Sheng Cui

Enteroviruses comprise numerous pathogens causing severe diseases that threaten public health. Nevertheless, effective antivirals against enterovirus remain lacking. Previous studies have revealed that disruption of picornaviral 2C oligomerization by targeting the PBD-Pocket interface is a potential antiviral strategy. In this study, We established a lead screening cascade to develop inhibitors targeting the 2C Pocket and identified compounds from two scaffold classes, Comp.2 and Comp.8, both of which inhibit the ATPase activity of 2C in vitro. While both compounds showed anti-EV71 activity in RD cells with EC50 values of 4.9 and 9.5 μM respectively, only Comp.8 showed antiviral activity in newborn mice. Serial viral passage under drug selection pressure identified that Comp.8 can suppress emergence of a pro-oligomerization mutation S318F near the 2C Pocket. Structure-activity relationship optimization of Comp.8 yielded Comp.8-2 with improved antiviral activity and favored pharmacokinetic profiles both in vitro and in vivo. Together, these findings identify a druggable hot spot within the 2C Pocket and a potential candidate for further development as an EV71 antiviral.

Crimean-Congo hemorrhagic fever virus (CCHFV), designated by the WHO as a priority pathogen under its R&D Blueprint for emerging epidemics, poses a major global health threat, yet licensed vaccines or specific antiviral treatments are lacking. As the sole viral enzyme responsible for genome replication and transcription, the CCHFV L protein is a large, multienzymatic protein, but its exceptional size (> 450 kDa) and extensive domain architecture have hindered structural analysis. Here, we present high-resolution cryo-electron microscopy structures of the full-length CCHFV L protein in its apo state and bound to the 5' viral RNA promoter. These structures reveal the largest polymerase known among Bunyavirales and demonstrate that the apo form adopts a highly flexible conformation in which multiple functional elements remain disordered. Binding of the 5' promoter RNA triggers extensive conformational rearrangements that organize these elements into a catalytically competent active site. We define a conserved 5' hook-binding mode and identify two previously unrecognized residues (K1545 and E1637) that form a constriction at the NTP entry channel, representing newly defined regulatory motifs J and K conserved across Bunyavirales. We further characterize an expanded pendant domain unique to nairoviruses that, although not essential for promoter binding, likely modulates template movement within the internal tunnel during RNA synthesis. Our results provide the first structural framework for a nairovirus polymerase, illuminate the mechanisms of CCHFV RNA synthesis, and establish a foundation for structure-guided antiviral development against this high-priority pathogen.

Shiftless (SFL) is a broad-spectrum inhibitor of programmed −1 ribosomal frameshift (−1 PRF) and exhibits various antiviral activities. Here, we characterized human SFL structurally and biochemically. The 2.0-angstrom resolution crystal structure of SFL reveals a boat-like module comprising an N-terminal helical bundle and three zinc fingers at the C terminus. A hyperphosphorylation loop (HPL) buried between the helical bundle and the zinc finger 1 harbors four phosphorylated residues (p-S249, p-T250 p-T253, and p-S256), which are important to protein folding. SFL forms monomers in solution and binds the HIV-1 −1 PRF sequence with nanomolar affinity ( K _D = 5.7 nanomolar). Disruption of HPL phosphorylation decreased the RNA binding affinity and undermined the SFL-mediated −1 PRF inhibition of various viruses. Proximity-dependent biotinylation identified three cellular Ser/Thr kinases—EEF2K, NEK9, and PBK—that phosphorylate SFL in cells. These findings shed light on the mechanisms underlying −1 PRF regulation by SFL and provide insights into the role of SFL in virus inhibition.

The Schlafen (SLFN) genes are induced by interferons, underscoring their roles in the immune response and viral replication inhibition. SLFN14, a member of SLFN family, is associated with multiple human diseases; however, neither its functions nor its disease mechanisms are fully understood. Herein, human SLFN14 biochemically is characterized, demonstrating that it specifically cleaves RNAs containing short duplex regions, such as hairpin RNAs and tRNAs, but does not have ATPase or helicase activity. Cryogenic electron microscopy structures of SLFN14 apoenzyme (2.84 Å) and SLFN14‐hairpin RNA complex (2.88 Å) are determined, revealing that SLFN14 assembles into a ring‐like dimer and dimerization is mainly mediated by hydrophobic contacts. Two N‐terminal RNase domains of SLFN14 are organized head‐to‐tail, forming an RNA‐binding groove that can accommodate a 12‐bp hairpin RNA. The hairpin RNA is recognized mainly through phosphate backbone interactions. Further, SLFN14 is shown to inhibits HIV‐1 pseudovirus replication. The anti‐HIV‐1 activity of SLFN14 is via codon‐usage‐dependent translational inhibition and impairment of the programmed ‐1 ribosomal frameshifting, with an efficiency comparable to that of Shiftless. Using tRNA PCR arrays, SLFN14 and SLFN11 are found to decrease both nuclear‐encoded and mitochondrial tRNAs in cells. Together, these results provide novel insights into the function of SLFN14 and its role in HIV‐1 restriction.

The NADase activity of the defense-associated sirtuins (DSRs) is activated by the phage tail tube protein (TTP). Herein, we report cryo-EM structures of a free-state Bacillus subtilis DSR2 tetramer and a fragment of the tetramer, a phage SPR tail tube, and two DSR2-TTP complexes. DSR2 contains an N-terminal SIR2 domain, a middle domain (MID) and a C-terminal domain (CTD). The DSR2 CTD harbors the α-solenoid tandem-repeats like the HEAT-repeat proteins. DSR2 assembles into a tetramer with four SIR2 clustered at the center, and two intertwined MID-CTD chains flank the SIR2 core. SPR TTPs self-assemble into a tube-like complex. Upon DSR2 binding, the D1 domain of SPR TTP is captured between the HEAT-repeats domains of DSR2, which conflicts with TTPs self-assembly. Binding of TTPs induces conformational changes in DSR2 tetramer, resulting in increase of the NAD⁺ pocket volume in SIR2, thus activates the NADase activity and leads to cellular NAD⁺ depletion.

The COVID-19 pandemic has underscored the importance of in-depth research into the proteins encoded by coronaviruses (CoV), particularly the highly conserved nonstructural CoV proteins (nsp). Among these, the nsp13 helicase of severe pathogenic MERS-CoV, SARS-CoV-2, and SARS-CoV is one of the most preserved CoV nsp. Utilizing single-molecule FRET, we discovered that MERS-CoV nsp13 unwinds DNA in distinct steps of about 9 bp when ATP is employed. If a different nucleotide is introduced, these steps diminish to 3-4 bp. Dwell-time analysis revealed 3-4 concealed steps within each unwinding process, which suggests the hydrolysis of 3-4 dTTP. Combining our observations with previous studies, we propose an unwinding model of CoV nsp13 helicase. This model suggests that the elongated and adaptable 1B-stalk of nsp13 may enable the 1B remnants to engage with the unwound single-stranded DNA, even as the helicase core domain has advanced over 3-4 bp, thereby inducing accumulated strain on the nsp13-DNA complex. Our findings provide a foundational framework for determining the unwinding mechanism of this unique helicase family.

The pathogen of COVID-19, SARS-CoV-2, has caused a severe global health crisis. So far, while COVID-19 has been suppressed, the continuous evolution of SARS-CoV-2 variants has reduced the effectiveness of vaccines such as mRNA-1273 and drugs such as Remdesivir. To uphold the effectiveness of vaccines and drugs prior to potential coronavirus outbreaks, it is necessary to explore the underlying mechanisms between biomolecules and nanodrugs. The experimental study reported that acrylamide fragments covalently attached to Cys145, the main protease enzyme (Mpro) of SARS-CoV-2, and occupied the substrate binding pocket, thereby disrupting protease dimerization. However, the potential mechanism linking them is unclear. The purpose of this work is to complement and validate experimental results, as well as to facilitate the study of novel antiviral drugs. Based on our experimental studies, we identified two acrylamide fragments and constructed corresponding protein-ligand complex models. Subsequently, we performed molecular dynamics (MD) simulations to unveil the crucial interaction mechanisms between these nanodrugs and SARS-CoV-2 Mpro. This approach allowed the capture of various binding conformations of the fragments on both monomeric and dimeric Mpro, revealing significant conformational dissociation between the catalytic and helix domains, which indicates the presence of allosteric targets. Notably, Compound 5 destabilizes Mpro dimerization and acts as an effective inhibitor by specifically targeting the active site, resulting in enhanced inhibitory effects. Consequently, these fragments can modulate Mpro’s conformational equilibrium among extended monomeric, compact, and dimeric forms, shedding light on the potential of these small molecules as novel inhibitors against coronaviruses. Overall, this research contributes to a broader understanding of drug development and fragment-based approaches in antiviral covalent therapeutics.