Nonstructural Protein 2C Research Articles

Stearoyl coenzyme A desaturase 1 (SCD1) regulates foot-and-mouth disease virus replication by modulating host cell lipid metabolism and viral protein 2C-mediated replication complex formation.

Many positive-stranded RNA viruses, including foot-and-mouth disease virus (FMDV), alter host membranes and lipid metabolism to create a suitable microenvironment for their survival and replication within host cells. In FMDV-infected cells, the endoplasmic reticulum membrane is remodeled, forming vesicular structures that rely heavily on increased free fatty acids, thereby linking lipid metabolism to the FMDV replication complex. Nonstructural FMDV protein 2C is crucial for this complex, while host cell enzyme stearoyl coenzyme A desaturase 1 (SCD1) is vital for lipid metabolism. We found that FMDV infection alters SCD1 expression in host cells. Inhibiting SCD1 expression or its enzymatic activity markedly decreases FMDV replication, while supplementing oleic acid, a catalytic product of SCD1, regulates FMDV replication. Additionally, SCD1 forms part of the FMDV replication complex and helps recruit 2C to a detergent-resistant membrane. Our study provides insights into the pathogenesis of FMDV and a potential novel drug target against the virus.

Chemoproteomics enables identification of coatomer subunit zeta-1 targeted by a small molecule for enterovirus A71 inhibition.

Human enterovirus A71 (EV-A71) is a significant etiological agent responsible for epidemics of hand, foot, and mouth disease (HFMD) in Asia-Pacific regions. There are presently no licensed antivirals against EV-A71, and the druggable target for EV-A71 remains very limited. The phenotypic hit 10,10'-bis(trifluoromethyl) marinopyrrole A derivative, herein termed MPA-CF3, is a novel potent small-molecule inhibitor against EV-A71, but its pharmacological target(s) and antiviral mechanisms are not defined. Here, quantitative chemoproteomics deciphered the antiviral target of MAP-CF3 as host factor coatomer subunit zeta-1 (COPZ1). Mechanistically, MPA-CF3 disrupts the interaction of COPZ1 with the EV-A71 nonstructural protein 2C by destabilizing COPZ1 upon binding. The destruction of this interaction blocks the coatomer-mediated transport of 2C to endoplasmic reticulum, and ultimately inhibits EV-A71 replication. Taken together, our study disclosed that MPA-CF3 can be a structurally novel host-targeting anti-EV-A71 agent, providing a structural basis for developing the COPZ1-targeting broad-spectrum antivirals against enteroviruses. The mechanistic elucidation of MPA-CF3 against EV-A71 may offer an alternative COPZ1-involved therapeutic pathway for enterovirus infection.

Leucoverdazyls as Novel Potent Inhibitors of Enterovirus Replication.

Enteroviruses (EV) are important pathogens causing human disease with various clinical manifestations. To date, treatment of enteroviral infections is mainly supportive since no vaccination or antiviral drugs are approved for their prevention or treatment. Here, we describe the antiviral properties and mechanisms of action of leucoverdazyls-novel heterocyclic compounds with antioxidant potential. The lead compound, 1a, demonstrated low cytotoxicity along with high antioxidant and virus-inhibiting activity. A viral strain resistant to 1a was selected, and the development of resistance was shown to be accompanied by mutation of virus-specific non-structural protein 2C. This resistant virus had lower fitness when grown in cell culture. Taken together, our results demonstrate high antiviral potential of leucoverdazyls as novel inhibitors of enterovirus replication and support previous evidence of an important role of 2C proteins in EV replication.

3Cpro of FMDV inhibits type II interferon-stimulated JAK-STAT signaling pathway by blocking STAT1 nuclear translocation

Foot-and-mouth disease virus (FMDV) has developed various strategies to antagonize the host innate immunity. FMDV Lpro and 3Cpro interfere with type I IFNs through different mechanisms. The structural protein VP3 of FMDV degrades Janus kinase 1 to suppress IFN-γ signaling transduction. Whether non-structural proteins of FMDV are involved in restraining type II IFN signaling pathways is unknown. In this study, it was shown that FMDV replication was resistant to IFN-γ treatment after the infection was established and FMDV inhibited type II IFN induced expression of IFN-γ-stimulated genes (ISGs). We also showed for the first time that FMDV non-structural protein 3C antagonized IFN-γ-stimulated JAK-STAT signaling pathway by blocking STAT1 nuclear translocation. 3Cpro expression significantly reduced the ISGs transcript levels and palindromic gamma-activated sequences (GAS) promoter activity, without affecting the protein level, tyrosine phosphorylation, and homodimerization of STAT1. Finally, we provided evidence that 3C protease activity played an essential role in degrading KPNA1 and thus inhibited ISGs mRNA and GAS promoter activities. Our results reveal a novel mechanism by which an FMDV non-structural protein antagonizes host type II IFN signaling.

Development of Enterovirus Antiviral Agents That Target the Viral 2C Protein.

The Enterovirus (EV) genus includes several important human and animal pathogens. EV-A71, EV-D68, poliovirus (PV), and coxsackievirus (CV) outbreaks have affected millions worldwide, causing a range of upper respiratory, skin, and neuromuscular diseases, including acute flaccid myelitis, and hand-foot-and-mouth disease. There are no FDA-approved antiviral therapeutics for these enteroviruses. This study describes novel antiviral compounds targeting the conserved non-structural viral protein 2C with low micromolar to nanomolar IC50 values. The selection of resistant mutants resulted in amino acid substitutions in the viral capsid protein, implying these compounds may play a role in inhibiting the interaction of 2C and the capsid protein. The assembly and encapsidation stages of the viral life cycle still need to be fully understood, and the inhibitors reported here could be useful probes in understanding these processes.

Whole-genome sequencing of foot-and-mouth disease virus serotype O/PanAsia-2/QOM-15 and comparison of its VP1-encoding region with two vaccine strains.

Despite widespread vaccination against foot-and-mouth disease, many outbreaks still occur in endemic areas. We attempted to determine the genetic and antigenic properties of the O/PanAsia-2/QOM-15 foot-and-mouth disease virus new vaccine strain. Thus, whole-genome sequencing was used to identify vulnerable pinpoint sites across the genome. The VP1 sequence (1D gene) of the O/PanAsia-2/QOM-15 viral genome was then compared to the VP1 sequences of two previously used vaccine strains, O/PanAsia (JQ321837) and O/PanAsia-2 (JN676146). The antigenic relationship of these three viruses was calculated by the two dimensional-virus neutralization test. At the nucleotide level, 47 single variants were identified, of which 19.00% were in the 5' untranslated region (UTR), 79.00% in the polyprotein region, and 2.00% in the 3' UTR region. Approximately half of the single nucleotide polymorphisms that have occurred in 1D gene resulted in amino acid (AA) substitutions in the VP1 structure. The single nucleotide polymorphisms also caused AA substitutions in other structural proteins, including VP2 and VP3, and some non-structural proteins (Lpro, 2C, and 3A). The O/PanAsia-2/QOM-15 shared higher sequence similarity with O/PanAsia-2 (91.00%) compared to O/PanAsia (87.30%). Evaluating r-value showed that the antigenic relationship of O/PanAsia-2/QOM-15 with O/PanAsia-2 (29.00%) was greater than that of the O/PanAsia (24.00%); however, all three viruses were immunologically distinct. After 10 years, the alteration of virus antigenicity and the lack of detectable adaptive pressure on VP1 sequence suggest that studying genetic dynamics beyond the VP1 region is necessary to evaluate FMDV pathogenicity and vaccine failure.

Heat Shock Protein 60 Is Involved in Viral Replication Complex Formation and Facilitates Foot and Mouth Virus Replication by Stabilizing Viral Nonstructural Proteins 3A and 2C.

ABSTRACTThe maintenance of viral protein homeostasis depends on the machinery of the infected host cells, giving us an insight into the interplay between host and virus. Accumulating evidence suggests that heat shock protein 60 (HSP60), as one molecular chaperone, is involved in regulating virus infection. However, the role of HSP60 during foot-and-mouth disease virus (FMDV) replication and its specific mechanisms have not been reported. We demonstrate that HSP60 modulates the FMDV life cycle. HSP60 plays a role at the postentry stage of the viral life cycle, including RNA replication and mRNA translation; however, HSP60 does not affect viral replication of Seneca Valley virus (SVA) or encephalomyocarditis virus (EMCV). We found that HSP60 is involved in FMDV replication complex (RC) formation. Furthermore, our results indicate that HSP60 interacts with FMDV nonstructural proteins 3A and 2C, key elements of the viral replication complex. We also show that HSP60 regulates the stability of 3A and 2C via caspase-dependent and autophagy-lysosome-dependent degradation, thereby promoting FMDV RNA synthesis and mRNA translation mediated by the RC. Additionally, we determined that the apical domain of HSP60 is responsible for interacting with 3A and 2C. The N terminus of 3A and ATPase domain of 2C are involved in binding to HSP60. Importantly, HSP60 depletion potently reduced FMDV pathogenicity in infected mice. Altogether, this study demonstrates a specific role of HSP60 in promoting FMDV replication. Furthermore, targeting host HSP60 will help us design the FMDV-specific antiviral drugs.

Biochemical and structural characterization of hepatitis A virus 2C reveals an unusual ribonuclease activity on single-stranded RNA.

The HAV nonstructural protein 2C is essential for virus replication; however, its precise function remains elusive. Although HAV 2C shares 24–27% sequence identity with other 2Cs, key motifs are conserved. Here, we demonstrate that HAV 2C is an ATPase but lacking helicase activity. We identified an ATPase-independent nuclease activity of HAV 2C with a preference for polyuridylic single-stranded RNAs. We determined the crystal structure of an HAV 2C fragment to 2.2 Å resolution, containing an ATPase domain, a region equivalent to enterovirus 2C zinc-finger (ZFER) and a C-terminal amphipathic helix (PBD). The PBD of HAV 2C occupies a hydrophobic pocket (Pocket) in the adjacent 2C, and we show the PBD–Pocket interaction is vital for 2C functions. We identified acidic residues that are essential for the ribonuclease activity and demonstrated mutations at these sites abrogate virus replication. We built a hexameric-ring model of HAV 2C, revealing the ribonuclease-essential residues clustering around the central pore of the ring, whereas the ATPase active sites line up at the gaps between adjacent 2Cs. Finally, we show the ribonuclease activity is shared by other picornavirus 2Cs. Our findings identified a previously unfound activity of picornavirus 2C, providing novel insights into the mechanisms of virus replication.

An anti-picornaviral strategy based on the crystal structure of foot-and-mouth disease virus 2C protein.

The foot-and-mouth disease virus (FMDV) 2C protein shares conserved motifs with enterovirus 2Cs despite low sequence identity. Here, we determine the crystal structure of an FMDV 2C fragment to 1.83Å resolution, which comprises an ATPase domain, a region equivalent to the enterovirus 2C zinc-finger (ZFER), and a C-terminal domain harboring a loop (PBL) that occupies a hydrophobic cleft (Pocket) in an adjacent 2C molecule. Mutations at ZFER, PBL, and Pocket affect FMDV 2C ATPase activity and are lethal to FMDV infectious clones. Because the PBL-Pocket interaction between FMDV 2C molecules is essential for its functions, we design an anti-FMDV peptide derived from PBL (PBL-peptide). PBL-peptide inhibits FMDV 2C ATPase activity, binds FMDV 2C with nanomolar affinity, and disrupts FMDV 2C oligomerization. FMDV 2C targets lipid droplets (LDs) and induces LD clustering in cells, and PBL-peptide disrupts FMDV 2C-induced LD clustering. Finally, we demonstrate that PBL-peptide exhibits anti-FMDV activity in cells.

The nonstructural protein 2C of Coxsackie B virus has RNA helicase and chaperoning activities.

RNA-remodeling proteins, including RNA helicases and chaperones, play vital roles in the remodeling of structured RNAs. During viral replication, viruses require RNA-remodeling proteins to facilitate proper folding and/or re-folding the viral RNA elements. Coxsackieviruses B3 (CVB3) and Coxsackieviruses B5 (CVB5), belonging to the genus Enterovirus in the family Picornaviridae, have been reported to cause various infectious diseases such as hand-foot-and-mouth disease, aseptic meningitis, and viral myocarditis. However, little is known about whether CVB3 and CVB5 encode any RNA remodeling proteins. In this study, we showed that 2C proteins of CVB3 and CVB5 contained the conserved SF3 helicase A, B, and C motifs, and functioned not only as RNA helicase that unwound RNA helix bidirectionally in an NTP-dependent manner, but also as RNA chaperone that remodeled structured RNAs and facilitated RNA strand annealing independently of NTP. In addition, we determined that the NTPase activity and RNA helicase activity of 2C proteins of CVB3 and CVB5 were dependent on the presence of divalent metallic ions. Our findings demonstrate that 2C proteins of CVBs possess RNA-remodeling activity and underline the functional importance of 2C protein in the life cycle of CVBs.

DDX21, a Host Restriction Factor of FMDV IRES-Dependent Translation and Replication.

In cells, the contributions of DEAD-box helicases (DDXs), without which cellular life is impossible, are of utmost importance. The extremely diverse roles of the nucleolar helicase DDX21, ranging from fundamental cellular processes such as cell growth, ribosome biogenesis, protein translation, protein–protein interaction, mediating and sensing transcription, and gene regulation to viral manipulation, drew our attention. We designed this project to study virus–host interactions and viral pathogenesis. A pulldown assay was used to investigate the association between foot-and-mouth disease virus (FMDV) and DDX21. Further insight into the DDX21–FMDV interaction was obtained through dual-luciferase, knockdown, overexpression, qPCR, and confocal microscopy assays. Our results highlight the antagonistic feature of DDX21 against FMDV, as it progressively inhibited FMDV internal ribosome entry site (IRES) -dependent translation through association with FMDV IRES domains 2, 3, and 4. To subvert this host helicase antagonism, FMDV degraded DDX21 through its non-structural proteins 2B, 2C, and 3C protease (3Cpro). Our results suggest that DDX21 is degraded during 2B and 2C overexpression and FMDV infection through the caspase pathway; however, DDX21 is degraded through the lysosomal pathway during 3Cpro overexpression. Further investigation showed that DDX21 enhanced interferon-beta and interleukin-8 production to restrict viral replication. Together, our results demonstrate that DDX21 is a novel FMDV IRES trans-acting factor, which negatively regulates FMDV IRES-dependent translation and replication.

Antiviral Peptides Targeting the Helicase Activity of Enterovirus Nonstructural Protein 2C.

Enteroviruses belong to the genus Enterovirus of the family Picornaviridae and include four human enterovirus groups (EV-A to -D): the epidemic of enteroviruses such as human enterovirus A71 (EV-A71) and coxsackievirus A16 (CVA16) is a threat to global public health. Enteroviral protein 2C is the most conserved nonstructural protein among all enteroviruses and possesses RNA helicase activity that plays pivotal roles during enteroviral life cycles, which makes 2C an attractive target for developing antienterovirus drugs. In this study, we designed a peptide, named 2CL, based on the structure of EV-A71 2C. This peptide effectively impaired the oligomerization of EV-A71 2C protein and inhibited the RNA helicase activities of 2C proteins encoded by EV-A71 and CVA16, both of which belong to EV-A, and showed potent antiviral efficacy against EV-A71 and CVA16 in cells. Moreover, the 2CL treatment elicited a strong in vivo protective efficacy against lethal EV-A71 challenge. In addition, the antiviral strategy of targeting the 2C helicase activity can be applied to inhibit the replication of EV-B. Either 2CL or B-2CL, the peptide redesigned based on the 2CL-corresponding sequence of EV-Bs, could exert effective antiviral activity against two important EV-Bs, coxsackievirus B3 and echovirus 11. Together, our findings demonstrated that targeting the helicase activity of 2C with a rationally designed peptide is an efficient antiviral strategy against enteroviruses, and 2CL and B-2CL show promising clinical potential to be further developed as broad-spectrum antienterovirus drugs.IMPORTANCE Enteroviruses are a large group of positive-sense single-stranded RNA viruses and include numerous human pathogens, such as enterovirus A71 (EV-A71), coxsackieviruses, and echoviruses. However, no approved EV antiviral drugs are available. Enteroviral 2C is the most conserved nonstructural protein among all enteroviruses and contains the RNA helicase activity critical for the viral life cycle. Herein, according to the structure of EV-A71 2C, we designed a peptide that effectively inhibited the RNA helicase activities of EV-A71- and coxsackievirus A16 (CVA16)-encoded 2C proteins. Moreover, this peptide exerted potent antiviral effects against EV-A71 and CVA16 in cells and elicited therapeutic efficacy against lethal EV-A71 challenge in vivo Furthermore, we demonstrate that the strategy of targeting the 2C helicase activity can be used for other relevant enteroviruses, including coxsackievirus B3 and echovirus 11. In summary, our findings provide compelling evidence that the designed peptides targeting the helicase activity of 2C could be broad-spectrum antivirals for enteroviruses.

Identification of novel interactions between host and non-structural protein 2C of foot-and-mouth disease virus.

The 2C protein of foot-and-mouth disease virus (FMDV) is reported to play a critical role in the virus replication complex and modulating the host's immune response. However, the underlying molecular intricacies of subversion of cellular machinery remains poorly understood, thus emphasizing the need to study 2C-host interactions. In this study, we identified the host proteins interacting with the 2C using yeast-two hybrid (Y2H) approach, which is one of the most recognized, high-throughput tools to study protein-protein interactions. The FMDV-2C bait was characterized for auto-activation, toxicity, and expression and was found to be suitable for mating with cDNA library. On preliminary screening a total of 32 interacting host proteins were identified which were reduced to 22 on subsequent confirmation with alternative yeast based assays. Amongst these, NMI/2C interaction has been reported earlier by Wang et al. (2012) and remaining 21 are novel interactions. The Reactome analysis has revealed the role of the identified host proteins in cellular pathways exploited by 2C during FMDV replication. We also confirmed interaction of MARCH7, an E3 ubiquitin ligase with 2C using mammalian two-hybrid system and co-immunoprecipitation. This study leads to the identification of novel 2C interacting host proteins which enhance our understanding of 2C-host interface and may provide checkpoints for development of potential therapeutics against FMDV.

1H,13C and 15N chemical shift assignments of the SUD domains of SARS-CoV-2 non-structural protein 3c: "The SUD-M and SUD-C domains".

SARS-CoV-2 RNA, nsP3c (non-structural Protein3c) spans the sequence of the so-called SARS Unique Domains (SUDs), first observed in SARS-CoV. Although the function of this viral protein is not fully elucidated, it is believed that it is crucial for the formation of the replication/transcription viral complex (RTC) and of the interaction of various viral “components” with the host cell; thus, it is essential for the entire viral life cycle. The first two SUDs, the so-called SUD-N (the N-terminal domain) and SUD-M (domain following SUD-N) domains, exhibit topological and conformational features that resemble the nsP3b macro (or “X”) domain. Indeed, they are all folded in a three-layer α/β/α sandwich structure, as revealed through crystallographic structural investigation of SARS-CoV SUDs, and they have been attributed to different substrate selectivity as they selectively bind to oligonucleotides. On the other hand, the C-terminal SUD (SUD-C) exhibit much lower sequence similarities compared to the SUD-N & SUD-M, as reported in previous crystallographic and NMR studies of SARS-CoV. In the absence of the 3D structures of SARS-CoV-2, we report herein the almost complete NMR backbone and side-chain resonance assignment (1H,13C,15N) of SARS-CoV-2 SUD-M and SUD-C proteins, and the NMR chemical shift-based prediction of their secondary structure elements. These NMR data will set the base for further understanding at the atomic-level conformational dynamics of these proteins and will allow the effective screening of a large number of small molecules as binders with potential biological impact on their function.

The Structure, Function, and Mechanisms of Action of Enterovirus Non-structural Protein 2C

Enteroviruses are a group of RNA viruses belonging to the family Picornaviridae. They include human enterovirus groups A, B, C, and D as well as non-human enteroviruses. Enterovirus infections can lead to hand, foot, and mouth disease and herpangina, whose clinical manifestations are often mild, although some strains can result in severe neurological complications such as encephalitis, myocarditis, meningitis, and poliomyelitis. To date, research on enterovirus non-structural proteins has mainly focused on the 2A and 3C proteases and 3D polymerase. However, another non-structural protein, 2C, is the most highly conserved protein, and plays a vital role in the enterovirus life cycle. There are relatively few studies on this protein. Previous studies have demonstrated that enterovirus 2C is involved in virus uncoating, host cell membrane rearrangements, RNA replication, encapsidation, morphogenesis, ATPase, helicase, and chaperoning activities. Despite ongoing research, little is known about the pathogenesis of enterovirus 2C proteins in viral replication or in the host innate immune system. In this review, we discuss and summarize the current understanding of the structure, function, and mechanism of the enterovirus 2C proteins, focusing on the key mutations and motifs involved in viral infection, replication, and immune regulation. We also focus on recent progress in research into the role of 2C proteins in regulating the pattern recognition receptors and type I interferon signaling pathway to facilitate viral replication. Given these functions and mechanisms, the potential application of the 2C proteins as a target for anti-viral drug development is also discussed. Future studies will focus on the determination of more crystal structures of enterovirus 2C proteins, which might provide more potential targets for anti-viral drug development against enterovirus infections.

Enterovirus 2C Protein Suppresses IKKα Phosphorylation by Recruiting IKKβ and IKKα into Viral Inclusion Bodies.

The nuclear factor-kappa B (NF-κB) signaling network constitutes a first line of defense against the invading viruses. However, viruses also adopted multiple strategies to interfere with NF-κB activation. Enterovirus 71 (EV71), in the family Picornaviridae, has become the main pathogen responsible for hand, foot, and mouth disease. Recent studies have reported that the nonstructural protein 2C of EV71 inhibits TNF-α induced NF-κB activation by suppressing IKKβ phosphorylation. In our study, we found that 2C can form inclusion bodies (IBs) in infected and transfected cells. Furthermore, 2C was able to sequester IKKβ into IBs through direct interaction with IKKβ. Although 2C did not directly interact with IKKα, viral protein 2C was able to sequester the IKKα into the IBs mediated by IKKβ. Our in vitro data further demonstrated that EV71 2C could suppress IKKα phosphorylation. These all together support a novel mechanism for EV71 to escape from NF-κB response, in which the phosphorylation of IKKα was suppressed by being recruited into viral IBs in the presence of 2C and IKKβ.

Rational design of highly potent broad-spectrum enterovirus inhibitors targeting the nonstructural protein 2C.

There is a great need for antiviral drugs to treat enterovirus (EV) and rhinovirus (RV) infections, which can be severe and occasionally life-threatening. The conserved nonstructural protein 2C, which is an AAA+ ATPase, is a promising target for drug development. Here, we present a structure-activity relationship study of a previously identified compound that targets the 2C protein of EV-A71 and several EV-B species members, but not poliovirus (PV) (EV-C species). This compound is structurally related to the Food and Drug Administration (FDA)-approved drug fluoxetine-which also targets 2C-but has favorable chemical properties. We identified several compounds with increased antiviral potency and broadened activity. Four compounds showed broad-spectrum EV and RV activity and inhibited contemporary strains of emerging EVs of public health concern, including EV-A71, coxsackievirus (CV)-A24v, and EV-D68. Importantly, unlike (S)-fluoxetine, these compounds are no longer neuroactive. By raising resistant EV-A71, CV-B3, and EV-D68 variants against one of these inhibitors, we identified novel 2C resistance mutations. Reverse engineering of these mutations revealed a conserved mechanism of resistance development. Resistant viruses first acquired a mutation in, or adjacent to, the α2 helix of 2C. This mutation disrupted compound binding and provided drug resistance, but this was at the cost of viral fitness. Additional mutations at distantly localized 2C residues were then acquired to increase resistance and/or to compensate for the loss of fitness. Using computational methods to identify solvent accessible tunnels near the α2 helix in the EV-A71 and PV 2C crystal structures, a conserved binding pocket of the inhibitors is proposed.

Virucidal activity and the antiviral mechanism of acidic polysaccharides against Enterovirus 71 infection in vitro.

Enterovirus 71 (EV71) is the predominant pathogen for severe hand, foot, and mouth disease (HFMD) in children younger than 5 years, and currently no effective drugs are available for EV71. Thus, there is an urgent need to develop new drugs for the control of EV71 infection. In this study, LJ04 was extracted from Laminaria japonica using diethylaminoethyl cellulose-52 with 0.4 mol/l NaCl as the eluent, and its virucidal activity was evaluated based on its cytopathic effects on a microplate. LJ04 is composed of fucose, galactose, and mannose and mainly showed good virucidal activity against EV71. The antiviral mechanisms of LJ04 were the direct inactivation of the virus, the blockage of virus binding, disruptions to viral entry, and weak inhibitory activity against the nonstructural protein 3C. The two most important findings from this study were that LJ04 inhibited EV71 proliferation in HM1900 cells, which are a human microglia cell line, and that LJ04 can directly inactivate EV71 within 2 hr at 37°C. This study demonstrates for the first time the ability of a polysaccharide from L. japonica to inhibit viral and 3C activity; importantly, the inhibition of 3C might have a minor effect on the antiviral effect of LJ04. Consequently, our results identify LJ04 as a potential drug candidate for the control of severe EV71 infection in clinical settings.

Fluoxetine Inhibits Enterovirus Replication by Targeting the Viral 2C Protein in a Stereospecific Manner.

Enteroviruses (family Picornaviridae) comprise a large group of human pathogens against which no licensed antiviral therapy exists. Drug-repurposing screens uncovered the FDA-approved drug fluoxetine as a replication inhibitor of enterovirus B and D species. Fluoxetine likely targets the nonstructural viral protein 2C, but detailed mode-of-action studies are missing because structural information on 2C of fluoxetine-sensitive enteroviruses is lacking. We here show that broad-spectrum anti-enteroviral activity of fluoxetine is stereospecific concomitant with binding to recombinant 2C. (S)-Fluoxetine inhibits with a 5-fold lower 50% effective concentration (EC50) than racemic fluoxetine. Using a homology model of 2C of the fluoxetine-sensitive enterovirus coxsackievirus B3 (CVB3) based upon a recently elucidated structure of a fluoxetine-insensitive enterovirus, we predicted stable binding of (S)-fluoxetine. Structure-guided mutations disrupted binding and rendered coxsackievirus B3 (CVB3) resistant to fluoxetine. The study provides new insights into the anti-enteroviral mode-of-action of fluoxetine. Importantly, using only (S)-fluoxetine would allow for lower dosing in patients, thereby likely reducing side effects.

Phylogenetic and recombination analyses of two deformed wing virus strains from different honeybee species in China.

BackgroundDeformed wing virus (DWV) is one of many viruses that infect honeybees and has been extensively studied because of its close association with honeybee colony collapse that is induced by Varroa destructor. However, virus genotypes, sequence characteristics, and genetic variations of DWV remain unknown in China.MethodsTwo DWV strains were isolated from Jinzhou and Qinhuangdao cities in China, and were named China1-2017 (accession number: MF770715) and China2-2018 (accession number: MH165180), respectively, and their complete genome sequences were analyzed. To investigate the phylogenetic relationships of the DWV isolates, a phylogenetic tree of the complete open reading frame (ORF), structural protein VP1, and non-structural protein 3C+RdRp of the DWV sequences was constructed using the MEGA 5.0 software program. Then, the similarity and recombinant events of the DWV isolated strains were analyzed using recombination detection program (RDP4) software and genetic algorithm for recombination detection (GARD).ResultsThe complete genomic analysis showed that the genomes of the China1-2017 and China2-2018 DWV strains consisted of 10,141 base pairs (bp) and 10,105 bp, respectively, and contained a single, large ORF (China1-2017: 1,146–9,827 bp; China2-2018: 1,351–9,816 bp) that encoded 2,894 amino acids. The sequences were compared with 20 previously reported DWV sequences from different countries and with sequences of two closely related viruses, Kakugo virus (KV) and V. destructor virus-1. Multiple sequence comparisons revealed a nucleotide identity of 84.3–96.7%, and identity of 94.7–98.6% in amino acids between the two isolate strains and 20 reference strains. The two novel isolates showed 96.7% nucleotide identity and 98.1% amino acid identity. The phylogenetic analyses showed that the two isolates belonged to DWV Type A and were closely related to the KV-2001 strain from Japan. Based on the RDP4 and GARD analyses, the recombination of the China2-2018 strain was located at the 4,266–7,507 nt region, with Korea I-2012 as an infer unknown parent and China-2017 as a minor parent, which spanned the entire helicase ORF. To the best of our knowledge, this is the first study to the complete sequence of DWV isolated from Apis cerana and the possible DWV recombination events in China. Our findings are important for further research of the phylogenetic relationship of DWVs in China with DWV strains from other countries and also contribute to the understanding of virological properties of these complex DWV recombinants.