Qi Yang

Lonafarnib, an oral antiviral that targets the fusion glycoprotein of respiratory syncytial virus (RSV), has demonstrated efficacy in vitro and in vivo . However, because the RSV has evolved to become resistant to other fusion inhibitors, there is a concern that the same could occur for lonafarnib. Here, we identified resistance to lonafarnib in the RSV A2 strain and a recent clinical isolate, RSV ON1, via in vitro selection at scale. Cell‒cell fusion and recombinant live RSV analysis confirmed that the mutations at K394, K399, and T400 of the cysteine-rich region of the fusion protein mediate high-level resistance. Lonafarnib resistance mutations also confer cross-resistance to other fusion inhibitors of clinical interest. All-atom molecular dynamics simulations revealed that these resistance mutations confer reduced stability to the fusion protein, thereby diminishing its binding affinity with lonafarnib. To address this vulnerability proactively and increase the barrier to resistance development, we designed the first potent proteolysis-targeting chimera (PROTAC) fusion protein degrader, compound 0179841, which uses lonafarnib and cereblon as ligands. This PROTAC effectively inhibited RSV replication. Collectively, our findings indicate that RSV develops resistance to lonafarnib in the cysteine-rich region of the fusion protein. This work sheds light on the mechanisms by which RSV evolves resistance to lonafarnib and provides a foundation for the rational design of antivirals aimed at preventing resistance. IMPORTANCE: Respiratory syncytial virus (RSV) infection poses a substantial public health challenge. Resistance to several potent fusion inhibitors, which are currently in various stages of clinical development, can readily emerge. Through a drug repurposing screen, we identified lonafarnib as an RSV fusion inhibitor; however, concerns exist regarding the potential development of resistance. Here, large-scale in vitro selection experiments revealed specific mutations within the highly conserved cysteine-rich region of the fusion (F) protein that confer high-level lonafarnib resistance across diverse RSV strains. These resistance mutations also confer cross-resistance to other clinical-stage fusion inhibitors. Mechanistic investigations demonstrated that these mutations reduce F protein stability, thereby diminishing the binding affinity of lonafarnib. As a proof of concept for an alternative antiviral strategy, we rationally designed the first potent proteolysis-targeting chimera (PROTAC) F protein degrader, compound 0179841, by utilizing lonafarnib and cereblon ligands. This novel antiviral agent effectively inhibits RSV infection by inducing degradation of the F protein. This work elucidates the molecular basis of RSV resistance to lonafarnib and establishes a strategy for developing next-generation antivirals aimed at preempting resistance.

While current antivirals primarily target viral proteins, host-directed strategies remain underexplored. Here, we performed a genome-wide CRISPRi screening to identify the host protein, Hepatocyte Growth Factor-Regulated Tyrosine Kinase Substrate (HGS), as essential for the pan-coronaviruses infection both in vitro and in vivo. Mechanistically, HGS directly interacts with the viral membrane (M) protein, facilitating its trafficking to the ER-Golgi intermediate compartment (ERGIC) for virion assembly. Conversely, HGS deficiency caused M retention in the ER, blocking assembly. Leveraging this interaction, we designed M-derived peptides and screened over 5,000 FDA-approved drugs, identifying riboflavin tetrabutyrate (RTB). Both the peptides and RTB bind HGS and disrupt its interaction with the M protein, leading to M retention in the ER and subsequent blockade of virion assembly. These agents demonstrated broad anti-pan-coronavirus activity in vitro and in vivo. Collectively, our findings establish HGS as a druggable host target and identify RTB as a promising broad-spectrum antiviral candidate.

The entry of respiratory syncytial virus (RSV) into host cells is a multifaceted process involving viral adsorption, interaction of viral glycoproteins with cellular receptors, and utilization of various invasion pathways. Despite these complexities, our understanding of potential common pathways facilitating RSV entry remains limited. In this study, we demonstrate that endocytosis via cholesterol-rich lipid rafts is a common mechanism utilized by various RSV genotypes in different cell types. Specifically, RSV strains A2, B18537, and the currently epidemic ON1 strains all employ this mechanism to gain entry into human cell lines, including HEp-2, A549, and primary human bronchial epithelial cells. Cellular receptors binding to viral fusion glycoprotein were recruited to cholesterol-rich lipid rafts, leading to actin rearrangement and endosome formation, which facilitated viral entry. Furthermore, reducing cholesterol levels using methyl-β-cyclodextrin, simvastatin, or terbinafine inhibited RSV infection. Notably, combining simvastatin with an RSV fusion protein inhibitor (AK0529) resulted in enhanced antiviral effects both in vitro and in vivo . These findings expand our understanding of viral-host interaction and provide a novel therapeutic strategy for treating RSV infection. IMPORTANCE: Respiratory syncytial virus (RSV) is an important human pathogen that causes severe bronchiolitis and pneumonia in infants and young children. RSV entry host cells involve generally different invasion pathways and are multistep processes. However, our understanding of the associated common pathways for viral entry remains limited. Our study uncovers a pivotal role for cholesterol-rich lipid rafts in facilitating RSV entry across diverse host cells, a finding that advances our understanding of viral-host interactions and paves the way for novel antiviral strategies. By meticulously examining various RSV genotypes, we revealed shared mechanisms underlying viral entry, highlighting the significance of cholesterol regulation and its impact on infection inhibition. Our findings also demonstrate enhanced antiviral efficacy through a combined approach targeting both viral entry and cholesterol metabolism.

Respiratory syncytial virus (RSV) exploits host proteases to enhance its replication efficiency; however, the precise mechanisms remain unclear. Through high-throughput screening, we identified four matrix metalloproteinase 9 (MMP-9) inhibitors (including JNJ0966 and doxycycline hyclate) that suppress RSV infection in vitro and in vivo. Mechanistic studies revealed a proteolytic cascade wherein MMP-9 cleaves transglutaminase 2 (TGM2) at the PVP375↓VR site, generating an N-terminal fragment (1–375) that activates its protein disulfide isomerase (PDI) activity. This TGM2-dependent PDI activity catalyzes disulfide bond rearrangement in the RSV fusion glycoprotein (F), enabling F protein maturation, a prerequisite for membrane fusion and syncytium formation—key processes driving late-stage viral propagation. Genetic ablation of MMP-9 significantly attenuated RSV infectivity, while pharmacological inhibition reduced pulmonary viral loads and mitigated lung pathology in infected mice. Our study defines a unified MMP-9→TGM2→F axis as the core mechanism driving RSV replication and validates MMP-9 as a therapeutic target.

HCoV-HKU1, one of seven human coronaviruses (HCoVs) that have harmful effects on human health, accounts for a substantial portion of common cold cases and can cause severe respiratory diseases in certain populations. Currently, effective antiviral treatments against this virus are limited. Recently, TMPRSS2, a host protease long acknowledged for its role in priming the spike proteins of various CoVs and promoting viral entry, was identified as a functional receptor for HCoV-HKU1, opening an avenue for anti-HCoV-HKU1 therapy development. In this study, we elucidate the detailed molecular mechanism underlying the interaction between the HCoV-HKU1 receptor-binding domain (RBD) and TMPRSS2 via crystallography. Guided by these structural insights, we successfully develop two types of therapeutic antibodies against HCoV-HKU1. The first type neutralizes the RBD, potently disrupting its interaction with TMPRSS2 and preventing viral infection. The second type targets TMPRSS2, inhibiting its enzymatic activity and/or interfering with its binding to the RBD. The latter demonstrates broad-spectrum anti-CoV activity, as the enzymatic activity of TMPRSS2 is crucial for both HCoV-HKU1 infection and other CoV infections. Our findings provide crucial structural insights into the recognition of TMPRSS2 by HCoV-HKU1 and offer promising antibody-based strategies for combating HCoV-HKU1 and other CoV infections.

Human coronaviruses (CoV) cause respiratory infections that range from mild to severe. CoVs are a large family of viruses with considerable genetic heterogeneity and a multitude of viral types, making preventing and treating these viruses difficult. Comprehensive treatments that inhibit CoV infections fulfill a pressing medical need and may be immensely valuable in managing emerging and endemic CoV infections. As the main protease (M^pro) is highly conserved across many CoVs, this protease has been identified as a route for broad CoV inhibition. We utilize the advanced generative chemistry platform Chemistry42 for de novo molecular design and obtained novel small-molecule, non-peptide-like inhibitors targeting the SARS-CoV-2 M^pro. ISM3312 is identified as an irreversible, covalent M^pro inhibitor from extensive virtual screening and structure-based optimization efforts. ISM3312 exhibits low off-target risk and outstanding antiviral activity against multiple human coronaviruses, including SARS-CoV-2, MERS-CoV, 229E, OC43, NL63, and HKU1 independent of P-glycoprotein (P-gp) inhibition. Furthermore, ISM3312 shows significant inhibitory effects against Nirmatrelvir-resistant M^pro mutants, suggesting ISM3312 may contribute to reduced viral escape in these settings. Incorporating ISM3312 and Nirmatrelvir into antiviral strategy could improve preparedness and reinforce defenses against future coronavirus threats.

Nipah virus (NiV), a highly lethal zoonotic pathogen causing encephalitis and respiratory diseases with mortality rates up to 40–70%, faces research limitations due to its strict biosafety level 4 (BSL-4) containment requirements, hindering antiviral development. To address this, we generated two NiV minigenome replicons (Fluc- and EGFP-based) expressed via helper plasmids encoding viral N, P, and L proteins, enabling replication studies under BSL-2 conditions. The minigenome replicon recapitulated the cytoplasmic inclusion body (IB) formation observed in live NiV infections. We further demonstrated that IB assembly is driven by liquid–liquid phase separation (LLPS), with biochemical analyses identifying the C-terminal N core domain of the N protein, as well as N₀ and XD domains and the intrinsically disordered region (IDR) of the P protein, as essential structural determinants for LLPS-mediated IB biogenesis. The targeted siRNA silencing of the 5′ and 3′ untranslated regions (UTRs) significantly reduced replicon-derived mRNA levels, validating the regulatory roles of these regions. Importantly, the minigenome replicon demonstrated sensitivity to type I/II/III interferons and antivirals (remdesivir, azvudine, molnupiravir), establishing its utility for drug screening. This study provides a safe and efficient platform for investigating NiV replication mechanisms and accelerating therapeutic development, circumventing the constraints of BSL-4 facilities while preserving key virological features.

Human bocavirus 1 (HBoV1; family: Parvoviridae) causes a wide spectrum of respiratory diseases in children and gastroenteritis in adults. A lack of sensitive cell lines and efficient animal models hinders research on HBoV, including the development of anti-HBoV drugs or vaccines. Although the construction of a wild-type HBoV1 infectious clone has been reported, generating HBoV1 infectious clone carrying foreign reporter genes with suitable insertion sites in its genome while retaining replicative ability remains challenging. Here, HBoV1 infectious clones harboring the 11-amino-acid HiBiT tag at five distinct insertion sites were constructed and evaluated. Only the recombinant HBoV1 carrying the HiBiT tag in the N-terminus of the NS1 protein (HBoV1-HiBiT_NS1) displayed comparable characteristics to wild-type HBoV1 as determined via the analysis of viral DNA copy number, NanoLuc activity, viral protein expression, and the formation of replication intermediates. Notably, the replication kinetics of HBoV1-HiBiT_NS1 could be examined by monitoring NanoLuc activity, which was noted to be correlated with the viral DNA level. Additionally, we successfully applied HiBiT-tagged HBoV1 for the evaluation of antiviral drug activity and identified ivermectin (EC50 = 2.27 μM) as a potent anti-HBoV1 replication drug. Overall, our study demonstrated that the HBoV1-HiBiT_NS1 reporter can serve as a convenient platform for screening candidate drugs targeting HBoV1 replication and may also be useful for investigating the life cycle of the virus.

Human bocavirus 1 (HBoV1) has appeared as an emerging pathogen, causing mild to life-threatening respiratory tract infections, acute otitis media, and encephalitis in young children and immunocompromised individuals. The lack of cell lines suitable for culturing replicative viruses hinders research on HBoV1. Here, we characterized the susceptibility to HBoV1 of 29 human and 7 animal cell lines, and identified a permissive cell line, MA104. The complete HBoV1 life cycle was achieved in MA104 cells, including viral entry, complete replication, and infectious progeny virion production. Additionally, the suppression of the interferon pathway facilitated the viral genome replication in MA104 cells. RNA-sequencing showed that innate immunity, inflammation, the PI3K-Akt and MAPK signaling pathways, and the cellular membrane system were mobilized in response to HBoV1 infection. Overall, our study is the first to identify a cell line, MA104, that supports the complete HBoV1 life cycle, which will promote research on HBoV1 virology and pathogenesis and benefit drug and vaccine development. IMPORTANCE: HBoV1 is an emerging pathogen that mainly causes respiratory tract infections, while the lack of cell lines suitable for culture replicative viruses hindered research on HBoV1. Here, we identify a permissive cell line for HBoV1 infection, MA104, and reveal that the complete life cycle of HBoV1 was supported in MA104 cells. Our findings provide a suitable cell model for the study of HBoV1 and explore its application for antiviral drug evaluation, which is vital for research on HBoV1 virology and pathogenesis, as well as for drug and vaccine development.