A reliable experimental system is essential for advancing coronavirus biology and expediting countermeasure development. In this study, we report the construction of an infectious clone of human coronavirus OC43 (HCoV-OC43) using an in vitro ligation strategy. The clone-derived virus faithfully recapitulates the replication characteristics of the parental isolate in vitro. Leveraging this platform, we have developed a dual-reporter virus, OC43-mNG-Nluc, by replacing the viral accessory gene ns2 with a gene cassette encoding both nanoluciferase and fluorescent protein mNeonGreen. This construct enables dual-mode detection via chemiluminescence and fluorescence within a single assay. OC43-mNG-Nluc maintains replication kinetics identical to wild-type HCoV-OC43 in standard immortalized cell lines but exhibits marked attenuation in primary human airway epithelial cultures, underscoring the critical role of ns2 in viral replication within physiologically relevant systems. The dual-reporter virus also demonstrates excellent genetic and phenotypic stability following fifteen passages in vitro. Furthermore, we validate the proof-of-concept utility of OC43-mNG-Nluc in high-throughput antiviral screening, showcasing its advantages in streamlining and facilitating hit identification, triaging, and prioritization. Collectively, this study provides valuable insights into HCoV-OC43 biology and introduces a versatile and robust tool for coronavirus research and therapeutic development.

Poxviruses remain a significant global health concern, necessitating the development of novel antiviral strategies. Through high-throughput screening, we previously identified ciclopirox (CPX), an FDA-approved antifungal, as a hit that inhibits vaccinia virus (VACV) replication. Here, we further characterized its antiviral activity and mechanism of action using human primary fibroblasts. CPX significantly reduced VACV titers without reducing host cell viability, with an EC in the sub-micromolar range and a CC > 500 μM. Rescue experiments demonstrated that CPX inhibits viral replication primarily through chelation of intracellular Fe and, to a lesser extent, Fe, as evidenced by partial restoration of viral replication with ferric ammonium citrate supplementation. Furthermore, overexpression of the iron-dependent enzymes RRM2 and the VACV-encoded F4L reduced the inhibitory effect of CPX, indicating that these host and viral proteins are affected by CPX treatment. Moreover, CPX treatment suppressed cowpox virus and monkeypox (mpox) virus replication in vitro. It also reduced VACV titers in ex vivo mouse lung tissue. These findings highlight host iron metabolism as a critical determinant of poxvirus replication and identify CPX as a promising antiviral candidate against multiple orthopoxviruses.

Human respiratory syncytial virus (RSV) remains the leading viral cause of severe lower respiratory tract disease in infants and young children worldwide. Despite decades of research, RSV vaccine development remains hindered by the lack of animal models that accurately recapitulate pediatric susceptibility and allow rigorous assessment of protective efficacy and safety. Recent studies have identified the human insulin-like growth factor 1 receptor (hIGF1R) as a critical host receptor that facilitates RSV fusion and entry. Using a novel conditional knock-in mouse model, lung-specific hIGF1R expression achieved through Ad5 vector transduction enables rapid generation of a pediatric-relevant mouse model, offering a promising route to close this gap. Following intranasal challenge with RSV A2 (1.62 × 10 TCID), four-week-old Ad5-hIGF1R mice exhibited higher pulmonary viral loads, marked peribronchiolar and perivascular inflammation, interstitial thickening and a tendency toward alveolar wall coalescence compared with wild-type controls, thereby recapitulating severe pediatric RSV disease. Transcriptomic analysis revealed 12 chemokine genes, upregulated in RSV-infected lungs of Ad5-hIGF1R mice relative to mock controls, that are involved in immune-inflammatory pathways and may serve as practical biomarkers for detecting dysregulated host responses during vaccine or antiviral-drug assessment. Furthermore, A mid-dose prefusion F (pre-F) vaccination regimen significantly reduced viral loads and moderately attenuated neutrophil infiltration in the lungs of Ad5-hIGF1R mice. In summary, the young Ad5-hIGF1R mice demonstrate that hIGF1R expression enhances RSV replication and immunopathology in vivo. This model not only overcomes the limited RSV susceptibility of conventional young mice but also provides a platform for evaluating RSV vaccines.

Emerging viruses pose an ongoing threat to human health. While certain viral families are common sources of outbreaks, predicting the specific virus within a family that will cause the next outbreak or pandemic is not possible, creating an urgent need for broad spectrum antiviral drugs that are effective against a wide array of related viral pathogens. However, broad spectrum drug development is hindered by the lack of detailed knowledge of compound binding sites that are structurally and functionally conserved between viral family members and are essential for virus replication. To overcome this limitation, we developed an in silico approach that combines AI-driven protein structure prediction, computational fragment soaking, multiple sequence alignment, and protein stability calculations to identify highly conserved target sites that are both solvent-accessible and conserved. We applied this approach to the Togaviridae family, which includes emerging pandemic disease threats such as chikungunya and Venezuelan equine encephalitis virus for which there are currently no approved antiviral therapies. Our analysis identified multiple solvent accessible and structurally conserved pockets in the alphavirus non-structural protein 2 (nsP2) protease domain, which is essential for processing the viral replicase proteins. Mutagenesis of key solvent accessible and conserved residues identified novel pockets that are essential for the replication of multiple alphaviruses, validating these pockets as potential antiviral target sites for nsP2 inhibitors. These findings highlight the potential of artificial intelligence-informed modeling for revealing functionally conserved, accessible pockets as a means of identifying potential target binding sites for broadly active direct acting antivirals.

Positive-sense RNA virus infections induce vesicle formation within host cells to support RNA replication. By extracting and purifying foot-and-mouth disease virus (FMDV) replication complexes and comparing host cells and replication complexes through targeted lipidomics and proteomics analyses, we found that FMDV enriches host cell proteins and polyunsaturated fatty acids in viral replication complexes (VRCs) to facilitate their formation. On the basis of these findings, we propose a model in which VRCs progress from single-membrane vesicles to multi-membrane vesicles (MMVs) during FMDV replication, a process that requires coordinated contributions of host cell proteins and organelle membranes derived from multiple organelles. Our study showed that, as infection advances, FMDV converts single-membrane vesicles (SMVs) into MMVs, which aggregate to expand the surface area of the replication platform and enhance replication efficiency. These membrane structures function in FMDV replication; the endoplasmic reticulum undergoes curling and folding to support VRC assembly. Additionally, some VRCs possess outward-facing openings that permit material exchange. These findings reveal unexpected similarities between FMDV and distantly related positive-strand RNA viruses, suggesting that shared host cellular pathways are exploited to construct membrane-bound replication factories.

Human cytomegalovirus is an opportunistic pathogen responsible for severe infections in immunocompromised patients, the leading cause of congenital infections worldwide, and potentially implicated in carcinogenesis. The HCMV terminase complex (pUL56-pUL89-pUL51) has emerged as a key target for antiviral drug development. Letermovir, an antiviral agent targeting this complex, inhibits viral DNA packaging, but resistance-associated mutations have already been identified within subunits. Moreover, the precise mechanism of action of letermovir remains incompletely understood. We investigated interactions among terminase subunits in presence or absence of letermovir. Wild-type and mutant forms of these proteins (including resistance mutations V236M, L241P, L257I, C325Y, R369M in pUL56 and A95V in pUL51) were cloned into NanoBiT® PPI and pCI-neo vectors. Letermovir was added after transfection in HEK293T cells, and protein-protein interactions were assessed. Our results show letermovir does not disrupt interactions between wild-type terminase subunits. Resistance-associated mutations modulate the strength of these interactions, with certain mutations (such as pUL56 V236M and L257I) significantly enhancing or reducing binding. Notably, double mutants exhibited synergistic effects. Structural analysis using the AlphaFold3 platform revealed differences between the mutation site of pUL56 and its HSV-1 counterpart pUL28. A hypothetical 3D analysis based on the cryo-EM structure of the HSV-1 terminase complex showed that resistance mutations were oriented outside the complex. These findings suggest letermovir does not act by directly inhibiting interactions among HCMV terminase subunits. Analysis of resistance-associated mutations provides insight into the molecular basis of HCMV resistance to letermovir and may inform development of novel antiviral strategies targeting the terminase complex.

Antiviral susceptibility monitoring is integral to influenza surveillance conducted by CDC in collaboration with partners. Here, we outlined the algorithm and methods used for assessing antiviral susceptibility of viruses collected during 2023-2024 season. Virus specimens were provided by public health laboratories in the United States (US) and by laboratories in other countries that belong to the Pan American Health Organization. In the US, antiviral susceptibility surveillance conducted nationally is strengthened by sequence-only analysis of additional viruses collected at a state level. Viral genome sequence analysis was the primary approach to assess susceptibility to M2 blockers (n = 5123), neuraminidase (NA) inhibitors (n = 6874), and a polymerase acidic protein (PA) inhibitor (baloxavir, n = 6567). Over 99 % of type A viruses had M2-S31N that confers resistance to M2 blockers. Although oseltamivir-resistant viruses carrying NA-H275Y (N1 numbering) were rare (0.35 %), a cluster of four such viruses was identified in Haiti. Viruses with other NA mutations conferring reduced inhibition by NA inhibitor(s) were also detected sporadically. This includes a cluster of three influenza B viruses in Texas that shared a new mutation, NA-A245G conferring reduced inhibition by peramivir. Three viruses with reduced baloxavir susceptibility were identified, which had PA-I38T, PA-Y24C or PA-V122A; the latter two new mutations identified through augmented approach to sequence analysis. To monitor baseline susceptibility, supplementary in vitro testing was conducted on approximately 7 % of viruses using NA inhibition assay and cell culture-based assay IRINA. Implementation of Sequence First approach provided comprehensive and high throughput methodology for antiviral susceptibility assessment and reduced redundant phenotypic testing.

Human respiratory syncytial virus (RSV) causes pediatric bronchiolitis and severe respiratory illness in the elderly. Despite recent advancements in vaccines and antibody therapies, the search for antiviral agents remains a significant public health challenge. We designed nucleotide analogs (NAs) with ribose modifications to assess their incorporation by the RSV polymerase. Biochemical assays and structural modelling revealed that these NAs effectively disrupt RNA synthesis elongation. They act as chain-terminators via a unique mechanism mediated by the 4'-modification, whereas 2'-F alone has no effect and 1'-modification slows-down RNA synthesis. We evaluated the ability of the polymerase to discriminate between natural nucleotides and NAs through incorporation efficiency/competitive assays, correlating these findings with RSV replication inhibition in infected cell cultures. Our ranking of compounds indicates that cytidine analogs demonstrate the strongest antiviral activity, due to their phosphorylation efficiency and intracellular concentration relative to natural nucleotides as well as their ready incorporation into the growing RNA chain. 4'-modifications are accepted by the RSV polymerase due to structural differences between the active sites of (+) and (-) RNA virus polymerases.

Seasonal epidemics and pandemics caused by influenza A viruses still represent a main public health burden in the world. Influenza viruses replicate and transcribe their genome in the nucleus of the infected cells, two functions that are supported by the viral RNA-dependent RNA-polymerase (FluPol) through extensive structural rearrangements and differential interactions with host cell factors. To get insights into its functioning, we screened a phage-display library of biosynthetic proteins (named αReps and built on a rigid alpha-helicoidal HEAT-like scaffold) against the structurally invariant FluPol core and several flexibly-linked domains of the FluPol PB2 subunit. Several αReps specific of the cap binding domain [CBD], the 627-domain and the NLS domain of PB2 displayed FluPol inhibitory and virus neutralization activities when transiently expressed in the cytosol. Furthermore, intracellular ectopic inducible expression of the αReps C3 and F3 (specific of the CBD and the 627-domain, respectively) in influenza virus permissive cells blocked multiplication of viruses representative of the H1N1, H3N2 and H7N1 subtypes, even when induced at late times post-infection. Bispecific αReps constructs (C3-F3 and F3-C3) display a higher FluPol inhibitory activity than their monomeric counterparts. These results suggest that interfering with FluPol structural rearrangements may represent a promising strategy to block virus multiplication and to design new types of antivirals such as dual binders targeting distant sites on FluPol. Furthermore, we found that the 627-domain constitutes a new possible target for engineering influenza antivirals.

To evaluate baseline HBV quasispecies (QS) characteristics as a predictor of HBsAg seroconversion in peginterferon-alfa-2a-treated HBeAg-positive pediatric chronic hepatitis B (CHB).

The Ebola virus (EBOV) and the Rabies virus (RABV) are deadly infectious agents impacting human and animal health. Current prevention and control strategies mainly rely on vaccines and antibodies, highlighting the urgent need for effective, low-cost antivirals suitable for therapeutic options. Plant-derived bioactive compounds offer a promising natural source for such candidate antivirals. As a contribution to this antiviral approach, we have characterized the anti-EBOV and anti-RABV activity of a Cranberry extract (CE) endowed with a very high content of bioactive A2-type proanthocyanidin (PAC-A2). The CE inhibited the in vitro infection of both pseudoviruses expressing EBOV-GP or RABV-G glycoproteins and authentic EBOV and RABV. Attachment and entry assays revealed that the extract targets early phases of infection preventing attachment and entry. Noteworthy, synthetic PAC-A2 reproduced the antiviral activity observed with the whole CE. Mechanistic studies then revealed that the CE interacted directly with the ectodomain of EBOV-GP or the RABV-G, suggesting interference with their functions. In support to this hypothesis, fluorescence spectroscopy analysis showed a reduction in intrinsic fluorescence of both EBOV-GP and RABV-G after incubation with synthetic PAC-A2, thus confirming a direct interaction of the viral glycoproteins with PAC-A2. In silico docking simulations further sustained in vitro results by predicting the binding of PAC-A2 into the binding pocket of EBOV-GP and to the trimeric architecture of RABV-G. Together, these results suggest this cranberry extract and bioactive PAC-A2 as potential candidates to be further develop as novel antiviral agents for the prevention of EBOV and RABV infections.

Hepatitis B virus (HBV) drastically increases the risk of developing liver cirrhosis and hepatocellular carcinoma (HCC) in the ∼300 million people with chronic hepatitis B infections. HBV reproduces through an epigenetic circular genome, but can occasionally integrate into the host genome as a replication-defective form. These integrated forms have been reported to contribute to virus persistence and hepatocarcinogenesis. In this review, we highlight the effects of current and novel treatment under development on HBV DNA integrations and provide areas of potential research to develop more effective therapies that target the underlying drivers of persistence and pathogenesis.

Coxsackievirus B (CVB) infections pose a significant clinical burden due to their association with severe diseases such as myocarditis and aseptic meningitis, and effective antiviral therapies remain an unmet medical need. Here, we investigated the potential of repurposing doxepin hydrochloride (DH), a tricyclic antidepressant, as an antiviral agent against CVB. Our in vitro and in vivo experiments demonstrated that DH significantly inhibits CVB replication. Mechanistically, we elucidated that DH targets the host AXL kinase, thereby inhibiting its phosphorylation of the viral 2C protein at tyrosine 162. This disruption of 2C phosphorylation abrogates CVB-induced suppression of the host IKKβ/NF-κB signaling pathway, leading to the restoration of innate immune responses and enhanced production of pro-inflammatory cytokines IL-1β and IL-6. Furthermore, our findings unveiled a novel immune evasion mechanism employed by CVB, wherein AXL kinase modulates viral replication by regulating host immune signaling. These results highlight AXL as a critical host factor in CVB infection and provide a strong rationale for considering DH as a potential host-targeted therapeutic strategy for CVB-associated diseases, particularly in the absence of specific antiviral agents.

Defective interfering particles (DIPs), containing truncated defective viral genomes (DVGs), are natural byproducts of RNA virus replication with potent antiviral activity. We recently developed a virus-free, dengue virus (DENV)-based platform producing antiviral DIPs with DI290 DVG, termed DIP-DI290. In this study, we established a scalable purification and lyophilization workflow for creating thermostable DIP-DI290 formulations for long-term storage. DIP-DI290 particles were purified via tangential flow filtration and ceramic hydroxyapatite chromatography, then lyophilized and reconstituted for biological assessment. After three months of storage at -80 °C, 4 °C, or room temperature, reconstituted DIP-DI290 retained biological activity, significantly upregulating interferon-stimulated genes (ISGs) and suppressing DENV-2 replication. These findings demonstrate that lyophilization preserves DIP-DI290's antiviral efficacy across diverse storage conditions, supporting its development as a thermostable, field-deployable therapeutic platform for virus infections.

Nucleoside and nucleobase analog antiviral drugs are pivotal in antiviral therapy, but comprehensive methods to understand their cellular response mechanisms and genetic regulators are still lacking. Here, we show that Eμ-Myc; Arf mouse lymphoma cells, which are highly apoptosis-prone, enabled genome-wide CRISPR-Cas9 screening on such drugs to identify genes that modulate their efficacy. Using retroviral sgRNA libraries and MAGeCK analysis, we uncovered key regulators of drug transport, activation, and inactivation for these drugs. For ribavirin, adenosine kinase (ADK) and adenylsuccinate synthase (ADSS) were critical for nucleotide metabolism and bioactivation. Remdesivir uptake and activation depended on the transporter SLC29A3 and phosphoamidase HINT1, whereas favipiravir resistance was linked to NT5C2-mediated dephosphorylation. Viral replication assays in Huh7 cells validated that knockout of SLC29A3, HINT1, or NT5C2 significantly altered antiviral efficacy. This study delineates the genetic network governing nucleotide analog response, providing mechanistic insights and potential biomarkers for personalized antiviral therapy.

Small-molecule HBV RNA destabilizing agents, such as the dihydroquinolizinones (DHQs), were first disclosed in a patent filing in 2015 and in peer reviewed literature in 2018. These compounds inhibit Poly-adenylating Polymerases 5 and 7 (PAPD5/7) and represent a novel antiviral strategy and their ability to degrade hepatitis B surface antigen (HBsAg) in cell culture and animal models generated considerable excitement and commercial interest. However, extrahepatic toxicity observed in preclinical and Phase I studies led to the discontinuation of several development programs. The subsequent emergence of liver-targeted PAPD5/7 inhibitors with improved safety profiles has rekindled interest in this therapeutic approach. Yet, with the apparent success of other investigational antivirals in reducing HBsAg levels, such as siRNAs, antisense oligonucleotides, and in at least one example, capsid assembly modulators (CAMs), questions remain as to whether RNA destabilizers still have a role in managing chronic hepatitis B (CHB). This review describes the current status of PAPD5/7 inhibitor development, evaluates the advantages and limitations of the approach, and considers potential strategies for integrating this class of molecules with other HBV therapies.

Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory infections and hospitalization in young children, but also poses a significant threat to elderly, high-risk adults, and immuno-compromised patients. Despite major progress regarding vaccines and RSV prophylaxis, the current arsenal of RSV antivirals is very limited. A new series of conjugates of N1-(phosphonoalkyl)-1,2,3-triazoles and 6-bromoquinazoline-2,4-diones functionalized with the 2-, 3- and 4-nitrobenzyl, 3-fluorobenzyl or 3-chlorobenzyl at N3 position of quinazoline-2,4-dione moiety were synthesized and evaluated for their potential antiviral activity. Among all tested compounds, conjugates 1 cb and 1 fb showed the strongest anti-RSV activity (high nanomolar to low micromolar range), both in U87MG and HEp-2 cells and proved to be non-toxic toward the tested cell lines. In addition, compounds (1R,2S)-1ha and (1R,2S)-1hd exhibited moderate activity (high micromolar range) against zika virus in Huh7 cell cultures. The C1-epimeric phosphonates (1S,2S)-1ha and (1S,2S)-1 hb proved to be inactive against the tested viruses, while being highly cytotoxic toward uninfected HEL, Huh-7 and MDCK cell lines. Phosphonic acids derived from the respective diethoxyphosphonyl conjugates showed no activity against the viruses tested, probably due to their poor permeability through cell membranes. In conclusion, our study demonstrates the great potency of the design of conjugates of N1-(phosphonoalkyl)-1,2,3-triazoles and 6-bromoquinazoline-2,4-diones as effective anti-RSV agents and highlights the potential of this compound class as antiviral therapeutics, warranting further structural optimization and in vivo evaluation to develop more potent and selective anti-RSV agents.

Hepatitis D virus (HDV) is a small RNA virus that requires Hepatitis B Surface Antigen (HBsAg) for its envelope. Eight genotypes with more than 80% sequence homology and many subgenotypes have been described. Worldwide prevalence of chronic hepatitis delta (CHD) is estimated at about 5% of chronic hepatitis B cases, translating to 15-20 million individuals. The diagnosis of HDV infection involves presence of antibodies to hepatitis D antigen (anti-HDV antibodies). Anti-HDV total antibody indicates HDV exposure (not infection). To document infection; the patient needs to undergo PCR testing and only if PCR is positive should the diagnosis of HDV ongoing infection done. Testing for the antibodies should be performed in all HBsAg-positive persons. CHD is more severe and progressive than HBV mono-infection, with a higher risk of cirrhosis and hepatocellular carcinoma (HCC), transplantation and death. Pegylated interferon-alpha (pegIFN-a) has been used for treating CHD with only limited durable responses. A 48-week course of weekly subcutaneous injections of pegIFN-a suppresses HDV replication in approximately 20-30% of patients 24 weeks off therapy, with significant side effects. Bulevirtide (BLV) was approved by the European Medicines Agency (EMA) in 2020 for CHD and compensated liver disease. Since its approval, real-life data on the use of BLV have been accumulating, with most treated patients in Europe having advanced fibrosis or cirrhosis. Real life data efficacy is concordant to that seen in clinical trials, with many patients achieving significant reductions in HDV RNA levels and ALT normalization after several months of treatment, and favorable safety. However, HBsAg loss is relatively rare. Finite therapy of BLV, in combination with pegIFN-a, leads to significant durable response, with more than 30% of patients achieving HDV RNA undetectability off therapy. We need new finite therapies. Further real-world data and newer therapies are required for this severe disease.

Functional cure has been proposed to be the treatment endpoint of cure therapies in chronic hepatitis B (CHB), yet it is rarely achieved with monotherapy of novel virus-targeting agents or immunomodulators. Although translation inhibitors - small interfering RNAs (siRNAs) and antisense oligonucleotides can produce marked decline in hepatitis B surface antigen (HBsAg) levels, the response is often not sustained and HBsAg seroclearance rarely occur after treatment cessation, suggesting that pharmacological reduction in HBsAg level may be insufficient in restoring HBV-specific immune response. Increasing number of studies have adopted the combination approach with virus-directing agent(s) plus immunomodulator(s). To date, the most effective regimen involves the concurrent or sequential use of siRNA with PEG-IFNα for 48 weeks, with resultant off-therapy HBsAg seroclearance rates approaching 30%, and functional cure rates of up to 10%. Other immunomodulators studied in combination with siRNA such as toll-like receptor agonists, therapeutic vaccines, monoclonal hepatitis B surface antibodies, and immune checkpoint inhibitors are less effective. Almost all studies included NUC and only a few evaluated protocolized NUC withdrawal; thus, few studies have truly evaluated functional cure. Low baseline HBsAg level is the most reliable predictor of HBsAg seroclearance, with many trials exclusively enrolling patients with HBsAg level <200 to <1000 IU/mL. While recent studies have shown promise, further research is needed to determine the optimal classes of drugs to combine, duration of use for each drug and whether they should be used concurrently or sequentially, to meet the desired goal of 30% functional cure rate.

BK and JC Polyomavirus are closely related and establish persistence in infected subjects. Recent studies suggest that JC Polyomavirus replication prevents BK Polyomavirus-related pathologies in kidney transplant recipients. One potential mechanism of this competition could involve viral microRNAs cross-reacting, as they are highly homologous between species. In fact, bkv-miR-B1-3p is strictly identical to jcv-miR-J1-3p, whereas species-specific miRNAs bkv-miR-B1-5p and jcv-miR-J1-5p differ barely. Early detection of jcv-miR-J1-5p in urine significantly reduces the risk of BK Polyomavirus DNAemia in kidney transplant recipients in a case-control study including 39 patients (odds ratio [95 % Confidence Interval] = 0.00 [0.00-0.65], p = 0.012). In vitro modeling revealed that prior infection with JC Polyomavirus reduces the ability of BK Polyomavirus to grow in immortalized human renal proximal tubular epithelial cells, without significant expression of JC Polyomavirus proteins. The JC Polyomavirus-specific miRNA jcv-miR-J1-5p was discovered to decrease BK Polyomavirus TAg mRNA expression, without affecting early genome replication, like the known regulatory effect of BK Polyomavirus-encoded bkv-miR-B1-3p and bkv-miR-B1-5p. An archetypal strain of JC Polyomavirus engineered to quench miRNA maturation did not inhibit BK Polyomavirus infection, unlike the wild-type strain, confirming that the inhibitory effect of JC Polyomavirus is due to miRNAs. These results suggest that JC Polyomavirus-specific miRNA jcv-miR-J1-5p limits BK Polyomavirus infectivity and early TAg expression, with an intensity similar to BK Polyomavirus miRNAs. This mechanism might explain in vivo competition for viral replication between BK Polyomavirus and JC Polyomavirus infections.

Elucidation of the regulation mechanism of hepatitis B virus (HBV) replication will provide potential targets for the development of novel anti-HBV therapeutics. It has been reported that the N6-methyladenosine (m6A) modification of HBV RNA plays a crucial role in the HBV life cycle. However, the mechanisms underlying the regulation of this modification remain incompletely understood. In this study, combining loss- and gain-of-function genetic analyses, we defined the role of IGF2BP1, an m6A reader, in facilitating HBV replication. Mechanistic studies revealed that IGF2BP1 stabilizes HBV RNAs primarily by binding to m6A-modified A1907 sites through its KH3-4 domain, thereby enhancing viral replication. Furthermore, targeted inhibition of IGF2BP1 by Cucurbitacin B, a small molecule inhibitor of IGF2BP1, was shown to inhibit HBV replication in vitro and in vivo. Taken together, these findings identify IGF2BP1 as a critical host regulator of HBV RNA stability through an m6A-dependent manner and targeted inhibition of IGF2BP1 effectively attenuates viral replication, providing a promising strategy for anti-HBV drug development.