Development Of Antiviral Drugs Research Articles

Discovery of First-in-Class PROTAC Degraders of SARS-CoV-2 Main Protease.

We have witnessed three coronavirus (CoV) outbreaks in the past two decades, including the COVID-19 pandemic caused by SARS-CoV-2. Main protease (MPro), a highly conserved protease among various CoVs, is essential for viral replication and pathogenesis, making it a prime target for antiviral drug development. Here, we leverage proteolysis targeting chimera (PROTAC) technology to develop a new class of small-molecule antivirals that induce the degradation of SARS-CoV-2 MPro. Among them, MPD2 was demonstrated to effectively reduce MPro protein levels in 293T cells, relying on a time-dependent, CRBN-mediated, and proteasome-driven mechanism. Furthermore, MPD2 exhibited remarkable efficacy in diminishing MPro protein levels in SARS-CoV-2-infected A549-ACE2 cells. MPD2 also displayed potent antiviral activity against various SARS-CoV-2 strains and exhibited enhanced potency against nirmatrelvir-resistant viruses. Overall, this proof-of-concept study highlights the potential of targeted protein degradation of MPro as an innovative approach for developing antivirals that could fight against drug-resistant viral variants.

The dsRNA isolated from Escherichia coli infected with the MS2 bacteriophage induces the production of interferons

Viral infections pose a significant threat to public health, and the production of interferons represents one of the most critical antiviral innate immune responses of the host. Consequently, the screening and identification of compounds or reagents that induce interferon production are of paramount importance. This study commenced with the cultivation of host bacterium 15,597, followed by the infection of Escherichia coli with the MS2 bacteriophage. Utilizing the J2 capture technique, a class of dsRNA mixtures (MS2+15,597) was isolated from the E. coli infected with the MS2 bacteriophage. Subsequent investigations were conducted on the immunostimulatory activity of the MS2+15,597 mixture. The results indicated that the dsRNA mixtures (MS2+15,597) extracted from E. coli infected with the MS2 bacteriophage possess the capability to activate innate immunity, thereby inducing the production of interferon-β. These dsRNA mixtures can activate the RIG-I and TLR3 pattern recognition receptors, stimulating the expression of interferon stimulatory factors 3/7, which in turn triggers the NF-κB signaling pathway, culminating in the cellular production of interferon-β to achieve antiviral effects. This study offers novel insights and strategies for the development of broad-spectrum antiviral drugs, potentially providing new modalities for future antiviral therapies.

Potent human neutralizing antibodies against Nipah virus derived from two ancestral antibody heavy chains

Nipah virus (NiV) is a World Health Organization priority pathogen and there are currently no approved drugs for clinical immunotherapy. Through the use of a naïve human phage-displayed Fab library, two neutralizing antibodies (NiV41 and NiV42) targeting the NiV receptor binding protein (RBP) were identified. Following affinity maturation, antibodies derived from NiV41 display cross-reactivity against both NiV and Hendra virus (HeV), whereas the antibody based on NiV42 is only specific to NiV. Results of immunogenetic analysis reveal a correlation between the maturation of antibodies and their antiviral activity. In vivo testing of NiV41 and its mature form (41-6) show protective efficacy against a lethal NiV challenge in hamsters. Furthermore, a 2.88 Å Cryo-EM structure of the tetrameric RBP and antibody complex demonstrates that 41-6 blocks the receptor binding interface. These findings can be beneficial for the development of antiviral drugs and the design of vaccines with broad spectrum against henipaviruses.

Current Status of Potential Antiviral Drugs Derived from Plant, Marine, and Microbial Sources

Abstract: Natural substances have been the principal source of medications since antiquity. Natural goods are gaining popularity as a source of novel medications. This article investigates a variety of variables like plant, marine, and microbial sources that contribute to the growing interest in natural goods as a source of novel medications. Viruses have remained resistant to treatment and prevention for a longer period than other forms of life. Viral diseases can currently only be treated with a limited number of drugs. Significant research initiatives have been committed to identifying novel antiviral natural compounds to fight viruses that harm people, plants, insects, animals, fungi, and microbes. A recent study of the prevalence and sources of antiviral medications licensed by the Food and Drug Administration (FDA) has focused on natural products. Out of the estimated 250,000 higher plant species, only 5 to 15 percent have been thoroughly evaluated for the existence of bioactive substances in them, and the ability of the other species has hardly been investigated. This review aims to offer an overview of the crucial role played by natural products in the discovery and development of novel antiviral drugs with potent antiviral activity, including phytochemicals such as carbohydrates, coumarins, flavonoids, chromones, alkaloids, lignans, phenols, tannins, proteins, peptides, antiviral plant extracts, other marine, and microbial sources.

Retracted and republished from: "The current state of research on influenza antiviral drug development: drugs in clinical trial and licensed drugs".

Influenza viruses (IVs) threaten global human health due to the high morbidity, infection, and mortality rates. Currently, the influenza drugs recommended by the Food and Drug Administration are oseltamivir, zanamivir, peramivir, and baloxavir marboxil. These recommended antivirals are currently effective for major subtypes of IVs as the compounds target conserved domains in neuraminidase or polymerase acidic (PA) protein. However, this trend may gradually change due to the selection of antiviral drugs and the natural evolution of IVs. Therefore, there is an urgent need to develop drugs related to the treatment of influenza to deal with the next pandemic. Here, we summarized the cutting-edge research in mechanism of action, inhibitory activity, and clinical efficacy of drugs that have been approved and drugs that are still in clinical trials for influenza treatment. We hope this review will provide up-to-date and comprehensive information on influenza antivirals and generate hypotheses for screens and development of new broad-spectrum influenza drugs in the near future.

Coat protein of alfalfa mosaic alfamovirus (AMV) from Türkiye: genetic inference and in silico docking analysis for potential antiphytoviral purposes

In 2021, a study was conducted in the Denizli region of Türkiye to investigate the phylogenetic relationship and presence of alfalfa mosaic alfamovirus (AMV) infecting pepper plants exhibiting viral disease symptoms. A total of 57 samples were collected, of which twenty-four tested positive for AMV with reverse transcriptase polymerase chain reaction (RT-PCR) assays. Samples from pepper plants displaying virus symptoms gave positive bands on the agarose gel, while healthy plants yielded negative results. One of the positive samples was randomly selected, cloned and sequenced. This sequence of the Denizli AMV isolate (753 bp) was recorded in the GenBank database with accession number OQ845956. The nucleotide sequence showed a high nucleotide consensus of 97%-99% compared with the nucleotide sequences of the same variants from different origins in GenBank. According to the phylogenetic tree generated through the Neighbour Joining (NJ) method, this AMV isolate belongs to the same group as Iranian isolates from various of hosts. Furthermore, in silico docking analysis of the coat protein (CP) of the AMV isolate and promising 12 essential oil compounds was performed to enable potential antiviral drug development. Docking study showed that eucalyptol, eugenol and carvacrol can make important contributions to the advancement of drug-based strategies for the managing of plant viruses by interacting with the virus coat protein of high binding energies, -5.3, -5.2 and -5.0 kcal mol-1, respectively. Although the presence of AMV in Denizli province has been reported previously, this study reports the phylogenetic relationships and docking analysis of the new AMV isolate in pepper crops.

Retraction for Li et al., "The current state of research on influenza antiviral drug development: drugs in clinical trial and licensed drugs".

Retraction for Li et al., "The current state of research on influenza antiviral drug development: drugs in clinical trial and licensed drugs".

BAG6 inhibits influenza A virus replication by inducing viral polymerase subunit PB2 degradation and perturbing RdRp complex assembly.

The interaction between influenza A virus (IAV) and host proteins is an important process that greatly influences viral replication and pathogenicity. PB2 protein is a subunit of viral ribonucleoprotein (vRNP) complex playing distinct roles in viral transcription and replication. BAG6 (BCL2-associated athanogene 6) as a multifunctional host protein participates in physiological and pathological processes. Here, we identify BAG6 as a new restriction factor for IAV replication through targeting PB2. For both avian and human influenza viruses, overexpression of BAG6 reduced viral protein expression and virus titers, whereas deletion of BAG6 significantly enhanced virus replication. Moreover, BAG6-knockdown mice developed more severe clinical symptoms and higher viral loads upon IAV infection. Mechanistically, BAG6 restricted IAV transcription and replication by inhibiting the activity of viral RNA-dependent RNA polymerase (RdRp). The co-immunoprecipitation assays showed BAG6 specifically interacted with the N-terminus of PB2 and competed with PB1 for RdRp complex assembly. The ubiquitination assay indicated that BAG6 promoted PB2 ubiquitination at K189 residue and targeted PB2 for K48-linked ubiquitination degradation. The antiviral effect of BAG6 necessitated its N-terminal region containing a ubiquitin-like (UBL) domain (17-92aa) and a PB2-binding domain (124-186aa), which are synergistically responsible for viral polymerase subunit PB2 degradation and perturbing RdRp complex assembly. These findings unravel a novel antiviral mechanism via the interaction of viral PB2 and host protein BAG6 during avian or human influenza virus infection and highlight a potential application of BAG6 for antiviral drug development.

A Mini-Review on the Common Antiviral Drug Targets of Coronavirus.

Coronaviruses in general are a zoonotic pathogen with significant cross-species transmission. They are widely distributed in nature and have recently become a major threat to global public health. Vaccines are the preferred strategy for the prevention of coronaviruses. However, the rapid rate of virus mutation, large number of prevalent strains, and lag in vaccine development contribute to the continuing frequent occurrence of coronavirus diseases. There is an urgent need for new antiviral strategies to address coronavirus infections effectively. Antiviral drugs are important in the prevention and control of viral diseases. Members of the genus coronavirus are highly similar in life-cycle processes such as viral invasion and replication. These, together with the high degree of similarity in the protein sequences and structures of viruses in the same genus, provide common targets for antiviral drug screening of coronaviruses and have led to important advances in recent years. In this review, we summarize the pathogenic mechanisms of coronavirus, common drugs targeting coronavirus entry into host cells, and common drug targets against coronaviruses based on biosynthesis and on viral assembly and release. We also describe the common targets of antiviral drugs against coronaviruses and the progress of antiviral drug research. Our aim is to provide a theoretical basis for the development of antiviral drugs and to accelerate the development and utilization of commonly used antiviral drugs in China.

Deoxycholic acid inhibits ASFV replication by inhibiting MAPK signaling pathway

African swine fever (ASF) is an acute, febrile, highly contagious infection of pigs caused by the African swine fever virus (ASFV). The purpose of this study is to understand the molecular mechanism of ASFV infection and evaluate the effect of DCA on MAPK pathway, so as to provide scientific basis for the development of new antiviral drugs. The transcriptome analysis found that ASFV infection up-regulated the IL-17 and MAPK signaling pathways to facilitate viral replication. Metabolome analysis showed that DCA levels were up-regulated after ASFV infection, and that exogenous DCA could inhibit activation of the MAPK pathway by ASFV infection and thus inhibit viral replication. Dual-luciferase reporter assays were used to screen the genes of ASFV and revealed that I73R could significantly up-regulate the transcription level of AP-1 transcription factor in the MAPK pathway. Confocal microscopy demonstrated that I73R could promote AP-1 entry into the nucleus, and that DCA could inhibit the I73R-mediated nuclear entry of AP-1, inhibiting MAPK pathway, and I73R interacts with AP-1. These results indicated that DCA can inhibit ASFV-mediated activation of the MAPK pathway, thus inhibiting ASFV replication. This study provides a theoretical basis for research on ASF pathogenesis and for antiviral drug development.

Construction of recombinant fluorescent LSDV for high-throughput screening of antiviral drugs

Lumpy skin disease virus (LSDV) infection is a major socio-economic issue that seriously threatens the global cattle-farming industry. Here, a recombinant virus LSDV-ΔTK/EGFP, expressing enhanced green fluorescent protein (EGFP), was constructed with a homologous recombination system and applied to the high-throughput screening of antiviral drugs. LSDV-ΔTK/EGFP replicates in various kidney cell lines, consistent with wild-type LSDV. The cytopathic effect, viral particle morphology, and growth performance of LSDV-ΔTK/EGFP are consistent with those of wild-type LSDV. High-throughput screening allowed to identify several molecules that inhibit LSDV-ΔTK/EGFP replication. The strong inhibitory effect of theaflavin on LSDV was identified when 100 antiviral drugs were screened in vitro. An infection time analysis showed that theaflavin plays a role in the entry of LSDV into cells and in subsequent viral replication stages. The development of this recombinant virus will contribute to the development of LSDV-directed antiviral drugs and the study of viral replication and mechanisms of action.

Getah virus capsid protein undergoes co-condensation with viral genomic RNA to facilitate virion assembly

Getah virus (GETV) belongs to the Alphavirus genus in the Togaviridae family and is a zoonotic arbovirus causing disease in both humans and animals. The capsid protein (CP) of GETV regulates the viral core assembly, but the mechanism underlying this process is poorly understood. In this study, we demonstrate that CP undergoes liquid-liquid phase separation (LLPS) with the GETV genome RNA (gRNA) in vitro and forms cytoplasmic puncta in cells. Two regions of GETV gRNA (nucleotides 1–4000 and 5000-8000) enhance CP droplet formation in vitro and the lysine-rich Link region of CP is essential for its phase separation. CP(K/R) mutant with all lysines in the Link region replaced by arginines exhibits improved LLPS versus wild type (WT) CP, but CP(K/E) mutant with lysines substituted by glutamic acids virtually loses condensation ability. Consistently, recombinant virus mutant with CP(K/R) possesses significantly higher gRNA binding affinity, virion assembly efficiency and infectivity than the virus with WT-CP. Overall, our findings provide new insights into the understanding of GETV assembly and development of new antiviral drugs against alphaviruses.

The Accumulation of Phenyllactic Acid Impairs Host Glutamine Metabolism and Inhibits African Swine Fever Virus Replication: A Novel Target for the Development of Anti-ASFV Drugs.

African swine fever (ASF) is a highly contagious and hemorrhagic disease caused by infection with the African swine fever virus (ASFV), resulting in a mortality rate of up to 100%. Currently, there are no effective treatments and commercially available vaccines for ASF. Therefore, it is crucial to identify biochemicals derived from host cells that can impede ASFV replication, with the aim of preventing and controlling ASF. The ASFV is an acellular organism that promotes self-replication by hijacking the metabolic machinery and biochemical resources of host cells. ASFV specifically alters the utilization of glucose and glutamine, which are the primary metabolic sources in mammalian cells. This study aimed to investigate the impact of glucose and glutamine metabolic dynamics on the rate of ASFV replication. Our findings demonstrate that ASFV infection favors using glutamine as a metabolic fuel to facilitate self-replication. ASFV replication can be substantially inhibited by blocking glutamine metabolism. The metabolomics analysis of the host cell after late-stage ASFV infection revealed a significant disruption of normal glutamine metabolic pathways due to the abundant expression of PLA (phenyllactic acid). Pretreatment with PLA also inhibited ASFV proliferation and glutamine consumption following infection. The metabolomic analysis also showed that PLA pretreatment greatly slowed down the metabolism of amino acids and nucleotides that depend on glutamine. The depletion of these building blocks directly hindered the replication of ASFV by decreasing the biosynthetic precursors produced during the replication of ASFV's progeny virus. These findings provide valuable insight into the possibility of pursuing the development of antiviral drugs against ASFV that selectively target metabolic pathways.

Neuroinvasive virus facilitates viral replication by employing lipid droplets to reduce arachidonic acid-induced ferroptosis

Lipids have been previously implicated in the lifecycle of neuroinvasive viruses. However, the role of lipids in programmed cell death and the relationship between programmed cell death and lipid droplets (LDs) in neuroinvasive virus infection remains unclear. Here, we found that the infection of neuroinvasive virus, such as rabies virus and encephalomyocarditis virus could enhance the LD formation in N2a cells, and decreasing LDs production by targeting diacylglycerol acyltransferase could suppress viral replication. The lipidomics analysis revealed that arachidonic acid (AA) was significantly increased after reducing LD formation by restricting diacylglycerol acyltransferase, and AA was further demonstrated to induce ferroptosis to inhibit neuroinvasive virus replication. Moreover, lipid peroxidation and viral replication inhibition could be significantly alleviated by a ferroptosis inhibitor, ferrostatin-1, indicating that AA affected neuroinvasive virus replication mainly through inducing ferroptosis. Furthermore, AA was demonstrated to activate the acyl-CoA synthetase long-chain family member 4-lysophosphatidylcholine acyltransferase 3-cytochrome P450 oxidoreductase axis to induce ferroptosis. Our findings highlight novel cross-talks among viral infection, LDs, and ferroptosis for the first time, providing a potential target for antiviral drug development.

Potent small molecule inhibitors against the 3C protease of foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is one of the most devastating diseases of livestock which can cause significant economic losses, especially when introduced to FMD-free countries. FMD virus (FMDV) belongs to the family Picornaviridae and is antigenically heterogeneous with seven established serotypes. The prevailing preventive and control strategies are limited to restriction of animal movement and elimination of infected or exposed animals, which can be potentially combined with vaccination. However, FMD vaccination has limitations including delayed protection and lack of cross-protection against different serotypes. Recently, antiviral drug use for FMD outbreaks has increasingly been recognized as a potential tool to augment the existing early response strategies, but limited research has been reported on potential antiviral compounds for FMDV. FMDV 3C protease (3Cpro) cleaves the viral-encoded polyprotein into mature and functional proteins during viral replication. The essential role of viral 3Cpro in viral replication and the high conservation of 3Cpro among different FMDV serotypes make it an excellent target for antiviral drug development. We have previously reported multiple series of inhibitors against picornavirus 3Cpro or 3C-like proteases (3CLpros) encoded by coronaviruses or caliciviruses. In this study, we conducted structure-activity relationship studies for our in-house focused compound library containing 3Cpro or 3CLpro inhibitors against FMDV 3Cpro using enzyme and cell-based assays. Herein, we report the discovery of aldehyde and α-ketoamide inhibitors of FMDV 3Cpro with high potency. These data inform future preclinical studies that are related to the advancement of these compounds further along the drug development pathway.IMPORTANCEFood-and-mouth disease (FMD) virus (FMDV) causes devastating disease in cloven-hoofed animals with a significant economic impact. Emergency response to FMD outbreaks to limit FMD spread is critical, and the use of antivirals may overcome the limitations of existing control measures by providing immediate protection for susceptible animals. FMDV encodes 3C protease (3Cpro), which is essential for virus replication and an attractive target for antiviral drug discovery. Here, we report a structure-activity relationship study on multiple series of protease inhibitors and identified potent inhibitors of FMDV 3Cpro. Our results suggest that these compounds have the potential for further development as FMD antivirals.

Kernel Bayesian nonlinear matrix factorization based on variational inference for human–virus protein–protein interaction prediction

Identification of potential human–virus protein–protein interactions (PPIs) contributes to the understanding of the mechanisms of viral infection and to the development of antiviral drugs. Existing computational models often have more hyperparameters that need to be adjusted manually, which limits their computational efficiency and generalization ability. Based on this, this study proposes a kernel Bayesian logistic matrix decomposition model with automatic rank determination, VKBNMF, for the prediction of human–virus PPIs. VKBNMF introduces auxiliary information into the logistic matrix decomposition and sets the prior probabilities of the latent variables to build a Bayesian framework for automatic parameter search. In addition, we construct the variational inference framework of VKBNMF to ensure the solution efficiency. The experimental results show that for the scenarios of paired PPIs, VKBNMF achieves an average AUPR of 0.9101, 0.9316, 0.8727, and 0.9517 on the four benchmark datasets, respectively, and for the scenarios of new human (viral) proteins, VKBNMF still achieves a higher hit rate. The case study also further demonstrated that VKBNMF can be used as an effective tool for the prediction of human–virus PPIs.

Construction of an infectious clone for enterovirus A89 and mutagenesis analysis of viral infection and cell binding.

Enterovirus A89 (EV-A89) is an unconventional strain belonging to the Enterovirus A species. Limited research has been conducted on EV-A89, leaving its biological and pathogenic properties unclear. Developing reverse genetic tools for EV-A89 would help to unravel its infection mechanisms and aid in the development of vaccines and anti-viral drugs. In this study, an infectious clone for EV-A89 was successfully constructed and recombinant enterovirus A89 (rEV-A89) was generated. The rEV-A89 exhibited similar characteristics such as growth curve, plaque morphology, and dsRNA expression with parental strain. Four amino acid substitutions were identified in the EV-A89 capsid, which were found to enhance viral infection. Mechanistic studies revealed that these substitutions increased the virus's cell-binding ability. Establishing reverse genetic tools for EV-A89 will significantly contribute to understanding viral infection and developing anti-viral strategies.IMPORTANCEEnterovirus A species contain many human pathogens and have been classified into conventional cluster and unconventional cluster. Most of the research focuses on various conventional members, while understanding of the life cycle and infection characteristics of unconventional viruses is still very limited. In our study, we constructed the infectious cDNA clone and single-round infectious particles for the unconventional EV-A89, allowing us to investigate the biological properties of recombinant viruses. Moreover, we identified key amino acids residues that facilitate EV-A89 infection and elucidate their roles in enhancing viral binding to host cells. The establishment of the reverse genetics system will greatly facilitate future study on the life cycle of EV-A89 and contribute to the development of prophylactic vaccines and anti-viral drugs.

Function and Mechanism of Antiviral Wasp Venom Peptide Protopolybia-MP III and Its Derivatives against HSV-1.

Viruses are one of the leading causes of human disease, and many highly pathogenic viruses still have no specific treatment drugs. Therefore, producing new antiviral drugs is an urgent matter. In our study, we first found that the natural wasp venom peptide Protopolybia-MP III had a significant inhibitory effect on herpes simplex virus type 1 (HSV-1) replication in vitro by using quantitative real-time PCR (qPCR), Western blotting, and plaque-forming assays. Immunofluorescence analysis showed Protopolybia-MP III could enter cells, and it inhibited multiple stages of the HSV-1 life cycle, including the attachment, entry/fusion, and post-entry stages. Furthermore, ultracentrifugation and electron microscopy detected that Protopolybia-MP III significantly suppressed HSV-1 virion infectivity at different temperatures by destroying the integrity of the HSV-1 virion. Finally, by comparing the antiviral activity of Protopolybia-MP III and its mutants, a series of peptides with better anti-HSV-1 activity were identified. Overall, this work found the function and mechanism of the antiviral wasp venom peptide Protopolybia-MP III and its derivatives against HSV-1 and laid the foundation for the research and development of wasp venom-derived antiviral candidate peptide drugs.

Regulatory role of interfacial adhesion and mechanical microenvironments in microbe-host interactions

A recent study published in Nature Communications showed that essential modulatory roles of interfacial adhesion and mechanical microenvironments such as geometric constraints and extracellular matrix stiffness, in microbe-host cell interactions. This study utilized single-cell force spectroscopy and RNA sequencing to gain insight into the intrinsic mechanisms by which the mechanical microenvironment regulates bacterial-host interactions and therefore reveal potential interventions against bacterial invasion. Meanwhile, the adhesion forces involved in the bacterial–host interactions were recognized as a new indicator for assessing the extent of bacterial infection. Taken together, these findings demonstrate that interfacial adhesion forces and mechanical microenvironments play a dominant role in modulating functions and behaviors of microorganisms and host cells, which also provide a mechanobiology-inspired idea for the development of subsequent drug-resistant antimicrobials and broad-spectrum antiviral drugs.

Prokaryotic Expression and Affinity Purification of DDX3 Protein.

DDX3 is a protein with RNA helicase activity that is involved in a variety of biological processes, and it is an important protein target for the development of broad-spectrum antiviral drugs, multiple cancers and chronic inflammation. The objective of this study is to establish a simple and efficient method to express and purify DDX3 protein in E. coli, and the recombinant DDX3 should maintain helicase activity for further tailor-made screening and biochemical function validation. DDX3 cDNA was simultaneously cloned into pET28a-TEV and pNIC28-Bsa4 vectors and transfected into E. coli BL21 (DE3) to compare one suitable prokaryotic expression system. The 6×His-tag was fused to the C-terminus of DDX3 to form a His-tagging DDX3 fusion protein for subsequent purification. Protein dissolution buffer and purification washing conditions were optimized. The His-tagged DDX3 protein would bind with the Ni-NTA agarose by chelation and collected by affinity purification. The 6×His-tag fused with N-terminal DDX3 was eliminated from DDX3 by TEV digestion. A fine purification of DDX3 was performed by gel filtration chromatography. The recombinant plasmid pNIC28-DDX3, which contained a 6×His-tag and one TEV cleavage site at the N terminal of DDX3 sequence, was constructed for DDX3 prokaryotic expression and affinity purification based on considering the good solubility of the recombinant His-tagging DDX3, especially under 0.5 mM IPTG incubation at 18°C for 18 h to obtain more soluble DDX3 protein. Finally, the exogenous recombinant DDX3 protein was obtained with more than 95% purity by affinity purification on the Ni-NTA column and removal of miscellaneous through gel filtration chromatography. The finely-purified DDX3 still retained its ATPase activity. A prokaryotic expression pNIC28-DDX3 system is constructed for efficient expression and affinity purification of bioactive DDX3 protein in E. coli BL21(DE3), which provides an important high-throughput screening and validation of drugs targeting DDX3.