5A interaction. These results suggested that ANXA2 might participate in CSFV production process by binding NS5A.

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Characterization of the C-terminal sequence of NS5A necessary for the assembly and production of classical swine fever virus infectious particles

Cited by 19 publications

References 26 publications

Rab18 binds to classical swine fever virus NS5A and mediates viral replication and assembly in swine umbilical vein endothelial cells

Rab18 binds to classical swine fever virus NS5A and mediates viral replication and assembly in swine umbilical vein endothelial cells

Guanylate-Binding Protein 1, an Interferon-Induced GTPase, Exerts an Antiviral Activity against Classical Swine Fever Virus Depending on Its GTPase Activity

Annexin A2 is involved in the production of classical swine fever virus infectious particles

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