Cleavage of poly(A)-binding protein by duck hepatitis A virus 3C protease

Sun, Di; Wang, Mingshu; Wen, Xingjian; Cheng, Anchun; Jia, Renyong; Sun, Kunfeng; Yang, Qiao; Wu, Ying; Zhu, Dekang; Chen, Shun; Liu, Mafeng; Zhao, Xinxin; Chen, Xiaoyue

doi:10.1038/s41598-017-16484-1

Cited by 40 publications

(27 citation statements)

References 51 publications

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“…For FMDV, different mutations in the cysteine residue at position 142 resulted in different reductions in the protease activity of the 3C protease compared to wild type (k cat /K m = 990 ± 20 M − 1 s − 1 ) [40]. However, studies on the DHAV 3C protease have been limited [41]. Here, we report the activity and localization of the DHAV 3C protease, which provides insight for a better understanding and further characterization of the 3C protease in DHAV infection.…”

Section: Introductionmentioning

confidence: 99%

Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization

Sun

Wang

Wen

et al. 2019

Virol J

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Background The picornaviral 3C protease mediates viral polyprotein maturation and multiple cleavages of host proteins to modulate viral translation and transcription. The 3C protease has been regarded as a valid target due to its structural similarity among different picornaviruses and minimal sequence similarity with host proteins; therefore, the development of potent inhibitors against the 3C protease as an antiviral drug is ongoing. Duck hepatitis A virus (DHAV) belongs to the Picornavidea family and is a major threat to the poultry industry. To date, little is known about the roles of the DHAV 3C protease plays during infection. Methods In this study, we compared the full-length DHAV 3C protein sequence with other 3C sequences to obtain an alignment for the construction of a phylogenetic tree. Then, we expressed and purified recombinant DHAV 3C protease in the BL21 expression system using nickel-NTA affinity chromatography. The optimization of the cleavage assay conditions and the kinetic analysis for DHAV 3C protease were done by in vitro cleavage assays with a fluorogenic peptide respectively. The inhibitory activity of rupintrivir against the DHAV 3C protease was further evaluated. The localization of the 3C protease in infected and transfected cells was determined using immunofluorescence and confocal microscopy. Results Under different expression conditions, the 3C protease was found to be highly expressed after induction with 1 mM IPTG at 16 °C for 10 h. We synthesized a fluorogenic peptide derived from the cleavage site of the DHAV polyprotein and evaluated the protease activity of the DHAV 3C protease for the first time. We used fluorimetric based kinetic analysis to determine kinetic parameters, and V max and K m values were determined to be 16.52 nmol/min and 50.78 μM, respectively. Rupintrivir was found to exhibit inhibitory activity against the DHAV 3C protease. Using polyclonal antibody and an indirect immunofluorescence microscopy assay (IFA), it was determined that the DHAV 3C protease was found in the nucleus during infection. In addition, the DHAV 3C protease can enter into the nucleus without the cooperation of viral proteins. Conclusions This is the first study to examine the activity of the DHAV 3C protease, and the activity of the DHAV 3C protease is temperature-, pH- and NaCl concentration- dependent. The DHAV 3C protease localizes throughout DHAV-infected cells and can enter into the nucleus in the absence of other viral proteins. The kinetic analysis was calculated, and the V max and K m values were 16.52 nmol/min and 50.78 μM, respectively, using the Lineweaver–Burk plot.

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Section: Introductionmentioning

confidence: 99%

Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization

Sun

Wang

Wen

et al. 2019

Virol J

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“…Duck hepatitis A virus type 1 (DHAV-1), a member of the genus Avihepatovirus with characteristics of the family Picornaviridae ( 1 – 8 ), was first reported in Long Island, New York in 1945. DHAV-1 poses a serious threat to the duck industry worldwide ( 9 , 10 ).…”

Section: Introductionmentioning

confidence: 99%

Transcriptomic Characterization of a Chicken Embryo Model Infected With Duck Hepatitis A Virus Type 1

et al. 2018

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Duck hepatitis A virus type 1 (DHAV-1) is one of the most common and lethal pathogens in young ducklings. Live-attenuated DHAV vaccine (CH60 strain) developed by passaging in chicken embryos provided effective immune protection for ducklings. However, the accurate mechanism for such adaption in chicken embryos is not fully revealed. Here, we utilize RNA-sequencing to perform global transcriptional analysis of DHAV-1-innoculated embryonated livers along with histopathological and ultrastructural analysis. This study revealed that infection with DHAV-1 strain CH60 is associated with enhanced type I and II interferon responses, activated innate immune responses, elevated levels of suppressor of cytokine signaling 1 and 3 (SOCS1 and SOCS3) accompanied with abnormalities in multiple metabolic pathways. Excessive inflammatory and innate immune responses induced by the CH60 strain are related to severe liver damage. Our study presents a comprehensive characterization of the transcriptome of chicken embryos infected with DHAV-CH60 and provides insight for in-depth exploration of viral adaption and virus–host interactions.

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“…The process occurs as follows: 43S preinitiation complex assembly, 43S preinitiation complex binding to mRNA, initiation codon (AUG) recognition, and 60S ribosomal subunit addition to form a complete initiation complex and initiate translation [4]. After the virus invades cells, it can hijack or disturb PABP, eIF4G, eIF4E, eIF2, etc., which are involved in classic cap-dependent translation initiation, and reduce [55,56]), blocking PKR activation (PRRSV [57], EMCV [58], IAV [59,60], and MERS-CoV [61]), competing for PKR phosphorylation substrates (myxoma virus [62]), degrading PKR by the lysosome or proteasome pathway (MAV-1 [63] and FMDV [64]) and cleaving PKR via viral proteases (EV71 [65]) the efficiency of intracellular mRNA recruitment by ribosomes [74][75][76][77]. Among these factors, eIF2 α subunit phosphorylation disrupts 43S subunit formation, leading to translation initiation cessation [78].…”

Section: Effect Of Eif2α Phosphorylation On Viral Replication Eif2α Pmentioning

confidence: 99%

The role of host eIF2α in viral infection

Liu¹,

Wang²,

Cheng³

et al. 2020

Virol J

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Background eIF2α is a regulatory node that controls protein synthesis initiation by its phosphorylation or dephosphorylation. General control nonderepressible-2 (GCN2), protein kinase R-like endoplasmic reticulum kinase (PERK), double-stranded RNA (dsRNA)-dependent protein kinase (PKR) and heme-regulated inhibitor (HRI) are four kinases that regulate eIF2α phosphorylation. Main body In the viral infection process, dsRNA or viral proteins produced by viral proliferation activate different eIF2α kinases, resulting in eIF2α phosphorylation, which hinders ternary tRNAMet-GTP-eIF2 complex formation and inhibits host or viral protein synthesis. The stalled messenger ribonucleoprotein (mRNP) complex aggregates under viral infection stress to form stress granules (SGs), which encapsulate viral RNA and transcription- and translation-related proteins, thereby limiting virus proliferation. However, many viruses have evolved a corresponding escape mechanism to synthesize their own proteins in the event of host protein synthesis shutdown and SG formation caused by eIF2α phosphorylation, and viruses can block the cell replication cycle through the PERK-eIF2α pathway, providing a favorable environment for their own replication. Subsequently, viruses can induce host cell autophagy or apoptosis through the eIF2α-ATF4-CHOP pathway. Conclusions This review summarizes the role of eIF2α in viral infection to provide a reference for studying the interactions between viruses and hosts.

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Cleavage of poly(A)-binding protein by duck hepatitis A virus 3C protease

Cited by 40 publications

References 51 publications

Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization

Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization

Transcriptomic Characterization of a Chicken Embryo Model Infected With Duck Hepatitis A Virus Type 1

The role of host eIF2α in viral infection

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