Development of a replication-defective mpox virus platform for fundamental and therapeutic research

Domain: biology
Published: 2025-12-15
Editorial outcome: minor_revision

Abstract
The recent global outbreaks of mpox highlight the urgent need for both fundamental research and antiviral development. However, studying the mpox virus (MPXV), with its large and complex genome, remains challenging due to the requirement for high-containment facilities. Here, we describe a strategy for de novo assembly of MPXV clade IIb genomes in bacterial artificial chromosomes using transformation-associated recombination cloning. Leveraging CRISPR-Cas9 and Lambda Red recombination, we engineer replication-defective MPXV particles with dual deletions of OPG96 (M2R) and OPG158 (A32.5L)—genes essential for virion assembly, that are capable of recapitulating key stages of the viral lifecycle. We apply this system to screen a compound library and identify G243-1720, a potent anti-poxvirus inhibitor with broad activity in vitro and in vivo. G243-1720 blocks the formation of extracellular enveloped virions and cell-cell spread. Resistance mutation selection, crystallographic analysis, analytical ultracentrifugation, and mass photometry reveal that, despite its distinct chemical structure, G243-1720 shares a mode of action with tecovirimat, both functioning by affecting dimerization of protein OPG57 (F13). Our findings underscore the potential of G243-1720 as a promising broad-spectrum anti-poxvirus lead compound and demonstrate the utility of replication-defective MPXV particles as a reliable platform for viral biology studies and antiviral development.

Introduction
Mpox virus (MPXV), the causative agent of the zoonotic disease mpox, is an enveloped double-stranded DNA virus belonging to the genus Orthopoxvirus of the family Poxviridae, which also includes variola virus (VARV), vaccinia virus (VACV), and cowpox virus (CPXV)1. MPXV strains are classified into two distinct clades: Clade I, found mainly in Central Africa (particularly the Democratic Republic of the Congo, DRC), is associated with severe clinical symptoms and substantial mortality (4–11%); Clade II, prevalent in West Africa, is characterized by milder symptoms and a mortality rate of less than 1%2,3. For over 50 years, MPXV outbreaks in Africa have been driven predominantly by zoonotic spillover and caused isolated, self-limiting outbreaks with poor human-to-human transmission4.

In 2022, MPXV clade IIb caused the first widespread community transmission of mpox outside Africa5,6, prompting the World Health Organization (WHO) to declare mpox a Public Health Emergency of International Concern (PHEIC). Subsequently, due to the rising number of cases caused by clade Ib, the WHO declared a second mpox PHEIC in 20247, following its earlier reprioritization of mpox as a neglected tropical disease with pandemic potential. This underscores the urgent need for effective prevention and treatment strategies8.

Poxviruses enter host cells via fusion with either the plasma membrane or the membrane of an intracellular acidified vesicle9. Following fusion of the viral envelope with the host membrane, the core—containing the ~200 kb viral genome (encoding nearly 200 proteins) and structural components (e.g., RNA polymerase)—is released into the cytoplasm10. The transcription system of the core then drives the expression of early genes, which facilitate immune evasion, genome release, and subsequent DNA replication. Newly synthesized genomes serve as templates for sequential expression of intermediate and late genes, initiating viral particle assembly11.

The earliest hallmark of particle assembly is the formation of rigid, crescent-shaped structures within viral factories12–15. These crescents require at least nine essential proteins, comprising three structural and six regulatory (e.g., L2 (now renamed orthopoxvirus gene 9616, OPG96), A30.5 (OPG158)) factors, collectively termed viral membrane assembly proteins (VMAPs). Among these, OPG96 and OPG158 are the smallest and critical for regulating crescent formation. The crescent extends with constant curvature and eventually closes to form immature virions (IV), which then mature into the infectious intracellular mature virus (IMV), also called mature virus (MV). IMV particles mediate cell-to-cell transmission but are released primarily upon cell lysis. A subset of IMV acquires a double-layer envelope from the trans-Golgi network or early endosomes, forming an intracellular enveloped virus (IEV), also called a wrapped virus (WV). IEV particles are transported along microtubules to the cell periphery, where they fuse with the plasma membrane to become cell-associated enveloped virus (CEV) on the cell surface. CEV can induce actin tail formation to propel itself towards neighboring cells for direct spread; alternatively, it may be released into the extracellular space as extracellular enveloped virus (EEV), also called enveloped virus (EV), to mediate systemic dissemination12–15.

The large and complex MPXV genome and the high biosafety containment (e.g., BSL-3) required to handle the live virus hinder studies on its molecular biology, pathogenesis, and antiviral drug development. Consequently, the development of tools such as replication-defective viruses—which enable genome manipulation and recapitulate viral infection under lower biosafety conditions—provides a valuable platform for scientific research.

Despite the global spread of MPXV, few drugs are approved for clinical use. Currently, only two therapeutics, tecovirimat (ST-246, TOPXX) and cidofovir or its prodrug brincidofovir (CMX001), are authorized for mpox treatment17. Tecovirimat dimerizes the orthopoxvirus protein P37 (also called F13) encoded by the F13L (OPG57) gene, to inhibit the formation of EEV particles and cell-cell spread18,19, but its efficacy remains unproven in clinical settings17,20,21. Cidofovir or brincidofovir can inhibit viral DNA polymerase activity22, but is associated with gastrointestinal and hepatic toxicity. Thus, the identification of antivirals against MPXV is important.

To establish a safe and reliable alternative to authentic MPXV for antiviral research, we assembled a circular, full-length MPXV genome within a bacterial artificial chromosome (BAC) by transformation-associated recombination (TAR) cloning in yeast. We deleted MPXV homologs of OPG96 (VACV L2R) and OPG158 (VACV A30.5L)—both essential for viral envelope formation23–25—to generate replication-defective MPXV (rdMPXV) particles that mimic the viral life cycle. Using this platform, we performed a proof-of-concept screen of a small-compound library and identified G243-1720, a promising antiviral lead compound and discovered it has a mechanism of action analogous to tecovirimat.

Results
Assembly of the MPXV genome lacking OPG96 (MPXV gene M2R)
Compared to clade I MPXV strains, MPXV clade IIb was associated with mild disease symptoms during the 2022 outbreak, and so was selected for the construction of rdMPXV particles. The entire viral genome was divided into 23 fragments (F1–F23), with 80–100 bp overlaps between adjacent fragments. The MPXV OPG96 (M2R) lies within fragment F10, and encodes a 92 aa protein that is highly conserved in orthopoxviruses and essential for viral envelope formation and subsequent morphogenesis of IMV (intracellular mature virion) and EEV (extracellular enveloped virion)23,26. Therefore, this gene was selected for deletion from the MPXV genome. As illustrated in Fig. 1A, adjacent fragments (grouped in sets of four), excluding F1, F2, and F11, were assembled by transformation-associated recombination (TAR) cloning in yeast to generate fragments B, C, E, F, and G.

The reporter expression cassette, mGreenLantern (mGreen)-P2A-Gaussia luciferase, under the control of the viral late promoter P11, was inserted together with the linearized yeast/E. coli shuttle plasmid pBAC, into the thymidine kinase (tk) gene (OPG101) within fragment F11, yielding fragment D. Fragments B to G were then assembled by TAR cloning to generate intermediate plasmid pBAC-IΔ96, which lacks OPG96 (Fig. 1A). The plasmid pBAC-IΔ96 exhibited stable maintenance in E. coli over 10 passages (Supplementary Fig. 1A), as confirmed by next-generation sequencing (NGS). No mutations or indels (insertions or deletions) were found in the viral genome or reporter region compared to the reference sequence (Supplementary Fig. 1B, C).

The remaining fragments F1 and F2 in plasmids were digested with the Type IIS restriction enzyme SapI and ligated in vitro to form fragment A. Fragment A was then inserted into the AscI-linearized pBAC-IΔ96, yielding the plasmid pBAC-MPXVΔ96, which contains the full-length MPXV genome lacking OPG96 (Fig. 1A).

Generation of replication-defective MPXV particles lacking OPG96
To rescue the replication-defective virus particles, wild-type CV-1 cells (CV-1-WT) were transduced with a lentivirus expressing OPG96 with codon-optimized coding sequences. The resulting stable cells (CV-1-96) were infected with fowlpox virus (FPV) to provide poxvirus transcriptional enzymes and then transfected with pBAC-MPXVΔ96 (Fig. 1B). The green fluorescence from the expression of mGreen reporter and cytopathic effect were monitored daily. On day 5 post-transfection, distinct viral plaques were observed (Fig. 1B), and the replication-defective MPXV particles lacking OPG96 (rdMPXVΔ96) were harvested.

With the deletion of OPG96, which encodes a protein crucial for virion assembly, the rdMPXVΔ96 particles could only enter and express virus proteins in CV-1-WT cells, but were unable to form progeny IMV or EEV to infect neighboring cells. As expected, only single mGreen-positive cells were detected in CV-1-WT when compared to CV-1-96 cells, which expressed OPG96 (Fig. 1B). The growth kinetics and Gaussia luciferase expression of rdMPXVΔ96 in CV-1-WT and CV-1-96 cells were measured. Abortive replication in CV-1-WT cells was observed, while the virus titer reached around 107 FFU in CV-1-96 cells (Fig. 1C). Although moderate increase of luciferase activity was detected in CV-1-WT cells, the significant increase in CV-1-96 cells suggested that rdMPXVΔ96 underwent multiple cycles of replication, expressing abundant luciferase (Fig. 1D). Transmission electron microscopy revealed structurally intact virions in CV-1-96 cells infected with rdMPXVΔ96 (Fig. 1E). For rdMPXVΔ96 infection in CV-1-WT cells, only short “spicule”-coated membrane arcs resembling crescent segments were detected around the large dense aggregates (Fig. 1E), which was consistent with the previous report describing a VACV mutant not expressing OPG96 (VACV gene L2R)23. Thus, these results demonstrated that rdMPXVΔ96 can only complete the entire lifecycle and propagate in OPG96 trans-complementing cells.

The defective replication property of rdMPXVΔ96 upon serial passaging was further confirmed in passage 1 (P1), P5, and P10 viruses, in which no viral amplification was detected in CV-1-WT cells (Supplementary Fig. 2A, B). PCR analysis of viral genomic DNA from P5 and P10 viruses revealed no indels in either the OPG96-deleted region or the reporter gene region (Supplementary Fig. 2C). Depth-of-coverage analysis of NGS reads also revealed no mutations or indels in the deleted OPG96 region and the whole genome compared to the rdMPXVΔ96 reference sequences (Supplementary Fig. 2D, E). Additionally, analysis of NGS reads indicated no introduction of FPV DNA into the P5 rdMPXVΔ96 genome (Supplementary Fig. 2F). The above results indicate that the replication-defective rdMPXVΔ96 particles exhibit stability in their trans-complementing cells.

Generation of replication-defective MPXV particles with dual deletions of OPG96 and OPG158 (MPXV gene A32.5L)
The VACV protein OPG158 (A30.5) interacts with OPG96 (L2), and the deletion of OPG158 also blocks the formation of the virion envelope, resulting in the failure to produce infectious viral particles24,25. To further strengthen safety, we constructed replication-defective virus particles lacking both OPG96 and OPG158 (MPXV gene A32.5L). The CRISPR/Cas9 gene editing in combination with the Lambda Red recombination system was employed in E. coli to achieve this goal27. As illustrated in Fig. 2A, starting from E. coli DH10B containing the plasmid pBAC-IΔ96, the plasmid pEcCas, which constitutively expresses Cas9 and inducibly expresses the Lambda Red system, was transformed into DH10B for genome editing and recombination. The sgRNA-expressing plasmid pEcgRNA (targeting OPG158) and a homologous repair DNA template containing only the flanking sequences of the OPG158 open reading frame (ORF) were then introduced into the cells. Cas9/sgRNA-mediated cleavage of OPG158 within pBAC-IΔ96, combined with Lambda Red-induced homology-directed repair, resulted in deletion of OPG158. After removing both pEcgRNA and pEcCas plasmids in E. coli, the plasmid with dual deletions of OPG96 and OPG158 was obtained, validated by NGS (Supplementary Fig. 3A–C), and designated as pBAC-IΔ96,158. Using the virus rescue method applied for rdMPXVΔ96, replication-defective MPXV with double deletions (rdMPXVΔ96,158) was rescued in CV-1 cells trans-complementing both codon-optimized MPXV OPG96 and OPG158 (CV-1-96,158).

Due to the dual-gene deletion, the rdMPXVΔ96,158 particles could theoretically only enter and express proteins in CV-1-WT and CV-1-96 cells, but be unable to form progeny virions unless propagated in the trans-complementing CV-1-96,158 cell line. This was confirmed by viral growth kinetics and Gaussia luciferase expression, which demonstrated that rdMPXVΔ96,158 could complete multi-cycle propagation in the CV-1-96,158 cells, but not in the CV-1-WT or CV-1-96 cells (Fig. 2B, C). Additionally, the defective replication of rdMPXVΔ96,158 was evaluated in dormice, a natural host for MPXV infection28,29. Over 14 days, comparable increases in body weight were observed in mock- and rdMPXVΔ96,158-inoculated mice, whereas a significant reduction in body weight was noted in mice challenged with wild-type MPXV (Fig. 2D). Furthermore, rdMPXVΔ96,158 did not exhibit productive replication in the lungs of dormice by day 14, in contrast to wild-type MPXV (Fig. 2E).

Transmission electron microscopy was used to examine virion assembly. Structurally intact virions were observed in rdMPXVΔ96,158-infected CV-1-96,158 cells, but not in both CV-1-WT and CV-1-96 cells (Fig. 2F), consistent with previous reports on VACVs lacking either gene23,25. Additionally, intact virions released from rdMPXVΔ96,158-infected CV-1-96,158 cells were detected (Fig. 2G).

The stability of replication-defective rdMPXVΔ96,158 was studied. After 10 passages in CV-1-96,158 cells, rdMPXVΔ96,158 maintained the single-cycle replication property in CV-1-WT and CV-1-96 cells (Supplementary Fig. 3D). PCR analysis of the genomic DNA from the P10 virus confirmed no indels in OPG96, OPG158, and reporter regions (Supplementary Fig. 3E). NGS analysis further revealed no mutations and indels in the deleted OPG96 and OPG158 regions, and the whole genome of the P5 and P10 rdMPXVΔ96,158 compared to the rdMPXVΔ96,158 reference sequences (Supplementary Fig. 3F–H). Thus, these results demonstrate that rdMPXVΔ96,158 exhibits favorable safety and stability. Because of dual-gene deletions, rdMPXVΔ96,158 possesses a higher safety profile compared to rdMPXVΔ96, which has only a single gene deleted.

Application of rdMPXVΔ96,158 particles in cell biology and antiviral assessment
To expand the application of replication-defective rdMPXVΔ96,158 particles, the susceptibility of commonly used cell lines to rdMPXVΔ96,158 infection was evaluated. As shown in Fig. 2H and Supplementary Fig. 4A, HaCaT, CV-1, Vero E6, BS-C-1, and HFF-1 cells were highly susceptible to infection, as indicated by expression of mGreen reporter, while 293T, Huh7, and SW13 cells exhibited low susceptibility. It is intriguing that the susceptibility of skin and fibroblast cells, such as HaCaT and HFF-1, may correlate with the MPXV tissue tropism. Previous studies have reported that heparan sulfate and chondroitin sulfate are involved in the attachment of VACV virions to the cell membrane30–33. The B3GAT3 gene, responsible for heparan sulfate and chondroitin sulfate biosynthesis34, was found to be important for MPXV infection, as its deletion reduced the infection efficiency of rdMPXVΔ96,158 in A549 cells (Fig. 2I and Supplementary Fig. 4B).

Next, the capacity of rdMPXVΔ96,158 particles for antiviral development was explored. In VACV, the specific neutralizing antibody 7D11 targeting the L1 viral protein (OPG95) can block infection35. This antibody also inhibited rdMPXVΔ96,158 infection with an EC50 of 0.83 μg/mL (Fig. 2J). Tecovirimat, which targets the orthopoxvirus P37 protein encoded by OPG57 (VACV F13L) and blocks wrapping of IMV, so that the intracellular enveloped virus (IEV, also called wrapped virus, WV) is not formed, and cidofovir, which targets viral DNA polymerase to inhibit DNA synthesis, are FDA-approved drugs for emergency use in MPXV patients17,36. Both drugs effectively inhibited rdMPXVΔ96,158 spread (tecovirimat) or replication (cidofovir), with EC50 values of 0.003 μM (tecovirimat) and 10.450 μM (cidofovir) in CV-1-96,158 cells (Fig. 2K–M). Similar results were observed in human keratinocytes (HaCaT) stably-expressing OPG96 and OPG158 (HaCaT-96,158) (Supplementary Fig. 4C–E). The antiviral activities of tecovirimat and cidofovir assessed using replication-defective rdMPXVΔ96,158 particles were consistent with previous reports using wild-type MPXV in HFF, HFK, Vero E6, or other cell lines (Supplementary Table 1)19,36–40.

Screening of antiviral compounds using the rdMPXVΔ96,158
As a proof-of-concept, we performed a high-throughput screen of a small-compound library using the rdMPXVΔ96,158 particles. Highly susceptible HaCat-96,158 cells were pre-treated with individual compounds at 10 μM for 1 h, followed by infection with rdMPXVΔ96,158 for 60 h in the presence of the compounds. The numbers of mGreen-positive cells and DAPI-stained nuclei were analyzed by high-content imaging to calculate the number of infected cells versus the total number of cells per well (Fig. 3A).

Compounds that demonstrated more than 90% inhibition of viral replication and maintained over 80% relative cell number were selected as candidates for further validation. Three candidate compounds were re-evaluated at concentrations of 10 μM and 2 μM to confirm their antiviral activity (Fig. 3B). Among these, Merimepodib, reported previously to inhibit MPXV replication, was identified41, a finding that underscores the reliability of our screening procedure.

Two compounds—G243-1720 and AG-1478—were selected to determine the CC50 and EC50 in HaCaT-96,158 cells. Results showed that G243-1720 had the lowest EC50 (0.23 μM) and the highest CC50 (>100 μM) among them (Fig. 3C). The compound G243-1720 consists of a methyl chlorobenzene moiety linked via an aminosulfonyl group to a bicyclic structure, which includes an azepane attached to a carbonyl group and a five-membered nitrogen-containing ring with two methyl groups and a methylated nitrogen atom (Fig. 3D). The compound G243-1720 is synthesized and authenticated (Supplementary Data 1).

Immunofluorescent images showed that G243-1720 significantly inhibited the rdMPXVΔ96,158 infection in both HaCaT-96,158 and CV-1-96,158 cells (Fig. 3E), with an EC50 of 0.142 μM in the latter case (Fig. 3F). Similarly, plaque assay indicated that G243-1720 has an EC50 of 0.194 μM in Vero E6 cells infected with wild-type MPXV (Fig. 3G), and 0.16 μM of G243-1720 reduced the number of genome copies by 60% (Fig. 3H). Overall, G243-1720 exhibited potent anti-MPXV activity with low cytotoxicity and was selected for further investigation.

To validate the efficacy of G243-1720 against other orthopoxviruses and its broad-spectrum activity in cell lines from multiple species, the inhibition of VACV replication in HaCaT, A549, CV-1, Vero E6, BHK-21, and MEF cells was examined. The results showed that G243-1720 effectively inhibits VACV replication in these cell lines, with EC50 values ranging from 0.15 to 1.5 μM (Fig. 3I and Supplementary Fig. 5A). Additionally, G243-1720 exhibited inhibitory effect on the replication of other orthopoxviruses like CPXV-BR, CPXV-E, CMLV, and RPXV, but not FPV (a member of the distantly related Avipoxvirus genus), HSV-1 (herpes simplex virus type 1), and the RNA viruses VSV (vesicular stomatitis virus) and SINV (Sindbis virus) (Fig. 3J–L and Supplementary Fig. 5B). These findings demonstrate that G243-1720 specifically inhibits the replication of orthopoxviruses.

Pharmacokinetics (PK) of G243-1720 were studied in ICR mice to assess the bioavailability (Supplementary Fig. 5C, D). The compound demonstrated rapid absorption, achieving a maximum plasma concentration (Cmax) of ~30,000 ng/mL within ~12 min (Tmax) after intraperitoneal (i.p., 22.5 mg/kg) or intragastric (i.g., 45 mg/kg) administration, with a half-life (T1/2) of 0.828 h or 1.85 h, respectively. Plasma compound concentrations remained above the antiviral EC50 value (0.194 μM, 84.97 ng/mL) for ~4 h (i.p.) or ~8 h (i.g.). Moreover, no body weight loss was observed in severe combined immunodeficiency (SCID) mice treated with G243-1720 twice daily for up to 7 days (Supplementary Fig. 5E).

To evaluate the antiviral efficacy of G243-1720 in vivo, SCID mice were inoculated intranasally with MPXV and subsequently were treated with the compound. Viral titers in the lung were determined at day 10 post-infection (Fig. 3M). Twice-daily administration of G243-1720 via the intraperitoneal (i.p., 22.5 mg/kg) or intragastric (i.g., 45 mg/kg) route reduced the median viral titers from 5.48 logs in the vehicle control to 3.28 logs and 3.92 logs, respectively. As a positive control, once-daily dosing of tecovirimat (i.g., 50 mg/kg) reduced the titers to 2.78 logs. Although G243-1720 demonstrated slightly lower potency than the FDA-approved tecovirimat, its effective antiviral activity against MPXV, combined with favorable pharmacokinetic properties, supports its potential as a promising candidate for further development.

G243-1720 inhibits the formation of EEVs and virus spread
To investigate how G243-1720 inhibits poxvirus infection, we first examined which stages of the viral life cycle the compound might act on. Using tecovirimat as a control, we added the compounds during the entry, post-entry, or entire infection period (Fig. 4A). In HaCaT-96,158 cells infected with rdMPXVΔ96,158, G243-1720 significantly inhibited infection at the post-entry or entire infection period but not during entry, a phenotype similar to tecovirimat treatment (Fig. 4B). Similarly, VACV infection in wild-type HaCaT (HaCaT-WT) cells showed comparable results (Fig. 4C). These data suggest that G243-1720 likely targets viral replication, assembly, or release.

To further explore the impact of G243-1720 on viral DNA replication and late gene expression, HaCaT-WT cells were treated with varying concentrations of G243-1720, tecovirimat, or cidofovir, followed by infection with rdMPXVΔ96,158. As indicated by the mGreen reporter, the percentages of virus-infected cells treated with these three compounds were comparable to those of the DMSO control (Fig. 4D). However, analysis of the mean fluorescence intensity of mGreen-positive cells revealed that only cidofovir treatment, which inhibits viral replication, showed decreased intensity (Fig. 4E). Similarly, only cidofovir reduced Gaussia luciferase activity (Fig. 4F). These results suggest that G243-1720 does not interfere with the DNA replication or late gene expression.

To determine whether G243-1720 affects viral particle assembly, particularly IMV formation, VACV-infected CV-1-WT cells were analyzed by transmission electron microscopy. Normal IMV morphology was observed in cells treated with DMSO, G243-1720, or tecovirimat, indicating that G243-1720 does not impair IMV assembly (Fig. 4G–I). Moreover, similar titers of infectious IMVs were detected in VACV-infected HaCaT-WT cells treated with G243-1720 or tecovirimat, albeit lower than those in the DMSO-treated control (Fig. 4J). Likewise, comparable titers were observed in HaCaT-96,158 cells infected with rdMPXVΔ96,158 after treatment with G243-1720 or tecovirimat (Fig. 4K). These results demonstrate that G243-1720 does not interfere with IMV assembly or infectivity.

Next, we asked whether G243-1720 impacts IEV formation and EEV release, which are critical for the long-range spread of the orthopoxviruses26. We first examined EEV production in the supernatant of CV-1-96,158 cells infected with rdMPXVΔ96,158 at 48 h post-infection under G243-1720 treatment. Plaque assays revealed that, compared to DMSO-treated control, both G243-1720 and tecovirimat significantly reduced the titer of extracellular virus (Fig. 4L). We also investigated the effect of G243-1720 on viral plaque size. After treatment with G243-1720 or tecovirimat, VACV-infected HaCaT-WT or rdMPXVΔ96,158-infected HaCaT-96,158 cells were overlaid with methylcellulose, and the plaque size was measured. As shown in Fig. 4M–O, both compounds inhibited cell-cell spread and plaque formation. These findings suggest that G243-1720 might act during IEV formation, its transport to the cell surface or virus release, sharing a mechanism similar to that of tecovirimat.

G243-1720 targets protein OPG57 (F13) of MPXV
To elucidate the potential target of G243-1720, we selected resistant viral mutants by serially passaging rdMPXVΔ96,158 under increasing G243-1720 concentrations (Supplementary Fig. 6A). Resistant mutations were enriched through treatment with varying doses of G243-1720 in HaCaT-96,158 and CV-1-96,158 cells. After 5 or 10 passages, MPXV genomes were sequenced and compared with the control virus passaged in DMSO-treated cells. In addition to non-specific mutations (Supplementary Fig. 6B), two G243-1720-specific mutations, I372N and D294V, were identified in the OPG57 protein (VACV F13) at P5 and P10 in HaCaT-96,158 cells, respectively (Fig. 5A and Supplementary Fig. 6B). Similarly, two single mutations, I372N and A290V, in OPG57 were detected in CV-1-96,158 cells at P5 (Fig. 5A and Supplementary Fig. 6C). Viruses carrying these OPG57 mutations exhibited resistance to high concentrations of G243-1720, as evidenced by enhanced cell spread compared to the DMSO-treated control (Fig. 5B and Supplementary Fig. 7A, B).

Tecovirimat targets the OPG57 protein (F13), and specific mutations (e.g., A290V, D294V, and I372N) confer resistance to its inhibitory effects42–45 (Fig. 5C). Similarly, another OPG57-targeting small-molecule, IMCBH, is rendered ineffective by mutations like D294V43,46 (Fig. 5C). Since the same three mutations emerged under G243-1720 selection, we hypothesized that G243-1720-resistant viruses would also exhibit cross-resistance to molecules like tecovirimat. As expected, tecovirimat failed to inhibit the cell spread of virus carrying the OPG57 I372N mutation selected by G243-1720 (Fig. 5D). Although G243-1720 shows a different chemical formula compared to tecovirimat and IMCBH (Supplementary Fig. 7C), the findings of resistance mutations suggest that G243-1720 may target the same viral protein (OPG57) and share similar active sites.

G243-1720 induces the dimerization of the OPG57 protein
Structural data and functional assays have shown that tecovirimat inhibits MPXV egress by inducing OPG57 dimerization18. To test whether G243-1720 uses the same mechanism, we purified a soluble version of OPG57 (sOPG57) and evaluated its oligomerization state in the presence of G243-1720 using analytical ultracentrifugation (AUC), mass photometry, and size-exclusion chromatography coupled with small-angle X-ray scattering (SEC-SAXS) (Fig. 6). Using AUC, we observed that 10 µM G243-1720 induces a shift of sOPG057 from a monomeric form (S=3.5) to a homodimer (S=4.6) like that formed by tecovirimat (S=4.8) (Fig. 6A). Similarly, using mass photometry, in presence of 10 µM G243-1720, we observed 50% of homodimers, versus 80% in presence of 10 µM tecovirimat and 5% in the absence of drugs. The introduction of escape mutants A290V and I372N separately reduced the level of G243-1720-induced dimerization by almost half, while the double mutant D294V/I372N rendered sOPG57 insensitive to G243-1720-induced dimerization (Fig. 6B). In line with these results, SEC-SAXS showed a monodisperse dimer population compatible with the crystallographic dimer (Fig. 6C, D and Supplementary Fig. 8A–C).

To confirm that G243-1720 binds to the same pocket as tecovirimat, cubic crystals of sOPG57 were obtained and then soaked in a solution containing 1 mM of G243-1720 for 18 h. The resulting maps revealed an additional electron density at the dimer interface consistent with the shape of G243-1720; however, the presence of asymmetry axis prevented unambiguous modeling of the molecule (Supplementary Fig. 8D–G). Overall, these findings confirmed that G243-1720 induces OPG57 dimerization by binding to the tecovirimat-binding pocket.

Methods
Animals and ethics
Male ICR mice (4–6 weeks old), male SCID mice (4–5 weeks old), and male dormice (10–12 weeks old) were housed under controlled environmental conditions (23 ± 2°C, 30–70% relative humidity, 15 air changes per hour). Animals were group-housed (5 mice per cage) in individually ventilated polycarbonate cages with sterile corn cob bedding and provided ad libitum access to sterile food and water. All animal experiments were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committees of the Changchun Veterinary Research Institute (IACUC approval no. AMMS-11-2023-041).

Cells and viruses
BS-C-1 (Cell Bank of the Chinese Academy of Sciences #SCSP-5506), CV-1, HaCaT, SW13, Huh7, Vero E6 (Cell Bank of the Chinese Academy of Sciences, #GNO17), HFF-1 (ATCC #SCRC-1041), U-2 OS (ATCC #HTB-96), BHK-21 (ATCC #CCL-10), HEK 293T (ATCC #CRL-3216), A549 (ATCC #CCL-185), and HeLa (ATCC #CCL-2) were cultured at 37°C in Dulbecco’s Modified Eagle’s Medium (Hyclone) supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, 1 mM sodium pyruvate, 1× non-essential amino acids, and 100 U/mL penicillin-streptomycin. All cell lines were routinely tested and confirmed free of mycoplasma contamination. VACV WR strain was propagated in CV-1 cells. Fowlpox virus (FPV) vaccine strain (DCEP25, Boehringer-Ingelheim) was propagated in primary chick embryo fibroblasts (CEF). MPXV IIb strain (GenBank: PP778666.1) was grown in Vero E6 cells. CPXV-Brighton Red strain (CPXV-BR), CPXV isolated from an elephant (CPXV-E), camelpox virus (CMLV) CMS strain, and rabbitpox virus (RPXV) Utrecht strain were propagated in RK13 cells. Herpes simplex virus type 1 (HSV-1) (kindly provided by Prof. Liang, Shanghai Institute of Immunity and Infection), SINV, and VSV were propagated in BHK-21 cells.

Transformation-associated recombination (TAR) cloning of plasmids
The complete genome of MPXV (MPXV-BY-IMB25241, GenBank: ON568298), with a length of 197,378 bp, was divided into 23 fragments (F1–F23). Adjacent fragments were designed with 80–100 bp homologous arms, and OPG96 (M2R) in fragment F10 was deleted. As illustrated in Fig. 1, every four adjacent fragments (except F1, F2, and F11) were assembled in Saccharomyces cerevisiae strain VL6-48 using the yeast/E. coli shuttle plasmid pBAC. Assembled plasmids were purified from VL6-48 and transformed into E. coli DH10B for amplification, resulting in plasmids designated pBAC-B, -C, -E, -F, and -G. For pBAC-D construction, the reporter cassette (mGreenLantern-P2A-Gaussia luciferase) under the control of the late viral promoter P11 was synthesized and inserted into OPG101 (J2R gene, thymidine kinase (tk)) of fragment F11. The modified F11 fragment was cloned into the shuttle plasmid. Fragments B–G (digested from pBAC-B to pBAC-G) were co-transformed with linearized pBAC-D into yeast. Assembled plasmids were purified and transformed into DH10B, yielding the final plasmid pBAC-IΔ96, which lacks F1, F2, and OPG96.

Yeast transformation using the LiAc/SS carrier DNA/PEG
Yeast transformation via lithium acetate was performed as described previously65. Briefly, VL6-48 cells were grown in 2× YPAD medium, washed, and resuspended in sterile water. A 100 μL yeast suspension was transferred to a microcentrifuge tube and mixed with transformation solution containing 36 μL lithium acetate (1.0 M), 240 μL PEG 3350 (50% w/v), 50 μL single-stranded carrier DNA (2.0 mg/mL), and 34 μL DNA (fragments and linearized shuttle vector). The mixture was heat-shocked at 42°C for 40 min to facilitate DNA uptake. Cells were harvested by centrifugation, resuspended in sterile water, plated on selective medium, and incubated at 30°C for 3–4 days. Positive clones were validated by PCR and sequencing. Plasmids were extracted and electroporated into E. coli DH10B for amplification.

CRISPR-based editing of the plasmid in E. coli
To knockout OPG158 (A32.5L) via homologous recombination, a DNA template encompassing only the upstream and downstream sequences of OPG158 was PCR-amplified and joined by overlapping-extension PCR. The spectinomycin resistance gene in the pEcgRNA vector (Addgene #166581) was replaced with an ampicillin resistance gene, and an sgRNA (5ʹ-TTTGGAACGTTTGATAAAAG-3ʹ) targeting OPG158 was cloned to generate pEcgRNA-sgOPG158. The plasmids pBAC-IΔ96 (constructed above) and pEcCas (Addgene #73227) were transformed into DH10B cells, and the Lambda Red recombination system was induced with 10 mM L-arabinose. The sgRNA-expressing plasmid pEcgRNA-sgOPG158 and the DNA recombination repair template (lacking OPG158 coding sequences) were co-electroporated into DH10B cells harboring pBAC-IΔ96 and pEcCas. Positive clones lacking OPG158 were identified by PCR and sequencing. After overnight culture in selection medium, the pEcgRNA-sgOPG158 and pEcCas plasmids were eliminated, resulting in DH10B cells containing only the plasmid pBAC-IΔ96,158 with dual deletions of OPG96 and OPG158.

Generation of stable cell lines
The codon-optimized OPG96 coding sequence was cloned into pLV-EF1α-IRES-puro (Addgene #85132), in which the puromycin resistance gene was replaced with mCherry. Similarly, the codon-optimized OPG158 coding sequence was cloned into the same plasmid, where puromycin resistance was replaced with mIFP. Lentiviruses were packaged by co-transfecting HEK 293T cells with psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259). Wild-type (WT) CV-1 or HaCaT cells were transduced with lentiviruses and sorted for mCherry+ or mCherry+/mIFP+ cells to establish stable cell lines: CV-1-OPG96 (CV-1-96), CV-1-OPG96-OPG158 (CV-1-96,158), HaCaT-OPG96 (HaCaT-96), and HaCaT-OPG96-OPG158 (HaCaT-96,158).

Assembly of full-length MPXV genome and virus rescue
Fragments F1 and F2 in plasmids were digested with SapI and ligated using T4 DNA ligase to form fragment A. Fragment A and the AscI-linearized pBAC-IΔ96 plasmid were assembled via Gibson assembly to generate the plasmid pBAC-MPXVΔ96, which contains the full-length MPXV genome lacking OPG96. Similarly, the pBAC-MPXVΔ96,158 plasmid lacking OPG96 and OPG158 was constructed in the pBAC-IΔ96,158 backbone. CV-1-96 or CV-1-96,158 stable cell lines were infected with FPV for 2 h. After washing, the pBAC-MPXVΔ96 or pBAC-MPXVΔ96,158 plasmid was transfected into the cells, and the presence of green fluorescence and cytopathic effect (CPE) was monitored daily. Supernatants and cells were harvested and subjected to three freeze-thaw cycles at −80°C to generate the P0 virus stock. The resulting replication-defective viral particles—with a single deletion of OPG96 (rdMPXVΔ96) or dual deletions of OPG96 and OPG158 (rdMPXVΔ96,158)—were designated accordingly.

Viral growth kinetics and luciferase assay
In 12-well plates, cells were infected with the virus at an MOI of 0.05 for 2 h, followed by a medium change to DMEM containing 2% FBS. At the indicated time points, cells and supernatants were collected together and subjected to three freeze-thaw cycles at −80°C to release the virus. To determine the titer, monolayers of CV-1-96 or CV-1-96,158 in 96-well plates were inoculated with serially diluted virus for 2 h and then overlaid with methylcellulose for 36 h. Cells were fixed with 4% paraformaldehyde (PFA) in PBS for 1 h and washed with PBS. Green fluorescent foci were counted. To measure Gaussia luciferase activity, only the supernatants were collected. The luciferase activity was determined using the Secrete-Pair Gaussia Luciferase Assay Kit (iGeneBio #LF061) according to the manufacturer’s instructions. Briefly, 10 μL of the supernatant was added to a 96-well plate, followed by 100 μL of assay reagent, which was gently pipetted to mix thoroughly. After incubation at room temperature for 1 min, luminescence was recorded using a FlexStation 3 (Molecular Devices) with an integration time of 1 s per well.

Electron microscopy
CV-1-WT and CV-1-96 cells were infected with rdMPXVΔ96 at an MOI of 1. CV-1-WT, CV-1-96, and CV-1-96,158 cells were infected with rdMPXVΔ96,158 at an MOI of 1. At 36 h post-infection, cells were scraped and fixed with 2.5% glutaraldehyde for 1 h. Samples were further fixed with 1% osmium tetroxide at 20°C for 2 h, dehydrated through a graded ethanol series, and infiltrated overnight with acetone. After polymerization at 60°C for 48 h, ultrathin sections were prepared using a Leica UC7 microtome. Sections were stained with uranyl acetate and lead citrate, dried, and imaged using a FEI Tecnai G2 20 Twin transmission electron microscope. Electron microscopy analysis of IMV formation was performed in CV-1-WT cells pre-treated for 1 h with DMSO, tecovirimat (0.2 μM), or G243-1720 (10 μM), followed by infection with VACV at an MOI of 0.5 for 36 h. Cells were subsequently collected, fixed, processed into electron microscopy samples, and visualized as described above.

Assessment of cell susceptibility
The cell lines from different species and organs were infected with rdMPXVΔ96,158 at an MOI of 0.5 for 48 h. Representative images were captured using a fluorescence microscope. To determine infection efficiency, cells were trypsinized, fixed with 2% paraformaldehyde (PFA) for 10 min, and analyzed by flow cytometry. To examine the role of heparan sulfate and chondroitin sulfate in MPXV infection, a B3GAT3-knockout clonal cell line was generated in A549 cells by introducing a ribonucleoprotein (RNP) complex containing Cas9 protein and crRNA. Following limiting dilution and expansion, clonal knockout cells were selected and validated by sequencing. The sgRNA targeting B3GAT3 was designed as: 5ʹ-CCAGAGCCCATACCTGGCAT-3ʹ.

Assessment of viral stability
Trans-complemented stable cells in 12-well plates were infected with replication-defective MPXV particles at an MOI of 0.01 for 2 h. After washing, cells were maintained in DMEM supplemented with 2% FBS to allow viral propagation for 96 h, followed by three freeze-thaw cycles at −80°C to release virions. Virus particles were then used to infect new cells at an MOI of 0.01 for passaging as described. Genomic DNA from passage 5 (P5) and passage 10 (P10) was extracted. Regions encompassing OPG96, OPG158, or the mGreenLantern-P2A-Gaussia luciferase reporter cassette were amplified for gel electrophoresis and Sanger sequencing. Genomic DNA was also subjected to Next-Generation Sequencing (NGS) to analyze mutations or contamination.

Plaque reduction neutralization (PRNT) assay
The neutralizing antibody 7D11 (Antibody System #RVV13903) targeting OPG95 (VACV L1) was serially diluted in DMEM containing 2% FBS, starting from 80 μg/ml in 5-fold increments. The diluted antibody was mixed in equal volumes with 2% FBS DMEM containing 240 focus-forming units (FFU) of rdMPXVΔ96,158 (final volume: 100 μL) and incubated at 37°C for 1 h. CV-1-96,158 cells in a 96-well plate were infected with the mixture for 2 h, overlaid with methylcellulose for 30 h, and fixed with 4% PFA. Plaques were quantified using an ImmunoSpot® Analyzer. Experiments involving rdMPXVΔ96,158 were performed in HaCaT-96,158 cells.

Compound screening
HaCat-96,158 cells in 96-well plates were pre-treated with individual small-molecule compounds from a library (3185 compounds; TargetMol, #LC00) at a final concentration of 10 µM for 1 h. Cells were then infected with rdMPXVΔ96,158 at an MOI of 0.001 in the presence of the compounds. After 60 h of infection, cells were fixed and stained with DAPI. Infection efficiency and relative cell number in each well were analyzed using the Operetta CLS High-Content Analysis System. Compounds showing >90% viral inhibition and >80% relative cell number were selected for validation at final concentrations of 10 µM and 2 µM using the same method.

Half-maximal effective concentration (EC50) of compounds
HaCaT-96,158 cells in 96-well plates were pre-treated with serial dilutions of compounds (starting at 100 µM) for 1 h. Without washing, cells were infected with rdMPXVΔ96,158 at an MOI of 0.001 in the presence of the compounds. After 60 h of infection, cells were fixed and stained with DAPI. The infection efficiency in each well was analyzed using the Operetta CLS High-Content Analysis System. Using GraphPad Prism, the compound concentration and viral infection efficiency in treated cells were first log-transformed (base 10), followed by nonlinear regression (curve fitting) to determine the EC50 value. Alternatively, the EC50 of compounds was determined by plaque assay. Briefly, confluent monolayers of Vero E6 cells in 6-well plates were pretreated with serial dilutions of compounds (starting at 60 µM) for 1 h, infected with wild-type MPXV (200 PFU) for 2 h, and overlaid with methylcellulose in the presence of the compounds. After 72 h of infection, cells were fixed, stained with crystal violet, and plaques were counted. To assess the inhibitory effect of compounds on different orthopoxviruses, Vero E6 cells were pretreated with G243-1720 (10 µM) or tecovirimat (1 µM) for 1 h, and infected with viruses for plaque assays (CPXV-BR or CPXV-E, 300 PFU, 72 h; RPXV, 500 PFU, 48 h; CMLV, 500 PFU, 120 h).

Half-maximal cytotoxic concentration (CC50) of compounds
HaCat-96,158 cells in 96-well plates were treated with serial dilutions of compounds (starting at 100 µM). After 60 h of treatment, cell viability was assessed using the CellTiter-Lumi™ Steady Luminescence Cell Viability Assay Kit (Beyotime #C0069L) according to the manufacturer’s instructions. Briefly, CellTiter-Lumi™ reagent was added to each well and mixed by shaking for 2 min. After incubation at room temperature for 10 min, luminescence was recorded using a FlexStation 3 (Molecular Devices) with an integration time of 0.5 s per well. The luminescence intensity in each well was normalized by dividing it by the fluorescence intensity of DMSO-treated control wells. Using GraphPad Prism, the compound concentration and its corresponding relative luminescence intensity were first log-transformed (base 10), followed by nonlinear regression (curve fitting) to determine the CC50 value.

Time of addition experiments
HaCaT-WT cells were seeded in 96-well plates. Full-time group: cells were pre-treated with compounds for 1 h, infected with VACV at an MOI of 0.002 for 2 h in the presence of compounds, washed, and cultured in 2% FBS DMEM with compounds for 24 h. Entry group: cells were pre-treated with compounds for 1 h, infected with VACV (MOI = 0.002) for 2 h with compounds, washed, and cultured in 2% FBS DMEM without compounds for 24 h. Post-entry group: cells were infected with VACV (MOI = 0.002) for 2 h, washed, then cultured in 2% FBS DMEM with compounds for 24 h. After treatment, cells were collected and analyzed for infection efficiency by flow cytometry. Experiments involving rdMPXVΔ96,158 were performed in HaCaT-96,158 cells.

Determination of viral replication and late gene expression
HaCaT-WT cells seeded in 96-well plates were pre-treated with compounds for 1 h. Cells were infected with rdMPXVΔ96,158 at an MOI of 0.5 for 2 h. After removing the viral inoculum by washing, cells were cultured in DMEM supplemented with 2% FBS and compounds. At 24 h post-infection, supernatants were collected, and Gaussia luciferase activity was measured using the Secreted Gaussia Luciferase Assay Kit (iGeneBio #LF061). Cells were harvested to determine the percentage of mGreen-positive cells and mean fluorescence intensity by flow cytometry.

Pharmacokinetics and toxicity of compounds in mice
Male ICR mice (4–6 weeks, 18–22 g) were used to determine the pharmacokinetics of the compounds. Compounds were dissolved in a vehicle solution containing 10% DMSO and 30% w/v HP-β-CD in 0.9% saline. Following intraperitoneal (22.5 mg/kg) or intragastric (45 mg/kg) administration, blood samples were collected into K2 EDTA-containing tubes via retro-orbital bleeding at 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h. Plasma was obtained by centrifugation at 3500 × g for 3 min. Compound concentrations were determined using the SCIEX Triple Quad 5500+ LC-MS/MS System.

For toxicity monitoring, severe combined immunodeficiency (SCID) mice aged 4-5 weeks were administered either vehicle control via intragastric administration twice daily, or G243-1720 via intraperitoneal (22.5 mg/kg) or intragastric (45 mg/kg) route twice daily. Body weight was measured on days 0, 3, and 7. G243-1720 was dissolved in a vehicle solution containing 10% DMSO and 30% w/v HP-β-CD in 0.9% saline.

Virus infection and antiviral efficacy in animals
To evaluate the replication of rdMPXVΔ96,158 in vivo, male dormice aged 10-12 weeks were inoculated intranasally with PBS (mock control), 5×105 focus-forming unit (FFU) of rdMPXVΔ96,158 particles, or 1×105 FFU of wild-type MPXV in a volume of 30 μl. Body weight was monitored over 14 days. Lung tissues were harvested on days 3, 7, and 14 post-infection for DNA extraction. Viral genomic copies were quantified by qPCR targeting OPG47 (F3L) using the following primers and probe: Fwd, 5ʹ-CTCATTGATTTTTCGCGGGATA-3ʹ; Rev, 5ʹ-GACGTACTCCTCCTCGTTGGT-3ʹ; Probe, 5ʹ-CATCAGAATCTGTAGGCCGT-3ʹ.

To assess the antiviral efficacy of G243-1720, male SCID mice aged 4–5 weeks were inoculated intranasally with 1x106 TCID50 of MPXV IIb strain (GenBank: PP778666.1) in a volume of 50 μl. Compounds were dissolved in a vehicle solution containing 10% DMSO and 30% w/v HP-β-CD in 0.9% saline. One hour post-infection, mice were given vehicle control via intragastric route twice daily, G243-1720 via intraperitoneal (22.5 mg/kg) or intragastric (45 mg/kg) administration twice daily, or tecovirimat (50 mg/kg) via intragastric injection once daily. At day 10 post-infection, mice were euthanized, and lung tissues were collected for viral titer determination. Tissue homogenates were serially diluted (10-fold) and inoculated onto Vero E6 monolayer in 96-well plates in a volume of 100 μl. Viral titers were calculated based on cytopathic effects using the method of Muench and Reed.

Determination of infectious intracellular mature virus (IMV) titer
HaCaT-WT cells seeded in 12-well plates were pre-treated with compounds for 1 h, followed by infection with VACV for 2 h. Unbound virus was washed off, and cells were cultured in DMEM containing 2% FBS and compounds. At specified time points, cells were washed twice with PBS and collected to determine the infectious titer of IMV. To determine the titer, CV-1-WT monolayers in 96-well plates were inoculated with serially diluted virus for 2 h and overlaid with methylcellulose for 30 h. Cells were fixed with 4% paraformaldehyde (PFA) for 1 h, washed with PBS, and the formation of green fluorescent foci was quantified. Experiments involving rdMPXVΔ96,158 were performed in HaCaT-96 or CV-1-96,158 cells.

Determination of infectious extracellular enveloped virus (EEV) titer
CV-1-96,158 cells in T-75 flasks were pre-treated with G243-1720 (10 μM) or tecovirimat (1 μM) in 2% FBS DMEM for 1 h, followed by infection with rdMPXVΔ96,158 at an MOI of 0.5 for 5 h. Cells were washed twice with PBS and cultured in 2% FBS DMEM with compounds for 48 h. Supernatants were collected by centrifugation at 500 × g for 10 min. IMVs in the supernatant were neutralized with VACV IMV neutralizing antibody (mAb 7D11, 50 μg/mL, targeting OPG95, L1), and used to infect CV-1-96,158 cells in a 96-well plate for 2 h. Cells were washed twice with PBS and cultured in 2% FBS DMEM with 1% methylcellulose for 36 h. After fixation with 4% PFA, plaques were quantified using an ImmunoSpot® Analyzer.

Cell–cell spread assay
HaCaT-WT cells were pre-treated with tecovirimat (0.2 μM) or G243-1720 (10 μM) for 1 h, followed by infection with VACV at an MOI of 0.0005 for 2 h. For replication-defective MPXV particles (rdMPXVΔ96,158), HaCaT-96,158 cells were pre-treated with compounds and infected at an MOI of 0.002 for 2 h. After washing to remove unbound virus, cells were overlaid with 1% methylcellulose in 2% FBS DMEM supplemented with the compounds. After 24 h (VACV) or 50 h (rdMPXVΔ96,158), cells were fixed with 4% PFA for 1 h, and images were captured using a fluorescence microscope. The area of mGreen-positive cells was analyzed using ImageJ.

Identification of G243-1720-resistant MPXV mutations
As illustrated in Fig. S5A, HaCaT-96,158 or CV-1-96,158 cells seeded in 12-well plates were infected with rdMPXVΔ96,158 for 2 h. The virus was washed off, and cells were cultured in 2% FBS DMEM containing increasing concentrations of G243-1720. At day 4 post-infection, virus from the highest compound concentration that still supported viral replication was passaged onto new cells in medium containing a higher concentration of G243-1720. This process was repeated for 10 passages. P5 and P10 viruses were sequenced by NGS, and G243-1720-specific mutations were identified by comparing them to control viruses passaged in DMSO-treated cells.

sOPG57 production, purification, and crystallization
Soluble OPG57 (VACV strain Western Reserve, Uniprot code: P04021) was produced in a pET-28a(+) vector (Novagen) with an N-terminal His- and Strep-tag followed by a thrombin cleavage site, as reported elsewhere18. The mutations C53A, A290V, I372N and the double mutation D294V/I372N were inserted into this plasmid. E. coli BL21 (DE3) cells (New England Biolabs) were transformed with these plasmids, and protein expression was induced overnight at 16°C with 0.25 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). Cells were harvested from 3 L of culture, resuspended in 40 mL cold TNE8 buffer (Tris-HCl 10 mM, pH 8, NaCl 150 mM, EDTA 1 mM) supplemented with one tablet of complete protease inhibitor (Roche) and then lysed by sonication. After removing the insoluble material by centrifugation at 20,000 xg (30 min, 4°C), the recombinant protein was purified using Strep-tag-based affinity chromatography in a StrepTrap HP 5 mL column (Cytiva). The protein was treated with 5 mM Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) (Thermo Scientific) for 10 min at room temperature, and aggregates were removed by size-exclusion chromatography in TN8 buffer (Tris-HCl 10 mM pH 8, NaCl 100 mM).

For crystallization, we removed the purification tags by digestion with 1.5 units of thrombin (Cytiva) per 0.1 mg of protein overnight at 4°C, treated the protein with 5 mM TCEP for 10 min at room temperature and purified the protein by size exclusion chromatography in TN8 buffer. The protein was concentrated to 12 mg/mL, crystallized using 1 M Na citrate, 0.1 M imidazole, pH 8 and the crystals were soaked for 18 h in the crystallization solution supplemented with 1 mM G243-1720 and 10% DMSO. Shorter soaking times produced less clear electron densities for G243-1720, probably due to lower occupancy. After soaking, the crystals were cryoprotected with 33% (v/v) glycerol and x-ray diffraction data were collected on beamlines PROXIMA-1 and PROXIMA-2 at the synchrotron SOLEIL (St Aubin, France). The diffraction images were integrated with XDS (version January 10, 2022)66 and the phases were determined using a crystallographic model of OPG57 (PDB ID: 9FHS) as a template using PHASER67. To obtain the final models, the structures were built iteratively and refined using phenix.refine (Phenix version 1.19.2-4158)68 and coot69 using isotropic B factor and TLS groups as refinement strategy. All the models were validated using Molprobity70. The crystallographic statistics are provided in Supplementary Table 2. To facilitate the modeling, the final map was corrected using a bulk-solvent mosaic model available in the PHENIX program (phenix.mosaic). The final map showed an additional electron density at the interface compatible with G243-1720. Because the crystal only diffracted to 3.4 Å, there is a 2-fold symmetry axis crossing the ligand binding pocket, and the occupancy of the ligand is not complete; the density did not show clear features, and we have not succeeded in placing the molecule in the density without ambiguity to study ligand/protein contacts. Coordinates and structure factors have been deposited in the Protein Data Bank under the accession code 9QQV. Figures showing the crystallographic models were generated with PyMol v3.0.3 (Schrödinger, LLC).

Mass photometry
Mass photometry (MP) experiments were done using the TwoMP instrument (Refeyn Ltd, Oxford, UK) using filtered (0.22 μm) TN 8 buffer (10 mM Tris-HCl, pH 8, 100 mM NaCl). Contrast-to-mass calibrations were performed using two references (BSA and urease, both purchased from Sigma Aldrich) diluted in TN 8 buffer, covering a mass range from 66 kDa to 272 kDa. Samples were loaded on clean and dry microscope coverslips assembled into silicone gaskets. Immediately prior to mass photometry measurements, 2 μL of sOPG57 protein was diluted alone or supplemented with the antivirals in 18 μL of TN 8 into the gasket hole and mixed twice. The final concentrations were 25 nM for sOPG57 and 10 μM for the antivirals. Data were collected using AcquireMP v2.3 (Refeyn Ltd), and movies of 2936 frames at a 49 Hz frame rate were recorded. The images were processed and analyzed using DiscoverMP v2.3 (Refeyn Ltd). To extract mole fractions (percentage of each species), all particle contrasts obtained from each movie were converted to mass and calculated mole fractions as the area of each Gaussian curve. The percentage of sOPG57 dimer was plotted using Prism Graphpad v9.0.2.

Analytical ultracentrifugation (AUC)
Sedimentation velocity experiments were carried out at 20°C in an Optima AUC analytical ultracentrifuge (Beckman Coulter) equipped with double-UV and Rayleigh interference detection. Purified sOPG57 proteins at 0.2 mg ml−1 in the absence or presence of tecovirimat or G243-1720 (10 μM) were centrifuged at 42,000 r.p.m. (23,600 g) using an AN60-Ti rotor and 12 mm thick double sector centrepieces. Absorbance and interference profiles were recorded every 5 min. Buffer viscosity (η = 1.018 cP) and density (ρ = 1.0054 g ml−1) at 20°C were estimated with SEDNTERP 1.09. Partial specific volumes at 20°C were estimated based on amino acid sequences using SEDNTERP 1.09 software. Data were analyzed with SEDFIT 16.171 using a continuous size distribution c(S) model. Theoretical sedimentations of the complex were generated using HydroPro 1072.

Size-exclusion chromatography coupled with small-angle X-ray scattering (SEC-SAXS)
Data were collected on the SWING beamline at Synchrotron Soleil (France) using the online HPLC system. The column was equilibrated in the preparation buffer containing 10 mM Tris, pH 8, 100 mM NaCl and 10 μM G243-1720. These experiments have been performed using sOPG57 digested with thrombin. sOPG57 samples at 4.6 mg ml−1 were prepared in a buffer and injected into a size exclusion column (Superdex 75 increase 5/150 mm), cooled at 15°C, eluting directly into the SAXS flow-through capillary cell at a flow rate of 200 µl min−1. The data were analyzed using FOXTROT and PRIMUS from ATSAS 3.273, from which Guinier was generated. Scattering curves were selected for stable Rg at the apex of the elution profile; the selected curves were averaged, and the buffer signal was subtracted. The scattering curve of the complex was extracted from the main peak using LC and Evolving Factor analysis in RAW 2.3.174. From these corrected scattering curves, the pair distribution function was computed using GNOM (version 5.0)75, and the normalized Kratky plot was generated. Using the structure of sOPG57/G243-1720 (PDB ID: 9QQV), the experimental curve was compared to the theoretical curve using CRYSOL (version 2.8.3)75. Ab initio models were generated with DAMMIN (version 5.3)76, and for each model, sedimentation characteristic was calculated with hydropro (version 10)72. The SAXS statistics are provided (Supplementary Tables 3 and 4).

Statistical analysis
Statistical significance was assigned when P-values were <0.05 using Prism Version 9 (GraphPad). Data analysis was determined by an ANOVA or unpaired t-test, depending on data distribution and the number of comparison groups.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Authors present a timely and extensive work. Here authors have developed a non-replicative Mpox viral system using\nCRISPR-Cas9 and recombinant methods, resulting in a platform technology with reduced the safety rating for Mpox work\nfrom BLS-3 to BSL-2. Further, authors utilized the platform to screen for anti-Mpox molecules and successfully identified two\nmolecules and provided. majority of the work that is developing the replication-defective Mpox system was done meticulous\nwith proper controls. I believe the final biophysical characterization of OPG57 protein-ligand interactions lacked a similar\nrigor. Nonetheless, I recommend the publication of this article upon careful consideration on other reviews and corrections\nmentioned below\n1. A brief description in the introduction for protein encoded by Mpox genome and highlight the function of relevant protein\nfunction for the study. Also, IMV and EEV are introduced in the results sections; it will readers if IMV and EEV stages of\nMpox cycle are highlighted briefly in introduction to contextualize Cidofovir and tecovirimat mode of action.\n2. OPG85 aslo seems to strongly mutate in both cell lines apart from OPG57, are any experiments carried out eliminate as a\ntarget for G243-1720, please comment on this\n2.Mass photometry is highly sensitive and niche techniques, if the antiviral molecules can induce dimerization of sOPG57,\nstraight forward experiments with size exclusion chromatography or Ultracentrifugation or even native-PAGE may have been\nused.\n4. For mapping the interaction site of G243-1720 methods such as amide-hydrogen deuterium exchange mass spectrometry\n(HDXMS) can be used to conclusively show the binding interface.\nMinor issues\n1.Line 151 “Transmission electron microscopy was used to study the assembly of virions.” Please rephrase it gives the\nimpression that mechanistic study using TEM was carried out instead authors used TEM imaging to observe whether the\nsystem produced intact particles or not. Line 205 also\n2. Showing a table comparing the IC50 values of antivirals and antibodies (7D11) from known studies for Mpox virus and\ncurrent study with replication defective particles (lines 235-240) will be helpful for readers, as supplementary data\n3. Panel 2E , should have mock data too ?\n4.”(PDB code = 9FHS)” to PDB ID: 9FHS\n5.plasmid availability for the research community should be addressed as per the journal requirements.\n6.Title should be modified to emphasize the replication-defective Mpox platform alone", "reviewer_2": "Very interesting work. The authors have developed a BAC-MPXV system with dual gene (OPG96 and OPG158) deletions,\nwhich under helper FPXV infection produced replication-defective MPXV (rdMPXV) to be used as a tool for MPXV virology\nstudies and drug screening under standard BSL-2 lab settings, as compared to the WT MPXV which requires BSL-3\ncontainment. The new virus system was confirmed for safety and applied to screening a TargetMol library of 3185\ncompounds in trans-complementing cells using a high-content imaging assay. From this pilot screen, one of the 3 initial hits,\ncompound G243-1720, showed sub-micromolar potency without cytotoxicity, and hence was further studied in a wide range\nof rigorous experiments, and was characterized as a potent, non-toxic and bioavailable, broad-spectrum antiviral against\northopox viruses, by mechanistically promoting the dimerization of p37 and blocking IMV wrapping. The paper is very well\nwritten, and the technical quality is very high. However, it’s less clear how significant the work really is.\nMajor points\n1) Although the extremely well-characterized new drug hit G243-1720 showed sub-micromolar potency in infected cells and\ndemonstrated efficacy in mice, it’s substantially much less potent than tecovirimat: a) in CV-1-96,158 cells infected with\nrdMPXVΔ96,158, G243-1720 (EC50 = 140 nM) was 47-fold less potent than tecovirimat (EC50 = 3 nM); b) in vivo efficacy:\nG243-1720 twice daily i.g. at 45 mg / kg produced much less viral titer reduction than tecovirimat once daily i.g. at 50 mg / kg\n(1.56 log for G243-1720 vs 2.7 log for tecovirimat).\n2) G243-1720 is mechanistically identical to tecovirimat with overlapping resistance profile, diminishing its development\nvalue.\n3) One purported value of the rdMPXV is the safety when screening compounds for inhibitors impacting replication in wt\ncells, which amounts to a single replication cycle assay. It’s unclear why only a limited screen in trans-complementing cells\nwas conducted. The latter is a multiple rounds replication assay which increases the risk of producing MPXV via\nrecombination.\n4) It’s unclear what advantages this system offers over VACV as a surrogate virus for compound screening and drug MOA\ncharacterization, as was the case in the development of tecovirimat. The assay is cumbersome, especially when the trans-\ncomplementing cells are used.\nMinor points\n5) IC50 should be EC50\n6) G243-1720 should be properly authenticated with 1H and 13C NMR, HRMS and HPLC. These analytical data should be\nincluded in the supplemental file.\n7) References: for many citations only the first author is listed—this is not appropriate.", "reviewer_3": "In this communication, the authors describe the construction of a replication-defective MPXV and use it to screen a small-\ncompound library for replication inhibitors. The most potent inhibitor, G243-1720, has similar biological activity and the same\nOPG57 target as Tecovorimat, though a different chemical structure. Like Tecovorimat, G243-1720 inhibited multiple\northopoxviruses including VACV. The work was very well done, exceptionally complete and the paper clearly written.\nNevertheless, the screening could have been done with VACV instead of the defective MPXV as a broad inhibitor is most\nadvantageous and G243-1720 does not seem to offer any advantage in potency over Tecovorimat. Additionally,\nspontaneous mutants that resist the latter are also resistant to G243-1720.\nSpecific comments\n1. The deletion of two essential genes from MPXV, which increases safety, and establishment of a complementing cell line\nwere well described.\n2. Is there an identifier or catalog number for the TargetMol compound library?\n3. Does G243-1720 inhibit members of other poxvirus genera? For example, inhbitors of parapoxviruses would be useful.", "reviewer_4": "The generation of a replication defective model of MPXV infection is noteworthy and will likely prove to be a valuable tool to\nsupport the identification of antiviral compounds. This will be an important addition as it will allow additional laboratories to\ncomplete research against MPXV infections.\nOverall, the work that has been completed is sound and the conclusions are well-supported by the data.\nThe statistical analyses appear to be appropriate and enough replicates have been completed to instill confidence in the\nresults.\nThe methodology is clear and meets the standards in the field. There is enough information provided to accurately allow\nother researchers to reproduce this work.\nThere are a few small things which could be clarified to benefit the readers.\n1. The authors demonstrate efficient infection of rdMPXV in several different cell lines, however, screening of antiviral\ncompounds was only completed in HaCat-96,158 cells. The rationale for choosing this cell line over the others is not clear\nand a rationale would help the readers understand why other cell lines were not utilized.\n2. The authors identified Merimepodib as an inhibitor of MPXV during the screening assays. It is not clear whether this\ncompound was an intentional positive control or whether it was detected randomly in the compounds that were tested.\n3. The legend for Figure 6A does not specify what statistical analysis was used to compare the dimerization activity of F243-\n1720."}, "author_response": "We sincerely thank the Reviewer's evaluation and feedback of our manuscript.\n1. A brief description in the introduction for protein encoded by Mpox genome and\nhighlight the function of relevant protein function for the study. Also, IMV and EEV are\nintroduced in the results sections; it will readers if IMV and EEV stages of Mpox cycle are\nhighlighted briefly in introduction to contextualize Cidofovir and tecovirimat mode of action.\nWe agree with the Reviewer that providing this background information will improve the\nclarity. We have added the following description in the “Introduction” section of the revised\nmanuscript.\n“Poxviruses enter host cells via fusion with either the plasma membrane or the membrane\nof an intracellular acidified vesicle1. Following fusion of the viral envelope with the host\nmembrane, the core—containing the ~200 kb viral genome (encoding nearly 200 proteins)\nand structural components (e.g., RNA polymerase)—is released into the cytoplasm2. The\ntranscription system of the core then drives the expression of early genes, which facilitate\nimmune evasion, genome release, and subsequent DNA replication. Newly synthesized\ngenomes serve as templates for sequential expression of intermediate and late genes,\ninitiating viral particle assembly3.\nThe earliest hallmark of particle assembly is the formation of rigid, crescent-shaped\nstructures within viral factories4, 5, 6, 7. These crescents require at least nine essential\nproteins, comprising three structural and six regulatory (eg., L2 (now renamed\northopoxvirus gene 96, OPG96), A30.5 (OPG158)) factors, collectively termed viral\nmembrane assembly proteins (VMAPs). Among these, OPG96 and OPG158 are the\nsmallest, and critical for regulating crescent formation. The crescent extends with\nconstant curvature and eventually closes to form immature virions (IV), which then\nmatures into the infectious intracellular mature virus (IMV), also called mature virus (MV).\nIMV particles mediates cell-to-cell transmission but are released primarily upon cell lysis.\nA subset of IMV acquires a double-layer envelope from the trans-Golgi network or early\nendosomes, forming intracellular enveloped virus (IEV), also called wrapped virus (WV).\nIEV particles are transported along microtubules to the cell periphery, where they fuse\nwith the plasma membrane to become cell-associated enveloped virus (CEV) on the cell\nsurface. CEV can induce actin tail formation to propel itself towards neighboring cells for\ndirect spread; alternatively, it may be released into extracellular space as extracellular\nenveloped virus (EEV), also called enveloped virus (EV), to mediate systemic\ndissemination4, 5, 6, 7.”\n2. OPG85 aslo seems to strongly mutate in both cell lines apart from OPG57, are any\nexperiments carried out eliminate as a target for G243-1720, please comment on this.\nWe thank the Reviewer for raising this question. Indeed, our sequencing data showed\nmutations in OPG85 (G1L in VACV) in addition to OPG57 (F13L in VACV) in both cell\nlines. We excluded it from the investigation based on the following information: a. OPG85,\na reported viral metalloprotease, is essential for viral particle transition from IV to IMV via\nits proteolytic activity, acting after core cleavage but before condensation and infectivity\nacquisition. Deletion of OPG85 arrests viral morphogenesis at the IV stage and reduces\ninfectious progeny by 98.9%8, 9; b. Electron microscopy revealed that G243-1720 does\nnot affect IMV morphogenesis (Figs. 4G–4I); c. Subsequent IMV titration in cells also\nshowed G243-1720 does not impair IMV infectivity (Figs. 4J–4K); d. Although OPG85\nF215S mutation emerged in viruses passaged in CV-1-96,158 and HaCaT-96,158 cells\nunder G243-1720 treatment, the mutation also occurred in DMSO-treated controls during\nresistance screening.\nBased on these observations, we conclude that the OPG85 F215S mutation arises from\nviral passaging (adaptive changes) rather than specific resistance to G243-1720, and\nexclude OPG85 as a candidate target for G243-1720.\n3.Mass photometry is highly sensitive and niche techniques, if the antiviral molecules can\ninduce dimerization of sOPG57, straight forward experiments with size exclusion\nchromatography or Ultracentrifugation or even native-PAGE may have been used.\nWe thank the Reviewer for the suggestion. To complement the mass photometry\nexperiments, we have now incorporated analytical ultracentrifugation (AUC) and SEC-\nSAXS data, which demonstrate that G243-1720 induces the dimerization of OPG57. In\nthe AUC experiments performed in the absence and presence of 10 µM G243-1720 and\ntecovirimat (as a positive control), we found that G243-1720 shifted the equilibrium from\nmonomer to homodimers, as tecovirimat does. In the same line, SEC-SAXS data\nconfirmed that G243-1720 promotes the dimerization of sOPG057, as the experimental\ncurve aligned well with the theoretical SAXS profile calculated using the crystallographic\ndimer. Overall, consistent with the results from the mass photometry experiment, we\nobserved that G243-1720 shifted the equilibrium in solution of OPG057 to the dimeric\nform in both the AUC and SEC-SAXS experiments. These findings are now presented in\nFigure 6 and Supplementary Figure 8, along with SAXS data and collection parameters\nincluded in Supplementary Tables 4 and 5.\n4. For mapping the interaction site of G243-1720 methods such as amide-hydrogen\ndeuterium exchange mass spectrometry (HDXMS) can be used to conclusively show the\nbinding interface.\nThanks for the suggestion. We are sorry that we are currently do not have access to\nHDXMS technique. To complement this, we have conducted crystallization, mass\nphotometry, as well as analytical ultracentrifugation and SEC-SAXS to assess the\ninteraction of G243-1720 with OPG57 protein, showing the compound-induced\ndimerization.\nMinor issues\n1.Line 151 “Transmission electron microscopy was used to study the assembly of virions.”\nPlease rephrase it gives the impression that mechanistic study using TEM was carried\nout instead authors used TEM imaging to observe whether the system produced intact\nparticles or not. Line 205 also\nAs requested, we have rephrased these sentences for accuracy: “Transmission electron\nmicroscopy revealed structurally intact virions in CV-1-96 cells…” shown in line 186 of\nthe revised manuscript.\n2. Showing a table comparing the IC50 values of antivirals and antibodies (7D11) from\nknown studies for Mpox virus and current study with replication defective particles (lines\n235-240) will be helpful for readers, as supplementary data\nThis is a helpful suggestion. We include a new Supplementary Table 1 that compiles the\nEC values for tecovirimat, cidofovir, and the antibody 7D11 from published studies using\n50\nwild-type MPXV, alongside the values we obtained with our replication-defective MPXV\nparticles.\nCompound Strains EC values (μM) Cell lines Reference\n50\ns Zaire 0.01 Vero 10\nZaire 0.008±0.0014 Vero E6 11\nMPXV-V78-I-3945 0.023±0.0026 BSC-40 12\nMPXV-V81-I-179 0.032±0.0061 BSC-40 12\nMPXV-2003-USA-039 0.036±0.0045 BSC-40 12\nTecovirimat MPXV-V77-I-823 0.030±0.0114 BSC-40 12\nMPXV-V1979-I-005 0.039±0.0016 BSC-40 12\noutbreak in 2022\nSP2833 0.006±0.0002 Vero E6 11\nrdMPXVΔ96,158 0.003 CV-1-96,158 this study\nrdMPXVΔ96,158 0.007 HaCat-96,158 this study\nZaire 27±11 Vero 76 14\nCidofovir\nZaire 5.722±0.2059 Vero E6 11\noutbreak in 2022\nSP2833 1.980±0.0579 Vero E6 11\nrdMPXVΔ96,158 10.45 CV-1-96,158 this study\nrdMPXVΔ96,158 52.45 HaCat-96,158 this study\nAntibody Strain EC value (μg/mL) Cell line referenc\n7D11\nrdMPXVΔ96,158 0.83 CV-1-96,158 this study\n3. Panel 2E, should have mock data too?\nWe apologize for this oversight. We did not include mock infection in Panel 2E when\ncomparing the replication of rdMPXV and authentic MPXV in dormice.\n4. (PDB code = 9FHS)” to PDB ID: 9FHS\nWe have corrected it.\n5.plasmid availability for the research community should be addressed as per the journal\nrequirements.\nThanks for the suggestion. We have added the statement in Data Availability section:\n\"Materials generated in this study are available upon request.\"\n6.Title should be modified to emphasize the replication-defective Mpox platform alone\nThanks for the suggestion. We agree and have modified the title as \"Development of a\nreplication-defective monkeypox virus platform for fundamental and therapeutic research\".\nReviewer #2 (Remarks to the Author):\nVery interesting work. The authors have developed a BAC-MPXV system with dual gene\n(OPG96 and OPG158) deletions, which under helper FPXV infection produced\nreplication-defective MPXV (rdMPXV) to be used as a tool for MPXV virology studies and\ndrug screening under standard BSL-2 lab settings, as compared to the WT MPXV which\nrequires BSL-3 containment. The new virus system was confirmed for safety and applied\nto screening a TargetMol library of 3185 compounds in trans-complementing cells using\na high-content imaging assay. From this pilot screen, one of the 3 initial hits,\ncompound G243-1720, showed sub-micromolar potency without cytotoxicity, and hence was further\nstudied in a wide range of rigorous experiments, and was characterized as a potent, non-\ntoxic and bioavailable, broad-spectrum antiviral against orthopox viruses, by\nmechanistically promoting the dimerization of p37 and blocking IMV wrapping. The paper\nis very well written, and the technical quality is very high. However, it’s less clear how\nsignificant the work really is.\nWe sincerely thank the Reviewer for the thoughtful and constructive feedback.\nMajor points\n1) Although the extremely well-characterized new drug hit G243-1720 showed sub-\nmicromolar potency in infected cells and demonstrated efficacy in mice, it’s substantially\nmuch less potent than tecovirimat: a) in CV-1-96,158 cells infected with rdMPXVΔ96,158,\nG243-1720 (EC50 = 140 nM) was 47-fold less potent than tecovirimat (EC50 = 3 nM); b)\nin vivo efficacy: G243-1720 twice daily i.g. at 45 mg / kg produced much less viral titer\nreduction than tecovirimat once daily i.g. at 50 mg / kg (1.56 log for G243-1720 vs 2.7 log\nfor tecovirimat).\n2) G243-1720 is mechanistically identical to tecovirimat with overlapping resistance\nprofile, diminishing its development value.\nWe sincerely thank the Reviewer for the insightful comments of 1 and 2 regarding the\ncomparative potency of G243-1720 and its shared target with tecovirimat.\nWe acknowledge that G243-1720 is less potent than the FDA-approved drug tecovirimat,\nboth in vitro and in vivo. We also acknowledge that its shared mechanism and resistance\nprofile with tecovirimat present a challenge for its direct clinical development.\nTecovirimat was the result of a massive screening (>350,000 compounds) followed by\nextensive medicinal chemistry optimization. In contrast, G243-1720 was identified from a\nmuch smaller, proof-of-concept screen of ~3,000 compounds using our novel rdMPXV\nplatform and has not undergone any chemical optimization. Despite this, its distinct\nchemical scaffold—different from both tecovirimat and IMCBH—and its sub-micromolar\npotency position it as a lead compound, not a final drug candidate. It provides a new\nchemical starting point for a future optimization to improve its bioavailability and efficacy.\nThe clinical performance of tecovirimat against MPXV has been more variable than\nanticipated, with one trial (PALM007) showing no significant benefit, potentially due to\nsuboptimal drug exposure. This underscores that the full therapeutic potential of F13L as\na target may not yet be realized. As only the third F13L-targeting compound ever identified,\nG243-1720 serves as a valuable chemical probe. Its unique structure and the resistant\nmutations it selects (e.g., I372N, A290V, D294V) provide new tools to study the structure\nand function of F13L, which could inform the development of next-generation inhibitors.\nFinally, the primary contribution of this work is the validation of the rdMPXV platform itself.\nThe efficient discovery of G243-1720 from a modest library demonstrates the power and\nreliability of our platform for high-throughput antiviral discovery in a clinically relevant,\nBSL-2 setting. In accordance with other Reviewers' suggestions, we have revised the\nmanuscript title to better highlight the establishment of this platform for fundamental and\ntherapeutic studies.\n3) One purported value of the rdMPXV is the safety when screening compounds for\ninhibitors impacting replication in wt cells, which amounts to a single replication cycle\nassay. It’s unclear why only a limited screen in trans-complementing cells was conducted.\nThe latter is a multiple rounds replication assay which increases the risk of producing\nMPXV via recombination.\nWe thank the Reviewer for raising this important regarding our screening strategy. We\nagree that using the rdMPXV particles in wild-type cells represents the safest single-cycle\nsystem. However, we chose to conduct the primary high-throughput screen in\ncomplementing cells (HaCaT-96,158) for the following reasons:\na. Our reporter system uses a viral late promoter to drive expression. In a single-\ncycle assay, this system cannot detect inhibitors of late-stage processes like virion\nassembly and egress—a class of compounds with therapeutic potential. A multi-\ncycle replication assay in complementing cells is essential to identify these\nvaluable inhibitors.\nb. A single-cycle assay presents challenges for a large-scale screen. A readout\nbased on abolition of signal is overly stringent, as even potent drugs like Cidofovir\ncannot achieve ideal inhibition in one cycle (Fig. 4D). While a readout based on\nreduced fluorescence intensity is possible, it is less robust and more susceptible\nto technical variation. The multi-cycle assay, with its wider dynamic range and\nsignal amplification, provides a much more reliable and reproducible platform for\nscreening thousands of compounds.\nFollowing the primary screen, we specifically employ the rdMPXV particles in wild-type\ncells for secondary assays to characterize hit compounds and differentiate their stage of\naction (e.g., entry/replication vs. post-replication).\nWe acknowledge the Reviewer's concern regarding recombination. The safety of our\nsystem is supported by our data demonstrating no replication in wild-type cells over\nmultiple passages (Supplementary Figure 2A, 2B, and 3D) and is consistent with the\nextensive established safety record of similar replication-defective vaccinia virus\nsystems16, 17, 18. We are confident the risk of generating replication-competent MPXV is\nminimal and effectively mitigated.\n4) It’s unclear what advantages this system offers over VACV as a surrogate virus for\ncompound screening and drug MOA characterization, as was the case in the\ndevelopment of tecovirimat. The assay is cumbersome, especially when the trans-\ncomplementing cells are used.\nWe thank the Reviewer for this question regarding the advantages of our rdMPXV\nplatform over the well-established VACV model.\nWhile VACV has been an invaluable surrogate model, it has undergone extensive\nlaboratory passaging, resulting in significant biological and pathogenic differences from\nthe circulating MPXV. Specifically, there are genes and proteins encoded by MPXV that\nare absent from VACV and which are potential targets for drug development. Given that\nthe ongoing global health threat is driven specifically by MPXV, our replication-defective\nMPXV system offers a direct and clinically relevant tool to study the authentic pathogen\nresponsible for the current outbreaks. This allows for a more accurate understanding of\nvirus-host interactions and therapeutic efficacy against the virus of primary concern.\nOur system is engineered to be both safe and representative. The dual-gene deletion\nensures a high safety profile, enabling work at a reduced biosafety level (BSL-2).\nImportantly, it faithfully recapitulates the entire viral lifecycle: it undergoes single-cycle\ninfection in wild-type cells and multi-round replication in complementing cells. As a proof\nof concept, the system accurately reproduced the known efficacy profiles of established\nanti-poxvirus agents (tecovirimat, cidofovir, 7D11 Ab), yielding EC₅₀ values consistent\nwith those reported for authentic viruses (we summarized in a table above, as suggested\nby Reviewer #1). This validates its reliability for high-throughput screening and\nmechanistic studies.\nThus, this platform does not seek to replace VACV but rather provides a specialized,\npowerful, and necessary tool for the research community to study MPXV biology and\naccelerate antiviral discovery with direct translational relevance to the ongoing mpox\noutbreak.\nMinor points\n5) IC50 should be EC50\nThank you for the suggestion. We have corrected “IC50” to “EC50” throughout the\nmanuscript.\n6) G243-1720 should be properly authenticated with 1H and 13C NMR, HRMS and HPLC.\nThese analytical data should be included in the supplemental file.\nThanks for the suggestion. We provided the synthesis route and analytical report as\nSupplementary Table 2, and stated this in lines 298-299. The structure of G243-1720 was\nverified using 1H nuclear magnetic resonance (1H-NMR) and electrospray ionization liquid\nchromatography-mass spectrometry (ESI-LCMS).\n7) References: for many citations only the first author is listed—this is not appropriate.\nWe apologize for this error. We have checked the citations in the text and corrected it.\nAs to reference list, it is automatically generated using Endnote software following the\njournal's formatting guidelines.\nReviewer #3 (Remarks to the Author):\nIn this communication, the authors describe the construction of a replication-defective\nMPXV and use it to screen a small-compound library for replication inhibitors. The most\npotent inhibitor, G243-1720, has similar biological activity and the same OPG57 target as\nTecovorimat, though a different chemical structure. Like Tecovirimat, G243-1720\ninhibited multiple orthopoxviruses including VACV. The work was very well done,\nexceptionally complete and the paper clearly written. Nevertheless, the screening could\nhave been done with VACV instead of the defective MPXV as a broad inhibitor is most\nadvantageous and G243-1720 does not seem to offer any advantage in potency over\nTecovirimat. Additionally, spontaneous mutants that resist the latter are also resistant to\nG243-1720.\nWe thank the Reviewer for the comments and concerns which was also raised by\nreviewer #2. We are pleased to provide the following clarifications.\nAs to the screening model, the primary goal of our study was to discover therapeutics\nspecifically against MPXV. While VACV is a valuable model virus, biological differences—\nsuch as host range and virulence—can limit the translational relevance of findings. Our\nreplication-defective MPXV platform allows for screening in a more clinically relevant\ncontext, enabling the direct identification of lead compounds against MPXV. We fully\nagree that VACV remains essential for validating broad-spectrum activity. Consequently,\nas part of our characterization of the lead compound G243-1720, we evaluated its efficacy\nagainst VACV and confirmed its potent, broad-spectrum anti-orthopoxviral activity (see\nFigure 3I and 3J).\nAs to the developmental prospects of G243-1720, we have provided a detailed response\nregarding the potential of G243-1720 to Reviewer #2. We agree that its shared\nmechanism with tecovirimat and current lower efficacy present challenges. However, we\nbelieve G243-1720 still retains value for three reasons: a. it represents a lead compound\nfor further medicinal chemistry optimization to improve its bioavailability and potency; b.\nit serves as a successful proof-of-concept for our rdMPXV platform in identifying antivirals\nthrough high-throughput screening; c. its distinct chemical scaffold makes it a valuable\nbiological probe for studying OPG57 / F13 protein structure and function.\nSpecific comments\n1. The deletion of two essential genes from MPXV, which increases safety, and\nestablishment of a complementing cell line were well described.\nWe thank the Reviewer for the positive feedback on our development of the replication-\ndefective MPXV platform.\n2. Is there an identifier or catalog number for the TargetMol compound library?\nThe compound library used was the TargetMol #LC00. We have added the catalog\nnumber to the Methods section in the revised manuscript.\n3. Does G243-1720 inhibit members of other poxvirus genera? For example, inhbitors of\nparapoxviruses would be useful.\nWe thank the Reviewer for this question. To evaluate the broad-spectrum potential of\nG243-1720, we have tested its efficacy against a panel of orthopoxviruses, including\nVACV, CPXV-BR, CPXV-E, CMLV, and RPXV, as presented in Figure 3I and 3J of the\nmanuscript. G243-1720 effectively inhibited all of these viruses.\nIn response to the reviewer's point regarding specificity, we also sought to test its activity\nagainst a more distantly related poxvirus. While parapoxviruses were unfortunately\nunavailable for testing, we evaluated fowlpox virus (FPV), a member of the distantly\nrelated Avipoxvirus genus. As expected for a F13-targeting compound, neither G243-\n1720 nor tecovirimat inhibited FPV replication, confirming the specificity of their\nmechanism for orthopoxviruses. In contrast, the viral DNA polymerase inhibitor cidofovir\nshowed activity at high concentrations (>10 µM), serving as a positive control. These new\ndata have been added to Supplementary Fig.5B in the revised manuscript.\nReviewer #4 (Remarks to the Author):\nThe generation of a replication defective model of MPXV infection is noteworthy and will\nlikely prove to be a valuable tool to support the identification of antiviral compounds. This\nwill be an important addition as it will allow additional laboratories to complete research\nagainst MPXV infections. Overall, the work that has been completed is sound and the\nconclusions are well-supported by the data. The statistical analyses appear to be\nappropriate and enough replicates have been completed to instill confidence in the results.\nThe methodology is clear and meets the standards in the field. There is enough\ninformation provided to accurately allow other researchers to reproduce this work.\nWe sincerely thank the Reviewer for the thorough and positive assessment of our\nmanuscript. We are particularly grateful for the Reviewer’s recognition of the potential\nvalue that our replication-defective MPXV platform holds the research community.\nThere are a few small things which could be clarified to benefit the readers.\n1. The authors demonstrate efficient infection of rdMPXV in several different cell lines,\nhowever, screening of antiviral compounds was only completed in HaCat-96,158 cells.\nThe rationale for choosing this cell line over the others is not clear and a rationale would\nhelp the readers understand why other cell lines were not utilized.\nWe thank the Reviewer for this question. Our decision to use HaCaT-96,158 cells for the\nprimary high-throughput screen was based on two rationales. First, HaCaT is an\nimmortalized human keratinocyte line, representing a physiologically relevant target for\nMPXV infection, which enhances the translational relevance of our findings. Second, as\nshown in Fig. 2H, parental HaCaT cells exhibited high susceptibility to rdMPXVΔ96,158\ninfection. The derived HaCaT-96,158 cell line thus provided a robust platform with a high\nsignal-to-noise ratio, supporting efficient multi-round viral replication, which is essential\nfor a reliable large-scale imaging-based screen. We have clarified this in the revised\nmanuscript (line 258-262, and 286).\n2. The authors identified Merimepodib as an inhibitor of MPXV during the screening\nassays. It is not clear whether this compound was an intentional positive control or\nwhether it was detected randomly in the compounds that were tested.\nWe thank the Reviewer for requesting this clarification. Merimepodib was not included as\nan intentional positive control; rather, it was identified as a hit from the unbiased\ncompound library screen. Its known mechanism of action (inhibition of Inosine-5′-\nmonophosphate dehydrogenase, IMPDH) along with its previously reported anti-MPXV\nactivity established it as a validating hit from our screening platform.\n3. The legend for Figure 6A does not specify what statistical analysis was used to\ncompare the dimerization activity of F243-1720.\nWe thank the Reviewer for pointing this out. The statistical analysis in Figure 6A (now\nFigure 6B in the revised manuscript) was an unpaired, two-tailed Student's t-test. We\nhave updated the figure legend in the revised manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Authors have addressed all the queries. I recommend the manuscript for publication"}, "author_response": ""}]

Decision letter