A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors

Matsunaga, Satoko; Masaoka, Takashi; Sawasaki, Tatsuya; Morishita, Ryo; Iwatani, Yoshinori; Tatsumi, Masashi; Endo, Yaeta; Yamamoto, Naoki; Sugiura, Wataru; Ryo, Akihide

doi:10.3389/fmicb.2015.01220

Cited by 6 publications

(7 citation statements)

References 30 publications

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“…In vitro wheat germ cell-free protein synthesis was carried out as previously described [ 17 , 18 , 19 ]. Protein synthesis of FLAG-SARS-CoV-2-nsp4/5-GST-biotin was performed using WEPRO7240G wheat germ extract (CellFree Sciences, Yokohama, Japan) in the bilayer translation reaction.…”

Section: Methodsmentioning

confidence: 99%

All-Trans Retinoic Acid Exhibits Antiviral Effect against SARS-CoV-2 by Inhibiting 3CLpro Activity

Morita

Miyakawa

Jeremiah

et al. 2021

Viruses

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The pandemic of COVID-19 caused by SARS-CoV-2 continues to spread despite the global efforts taken to control it. The 3C-like protease (3CLpro), the major protease of SARS-CoV-2, is one of the most interesting targets for antiviral drug development because it is highly conserved among SARS-CoVs and plays an important role in viral replication. Herein, we developed high throughput screening for SARS-CoV-2 3CLpro inhibitor based on AlphaScreen. We screened 91 natural product compounds and found that all-trans retinoic acid (ATRA), an FDA-approved drug, inhibited 3CLpro activity. The 3CLpro inhibitory effect of ATRA was confirmed in vitro by both immunoblotting and AlphaScreen with a 50% inhibition concentration (IC50) of 24.7 ± 1.65 µM. ATRA inhibited the replication of SARS-CoV-2 in VeroE6/TMPRSS2 and Calu-3 cells, with IC50 = 2.69 ± 0.09 µM in the former and 0.82 ± 0.01 µM in the latter. Further, we showed the anti-SARS-CoV-2 effect of ATRA on the currently circulating variants of concern (VOC); alpha, beta, gamma, and delta. These results suggest that ATRA may be considered as a potential therapeutic agent against SARS-CoV-2.

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Section: Methodsmentioning

confidence: 99%

All-Trans Retinoic Acid Exhibits Antiviral Effect against SARS-CoV-2 by Inhibiting 3CLpro Activity

Morita

Miyakawa

Jeremiah

et al. 2021

Viruses

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“…We introduced the wheat germ CFPS to provide a set of recombinant proteins from a cDNA library of human protein kinases. Our system can produce proteins from linear cDNA templates generated by PCR ( Sawasaki et al, 2007 ; Matsuoka et al, 2010 ; Matsunaga et al, 2015 ). Moreover, when a biotin ligation sequence is inserted into cDNAs by PCR, biotinylated proteins can be readily synthesized in the presence of biotin and biotin ligase.…”

Section: Discussionmentioning

confidence: 99%

H11/HSPB8 Restricts HIV-2 Vpx to Restore the Anti-Viral Activity of SAMHD1

et al. 2016

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Virus–host interactions play vital roles in viral replication and virus-induced pathogenesis. Viruses rely entirely upon host cells to reproduce progeny viruses; however, host factors positively or negatively regulate virus replication by interacting with viral proteins. The elucidation of virus–host protein interaction not only provides a better understanding of the molecular mechanisms by which host cells combat viral infections, but also facilitates the development of new anti-viral therapeutics. Identification of relevant host factors requires techniques that enable comprehensive characterization of virus–host protein interactions. In this study, we developed a proteomic approach to systematically identify human protein kinases that interact potently with viral proteins. For this purpose, we synthesized 412 full-length human protein kinases using the wheat germ cell-free protein synthesis system, and screened them for their association with a virus protein using the amplified luminescent proximity homogenous assay (AlphaScreen). Using this system, we attempted to discover a robust anti-viral host restriction mechanism targeting virus protein X (Vpx) of HIV-2. The screen identified H11/HSPB8 as a Vpx-binding protein that negatively regulates the stability and function of Vpx. Indeed, overexpression of H11/HSPB8 promoted the degradation of Vpx via the ubiquitin–proteasome pathway and inhibited its interaction with SAMHD1, a host restriction factor responsible for blocking replication of HIV. Conversely, targeted knockdown of H11/HSPB8 in human trophoblast cells, which ordinarily express high levels of this protein, restored the expression and function of Vpx, making the cells highly susceptible to viral replication. These results demonstrate that our proteomic approach represents a powerful tool for revealing virus–host interaction not yet identified by conventional methods. Furthermore, we showed that H11/HSPB8 could be a potential host regulatory factor that may prevent placental infection of HIV-2 during pregnancy.

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“…In vitro transcription and cell-free protein synthesis were performed as previously described ( Takai and Endo, 2010 ; Takai et al, 2010 ; Senchi et al, 2013 ; Matsunaga et al, 2014 , 2015 ). For cell-free protein synthesis, WEPRO7240H wheat germ extract (CellFree Sciences, Yokohama, Japan) was used in the bilayer translation method as previously described ( Matsunaga et al, 2014 ).…”

Section: Methodsmentioning

confidence: 99%

Development of Monoclonal Antibody and Diagnostic Test for Middle East Respiratory Syndrome Coronavirus Using Cell-Free Synthesized Nucleocapsid Antigen

et al. 2016

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Protein nativity is one of the most critical factors for the quality of antigens used as immunogens and the reactivities of the resultant antibodies. The preparation and purification of native viral antigens in conventional cell-based protein expression systems are often accompanied by technical hardships. These challenges are attributable mainly to protein aggregation and insolubility during expression and purification, as well as to very low expression levels associated with the toxicity of some viral proteins. Here, we describe a novel approach for the production of monoclonal antibodies (mAbs) against nucleocapsid protein (NP) of the Middle East respiratory syndrome coronavirus (MERS-CoV). Using a wheat germ cell-free protein synthesis system, we successfully prepared large amounts of MERS-CoV NP antigen in a state that was highly soluble and intact for immunization. Following mouse immunization and hybridoma generation, we selected seven hybridoma clones that produced mAbs with exclusive reactivity against MERS-CoV NP. Epitope mapping and subsequent bioinformatic analysis revealed that these mAbs recognized epitopes located within relatively highly conserved regions of the MERS-CoV amino-acid sequence. Consistently, the mAbs exhibited no obvious cross-reactivity with NPs derived from other related viruses, including SARS coronavirus. After determining the optimal combinations of these mAbs, we developed an enzyme-linked immunosorbent assay and a rapid immunochromatographic antigen detection test that can be reliably used for laboratory diagnosis of MERS-CoV. Thus, this study provides strong evidence that the wheat germ cell-free system is useful for the production of diagnostic mAbs against emerging pathogens.

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A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors

Cited by 6 publications

References 30 publications

All-Trans Retinoic Acid Exhibits Antiviral Effect against SARS-CoV-2 by Inhibiting 3CLpro Activity

All-Trans Retinoic Acid Exhibits Antiviral Effect against SARS-CoV-2 by Inhibiting 3CLpro Activity

H11/HSPB8 Restricts HIV-2 Vpx to Restore the Anti-Viral Activity of SAMHD1

Development of Monoclonal Antibody and Diagnostic Test for Middle East Respiratory Syndrome Coronavirus Using Cell-Free Synthesized Nucleocapsid Antigen

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