Structures and Corresponding Functions of Five Types of Picornaviral 2A Proteins

Yang, Xiaoyao; Cheng, Anchun; Wang, Mingshu; Jia, Renyong; Sun, Kunfeng; Pan, Kangcheng; Yang, Qiao; Wu, Ying; Zhu, Dekang; Chen, Shun; Liu, Mafeng; Zhao, Xinxin; Chen, Xiaoyue

doi:10.3389/fmicb.2017.01373

Cited by 49 publications

(51 citation statements)

References 143 publications

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“…Regarding DHAV, it does not possess an L protein but has three 2A proteins. Specifically, DHAV possesses an NPG/P autocleavage motif in 2A1, avrRpt2-induced gene 1 (AIG1) domain in 2A2, and H-NC motif in 2A3 protein [24,25]. It is likely that the 3C protease is an important putative protease in DHAV for polyprotein processing [26] (Table 1).…”

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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“…For instance, 2A protease from diverse enteroviruses and L protease from aphthoviruses cleave eIF4G 29 , 43 – 45 . Unlike the enteroviruses and aphthoviruses, DHAV 5′UTR-directed translation is insensitive to eIF4G cleavage 23 . The 3C protease in DHAV is the only protease for processing the polyprotein, and the EMCV 3C protease is likewise the only protease of EMCV.…”

Section: Discussionmentioning

confidence: 99%

“…However, the translation of DHAV might not be modulated by its 3′UTR sequence 20 . In addition, there are three structurally different 2A proteins in the polyprotein of DHAV 21 , including an aphthovirus-like 2A1 22 , a conserved avrRpt2-induced gene (AIGI) protein 2A2 21 , and a parechovirus-like 2A3 23 . Hence, 3C protease is the only viral protease in the DHAV polyprotein.…”

Section: Introductionmentioning

confidence: 99%

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

Sun

Wang

Wen

et al. 2017

Sci Rep

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During viral infections, some viruses subvert the host proteins to promote the translation or RNA replication with their protease-mediated cleavage. Poly (A)-binding protein (PABP) is a target for several RNA viruses; however, the impact of duck hepatitis A virus (DHAV) on PABP remains unknown. In this study, we demonstrated for the first time that DHAV infection stimulates a decrease in endogenous PABP and generates two cleavage fragments. On the basis of in vitro cleavage assays, an accumulation of PABP cleavage fragments was detected in duck embryo fibroblast (DEF) cell extracts incubated with functional DHAV 3C protease. In addition, DHAV 3C protease was sufficient for the cleavage of recombinant PABP without the assistance of other eukaryotic cellular cofactors. Furthermore, using site-directed mutagenesis, our data demonstrated a 3C protease cleavage site located between Q367 and G368 in duck PABP. Moreover, the knockdown of PABP inhibited the production of viral RNA, and the C-terminal domain of PABP caused a reduction in viral replication compared to the N-terminal domain. Taken together, these findings suggested that DHAV 3C protease mediates the cleavage of PABP, which may be a strategy to manipulate viral replication.

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“…The 2A protein is one of the non-structural proteins, and of all the proteins encoded by the genome it is the least conserved. Interestingly, several types of 2A have been reported based on their structures and corresponding functions ( Yang et al, 2017 ). In addition, some viruses encode multiple 2A-like sequences – a picornavirus related to duck hepatitis A virus 1 (DHAV-1) was shown to have a total of six 2A proteins, including four ribosome “skipping” 2A proteins ( Wang et al, 2014 ).…”

Section: Introductionmentioning

confidence: 99%

DHAV-1 2A1 Peptide – A Newly Discovered Co-expression Tool That Mediates the Ribosomal “Skipping” Function

et al. 2018

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Duck hepatitis A virus 1 (DHAV-1) belongs to the genus Avihepatovirus in the family Picornaviridae. Little research has been carried out on the non-structural proteins of this virus. This study reports that 2A1 protein, the first non-structural protein on the DHAV-1 genome, has a ribosomal “skipping” function mediated by a “-GxExNPGP-” motif. In addition, we prove that when the sequence is extended 10aa to VP1 from the N-terminal of 2A1, the ribosome “skips” completely. However, as the N-terminus of 2A is shortened, the efficiency of ribosomal “skipping” reduces. When 2A1 is shortened to 10aa, it does not function. In addition, we demonstrate that N18, P19 G20, and P21 have vital roles in this function. We find that the expression of upstream and downstream proteins linked by 2A1 is different, and the expression of the upstream protein is much greater than that of the downstream protein. In addition, we demonstrate that it is the nature of 2A1 that is responsible for the expression imbalance. We also shows that the protein “cleavage” is not due to RNA “cleavage” or RNA transcription abnormalities, and the expressed protein level is independent of RNA transcriptional level. This study provides a systematic analysis of the activity of the DHAV-1 2A1 sequence and, therefore, adds to the “tool-box” that can be deployed for the co-expression applications. It provides a reference for how to apply 2A1 as a co-expression tool.

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Structures and Corresponding Functions of Five Types of Picornaviral 2A Proteins

Cited by 49 publications

References 143 publications

Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization

Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization

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

DHAV-1 2A1 Peptide – A Newly Discovered Co-expression Tool That Mediates the Ribosomal “Skipping” Function

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