A novel point mutation (L70P) inactivates poliovirus 3C protease

Uma, Madala; Hegde, Sanjay R.; Rao, P P; Nagalekshmi, Karthika; Gauthami, S.; Kumar, Dilip; Hegde, Nagendra R.

doi:10.4149/av_2018_108

Cited by 4 publications

(3 citation statements)

References 28 publications

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“…For example, the 69th residue (Asp) of EV 3C has been identified as a novel and important site that is involved in protease activity and is a virulence determinant [55]. Furthermore, the 3C protease of PV lost protease activity when the 70th residue (Leu) was mutated to proline [56]. In this study, the 3C protease was obtained by prokaryotic expression, and its enzymatic activity was detected with a cleavage assay in vitro.…”

Section: Discussionmentioning

confidence: 98%

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: Discussionmentioning

confidence: 98%

Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization

Sun

Wang

Wen

et al. 2019

Virol J

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“…The PV 3C protein structure has a traditional trypsin-like fold (Figure 2) [36,50], despite having less than 10% sequence identity with the trypsin protease family. It includes six β-sheets that fold into two βbarrel subdomains arranged at right angles [36,51]. The surface groove connecting these subdomains serves as the substrate-binding site, housing the central catalytic triad in the active site [36].…”

Section: Catalytic and Rna-binding Sitesmentioning

confidence: 99%

Picornavirus 3C Proteins Intervene in Host Cell Processes through Proteolysis and Interactions with RNA

Mondal,

Sarvari,

Boehr

2023

Viruses

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The Picornaviridae family comprises a large group of non-enveloped viruses with enormous impact on human and animal health. The picornaviral genome contains one open reading frame encoding a single polyprotein that can be processed by viral proteases. The picornaviral 3C proteases share similar three-dimensional structures and play a significant role in the viral life cycle and virus–host interactions. Picornaviral 3C proteins also have conserved RNA-binding activities that contribute to the assembly of the viral RNA replication complex. The 3C protease is important for regulating the host cell response through the cleavage of critical host cell proteins, acting to selectively ‘hijack’ host factors involved in gene expression, promoting picornavirus replication, and inactivating key factors in innate immunity signaling pathways. The protease and RNA-binding activities of 3C are involved in viral polyprotein processing and the initiation of viral RNA synthesis. Most importantly, 3C modifies critical molecules in host organelles and maintains virus infection by subtly subverting host cell death through the blocking of transcription, translation, and nucleocytoplasmic trafficking to modulate cell physiology for viral replication. Here, we discuss the molecular mechanisms through which 3C mediates physiological processes involved in promoting virus infection, replication, and release.

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“…Adenoviruses are considered to be a safe and well-studied platform for antigen delivery (6)(7)(8), and have been used successfully to develop a COVID-19 vaccine (9). The chimpanzee Ad-vectored (Y25, a simian Ad-23) vaccine candidate (10) encoding the spike protein of MERS-CoV is protective in mice (11) and camels (12), and was shown to be safe and immunogenic in humans (13).…”

Section: Introductionmentioning

confidence: 99%

Preclinical evaluation of safety and immunogenicity of a primary series intranasal COVID-19 vaccine candidate (BBV154) and humoral immunogenicity evaluation of a heterologous prime-boost strategy with COVAXIN (BBV152)

et al. 2022

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Most if not all vaccine candidates developed to combat COVID-19 due to SARS-CoV-2 infection are administered parenterally. As SARS-CoV-2 is transmitted through infectious respiratory fluids, vaccine-induced mucosal immunity could provide an important contribution to control this pandemic. ChAd-SARS-CoV-2-S (BBV154), a replication-defective chimpanzee adenovirus (ChAd)-vectored intranasal (IN) COVID-19 vaccine candidate, encodes a prefusion-stabilized version of the SARS-CoV-2 spike protein containing two proline substitutions in the S2 subunit. We performed preclinical evaluations of BBV154 in mice, rats, hamsters and rabbits. Repeated dose toxicity studies presented excellent safety profiles in terms of pathology and biochemical analysis. IN administration of BBV154 elicited robust mucosal and systemic humoral immune responses coupled with Th1 cell-mediated immune responses. BBV154 IN vaccination also elicited potent variant (omicron) cross neutralization antibodies. Assessment of anti-vector (ChAd36) neutralizing antibodies following repeated doses of BBV154 IN administration showed insignificant titers of ChAd36 neutralizing antibodies. However, the immune sera derived from the same animals displayed significantly higher levels of SARS-CoV-2 virus neutralization (p<0.003). We also evaluated the safety and immunogenicity of heterologous prime-boost vaccination with intramuscular (IM) COVAXIN-prime followed by BBV154 IN administration. COVAXIN priming followed by BBV154 IN-booster showed an acceptable reactogenicity profile comparable to the homologous COVAXIN/COVAXIN or BBV154/BBV154 vaccination model. Heterologous vaccination of COVAXIN-prime and BBV154 booster also elicited superior (p<0.005) and cross variant (omicron) protective immune responses (p<0.013) compared with the homologous COVAXIN/COVAXIN schedule. BBV154 has successfully completed both homologous and heterologous combination schedules of human phase 3 clinical trials and received the restricted emergency use approval (in those aged above 18 years) from the Drugs Controller General of India (DCGI).

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A novel point mutation (L70P) inactivates poliovirus 3C protease

Cited by 4 publications

References 28 publications

Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization

Biochemical characterization of recombinant Avihepatovirus 3C protease and its localization

Picornavirus 3C Proteins Intervene in Host Cell Processes through Proteolysis and Interactions with RNA

Preclinical evaluation of safety and immunogenicity of a primary series intranasal COVID-19 vaccine candidate (BBV154) and humoral immunogenicity evaluation of a heterologous prime-boost strategy with COVAXIN (BBV152)

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