Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowii, and formononetin (FMN), an active constituent of SH003, exhibit anti-cancer and anti-oxidant properties. However, few studies have reported on the anti-melanoma activities of SH003 and FMN. This work aimed to elucidate the anti-melanoma effects of SH003 and FMN through the PD-1/PD-L1 pathway, using B16F10 cells and CTLL-2 cells. Results showed that SH003 and FMN reduced melanin content and tyrosinase activity induced by α-MSH. Moreover, SH003 and FMN suppressed B16F10 growth and arrested cells at the G2/M phase. SH003 and FMN also led to cell apoptosis with increases in PARP and caspase-3 activation. The pro-apoptotic effects were further enhanced when combined with cisplatin. In addition, SH003 and FMN reversed the increased PD-L1 and STAT1 phosphorylation levels induced by cisplatin in the presence of IFN-γ. SH003 and FMN also enhanced the cytotoxicity of CTLL-2 cells against B16F10 cells. Therefore, the mixture of natural products SH003 demonstrates therapeutic potential in cancer treatment by exerting anti-melanoma effects through the PD-1/PD-L1 pathway.Melanoma is the most invasive and lethal skin cancer. Recently, PD-1/PD-L1 pathway modulation has been applied to cancer therapy due to its remarkable clinical efficacy. SH003, a mixture of natural products derived from  Melanoma is the deadliest form of skin cancer, and it poses significant clinical and financial challenges . FollowiOxidative stress significantly impacts melanogenesis and subsequent melanoma formation . While mAstragalus membranaceus (Fisch.) Bunge, Angelica gigas Nakai, and Trichosanthes kirilowii (Maxim.), has been commonly used in East Asia. Studies have revealed that SH003 possesses anti-tumor effects in several cancer types such as breast [Astragalus membranaceus (Fisch.) Bunge [SH003, a combination of natural products derived from s breast , lung [1s breast , gastrics breast , cervicas breast , and pros breast , as wells breast . Additios breast ,20. Form.) Bunge , has bee.) Bunge ,23,24. F.) Bunge , colon [.) Bunge , and bre.) Bunge , and in .) Bunge ,28. Rega.) Bunge . HoweverAccordingly, we aimed to identify the anti-melanogenesis and anti-cancer activity of SH003 and FMN with emphasis on the immune checkpoint in the melanoma microenvironment. We investigated the potential of SH003 and FMN as anti-melanogenesis inhibitors and anti-cancer agents for combatting melanoma using B16F10 cells. Furthermore, the cytotoxic efficacy of cytotoxic T lymphocytes by SH003 and FMN was evaluated using CTLL-2 cells.Astragalus membranaceus (Fisch.) Bunge, Angelica gigas Nakai, and Trichosanthes kirilowii (Maxim.) in a 1:1:1 ratio as previously described [SH003 was extracted from escribed . FMN  and CTLL-2 cells  were cultured according to the manufacturer’s instructions. B16F10 cells were cultured in Dulbecco’s Modified Eagle’s Medium with 10% fetal bovine serum. CTLL-2 cells were cultured in RPMI 1640 medium containing 10% T-cell culture supplement with Con A  and 10% fetal bovine serum.5 cells) were treated with SH003, FMN, and α-melanocyte-stimulating hormone  for 72 h. For the melanin content assay, the cells were washed with phosphate-buffered saline and the resulting pellets were lysed with 150 μL of 1 N NaOH containing 10% dimethyl sulfoxide for 1 h at 80 °C. The cell suspension was quantified by means of a bicinchoninic acid protein assay kit . The melanin content in the quantified cell suspension was assessed by means of a colorimetric microplate reader . In the tyrosinase activity assay, the cells were lysed in 1% Triton X-100 for 2 h at 4 °C. The quantified pellets were then added to 10 μL of 10 mM L-DOPA  for 30 min at 37 °C. Tyrosinase activity was assessed by means of a microplate reader . The melanin content and tyrosinase activity were calculated as 100% of the α-MSH-treated group.The intracellular melanin content and tyrosinase activity were assessed with reference to previous works ,31,32. BCell viability was assessed by means of a 3--2,5-diphenyltetrazolium Bromide (MTT) assay referring to existing work . The absEach expression was detected with reference to existing work . The harCell cycle phase and DNA content were assessed using a propidium iodide staining and flow cytometry assay with a propidium iodide flow cytometry kit . The collected cells were fixed with 70% ice-cold ethanol and stored overnight at −20 °C. After washing with phosphate-buffered saline, the pellets were then re-suspended in phosphate-buffered saline containing 50 μg/mL of propidium iodide and 100 μg/mL of ribonuclease and incubated for 30 min at room temperature. The cells were then subjected to analysis using a FACSCalibur flow cytometer (BD Biosciences).Apoptotic cells were measured using a FITC Annexin V apoptosis detection kit with propidium iodide . The collected cells for apoptosis analysis were double-stained with FITC-conjugated Annexin V and propidium iodide in a binding buffer for 30 min. The analysis was performed using a FACSCalibur flow cytometer.® Green Master Mix (Thermo Fisher Scientific) on a Real-Time PCR System . Primers for genes  were synthesized at Bioneer, Inc.Total RNA was prepared by means of an extraction kit . Reverse transcription and qPCR were performed using a cDNA synthesis kit  and Power SYBR® 647 secondary antibodies (abcam).Immunofluorescent staining was conducted as previously described  with antTo determine the cytotoxic T lymphocyte activities of CTLL-2 cells, a cell-mediated cytotoxicity assay was conducted using a cell cytotoxicity assay kit (DoGenBio). CTLL-2 cells (effector cells) were cultured with B16F10 cells (target cells) at an effector-to-target cell ratio of 20:1. The cells were exposed to SH003 and FMN for 24 h. The lactate dehydrogenase release was analyzed in the cell-free supernatants. Additionally, a viability assay was performed using the MTT assay on the adhered viable target cells.post hoc test. A Student’s unpaired t-test was used to compare between two independent groups. A p-value below 0.05 was considered statistically significant .Data are shown as the mean ± standard error of the mean (SEM). Multiple comparisons were conducted using one-way analysis of variance followed by Tukey’s p < 0.05, p < 0.05, First, we assessed the effects of SH003 and FMN on melanin content in α-MSH-stimulated B16F10 cells. α-MSH is one of the most common reagents used to stimulate pigmentation . The α-Mp < 0.05). To further explore the molecular mechanism of this effect, we investigated the expression of signaling molecules by immunoblots. Both SH003 and FMN significantly enhanced the levels of cleaved PARP and caspase-3 compared with the control group in B16F10 cells, .It has been known that cisplatin is a good chemotherapy drug for the treatment of various tumors . To verip < 0.05, p < 0.05, p < 0.05, To assess the effect of SH003 and FMN on apoptosis in B16F10 cells, a cell apoptosis assay was carried out. As expected, SH003 and FMN significantly augmented the apoptosis of B16F10 cells compared with the control group . Subsequently, the mechanism for the modulation of PD-L1 expression was further explored. p < 0.05).IFN-γ is involved in tumor immune evasion by stimulating the expression of PD-L1 on tumor cells, suppressing the effector functions of cytotoxic T lymphocytes, and helping tumor cells evade immune destruction . Thus, wp < 0.05). Subsequently, CTLL-2 cells downregulated the cell viability of B16F10 cells, and SH003 and FMN further suppressed the cell viability of B16F10 cells decreased by CTLL-2 cells  Bunge, Angelica gigas Nakai, and Trichosanthes kirilowii (Maxim.), as the functional foods that comprise SH003, possess not only high nutritional value but also exhibit anti-cancer and anti-oxidant properties [Carcinogenesis is strongly associated with oxidative stress and nutritional status . The nutoperties ,42,43,44operties . Thus, tAstragalus membranaceus (Fisch.) Bunge, has been reported to have anti-melanogenesis effects in an animal model [Angelica gigas Nakai, has been reported to possess protective effects against hyperpigmentation by inhibiting melanin synthesis and tyrosinase activity of B16F10 melanoma cells [Angelica gigas Nakai, showed anti-melanogenesis effects by decreasing the melanin synthesis and tyrosinase activity of B16F10 melanoma cells [Trichosanthes kirilowii (Maxim.), as a regulatory component on melanin synthesis and tyrosinase activity of B16F10 melanoma cells. Tsao et al. [Trichosanthes kirilowii (Maxim.), displayed cytotoxic activity against melanoma cells. Thus, the variations might be observed because SH003, being a mixture of Astragalus membranaceus (Fisch.) Bunge, Angelica gigas Nakai, and Trichosanthes kirilowii (Maxim.), contains a range of active compounds such as calycosin, decursin, nodakenin, cucurbitacins, and trichosanthin, whereas FMN is just one of the active compounds in SH003.The variations were observed in the anti-melanogenic and anti-cancer effects between SH003 and FMN in our work. Calycosin, an isoflavone of al model . Decursima cells . Decursima cells . Nodakenma cells . Oh et ama cells  suggesteo et al.  revealedMelanin plays a critical role in melanoma pathology by promoting malignant transformation of melanocytes . The melPD-L1 expression in tumor cells correlates with tumor immune evasion and poor clinical outcomes . AccumulCisplatin augments PD-L1 expression in tumor cells ,37. ThusThe current work is the first to characterize the anti-melanogenesis and anti-melanoma properties of SH003 and FMN. The principal findings of this study are as follows: (i) SH003 and FMN treatments inhibit melanogenesis. (ii) SH003 and FMN promote apoptosis in B16F10 cells, accompanied by elevated levels of active PARP and caspase-3. (iii) The combination of cisplatin with SH003 or FMN enhances their respective pro-apoptotic effects. (iv) SH003 and FMN reduce PD-L1 expression by downregulating STAT1 phosphorylation. (v) SH003 and FMN augment the cytotoxicity of cytotoxic T lymphocytes against B16F10 cells. Therefore, SH003 has anti-melanoma properties, simultaneously enhancing the function of cytotoxic T lymphocytes. These results propose that the natural product mixture SH003 holds promise as a novel approach in the prevention of melanoma progression. However, future studies should explore the comparative advantages of SH003 and FMN in clinical application.


In May of 2020, the Centers for Disease Control and Prevention defined a new diagnosis, Multisystem Inflammatory Syndrome in Children (MIS-C), described as a Kawasaki-like illness and related to the SARS-CoV-2 virus. Patients with this syndrome met criteria for inflammation, along with multiple organ system involvement. Clinically distinguishing between Kawasaki Disease (KD) and MIS-C is challenging due to significant overlap between the two disease entities. Furthermore, approximately 25-50% of MIS-C cases also meet criteria for KD. Currently there is very limited data describing specific differences in laboratory markers between KD and MIS-C to guide clinicians in diagnosis and management. This study aims to assess if quantitative laboratory data markers help distinguish between KD and MIS-C.We conducted a retrospective study of patients hospitalized within two network pediatric hospitals with the diagnosis of MIS-C [M35.81] or Kawasaki Disease [M30.3] between January 1, 2019 and March 31, 2021. Children with an alternate diagnosis at discharge were excluded. Demographic characteristics and laboratory data were recorded and analyzed.p=0.001). Patients with KD presented with higher median white blood cell (WBC) count, absolute lymphocyte count, and platelet count compared to those with MIS-C (p< 0.001). A higher alanine aminotransferase (ALT) was also noted (p=0.01). Patients with MIS-C were noted to have higher median ferritin levels and C-reactive protein (CRP) (p< 0.001) (Table 1).A total of 91 pediatric patients were included in the study: 40 with MIS-C and 45 with KD. Patients with MIS-C were significantly older with a median age 5.3 years, compared to 2.4 years for KD (Both MIS-C and KD are autoinflammatory disease processes that have significant overlap. Identifying differences in initial laboratory values upon presentation can lead to earlier diagnosis. A normal ferritin level may indicate KD.All Authors: No reported disclosures


Coronaviruses (CoVs), including severe acute respiratory syndrome CoV (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2, produce double-stranded RNA (dsRNA) that activates antiviral pathways such as PKR and OAS/RNase L. To successfully replicate in hosts, viruses must evade such antiviral pathways. Currently, the mechanism of how SARS-CoV-2 antagonizes dsRNA-activated antiviral pathways is unknown. In this study, we demonstrate that the SARS-CoV-2 nucleocapsid (N) protein, the most abundant viral structural protein, is capable of binding to dsRNA and phosphorylated PKR, inhibiting both the PKR and OAS/RNase L pathways. The N protein of the bat coronavirus (bat-CoV) RaTG13, the closest relative of SARS-CoV-2, has a similar ability to inhibit the human PKR and RNase L antiviral pathways. Via mutagenic analysis, we found that the C-terminal domain (CTD) of the N protein is sufficient for binding dsRNA and inhibiting RNase L activity. Interestingly, while the CTD is also sufficient for binding phosphorylated PKR, the inhibition of PKR antiviral activity requires not only the CTD but also the central linker region (LKR). Thus, our findings demonstrate that the SARS-CoV-2 N protein is capable of antagonizing the two critical antiviral pathways activated by viral dsRNA and that its inhibition of PKR activities requires more than dsRNA binding mediated by the CTD.IMPORTANCE The high transmissibility of SARS-CoV-2 is an important viral factor defining the coronavirus disease 2019 (COVID-19) pandemic. To transmit efficiently, SARS-CoV-2 must be capable of disarming the innate immune response of its host efficiently. Here, we describe that the nucleocapsid protein of SARS-CoV-2 is capable of inhibiting two critical innate antiviral pathways, PKR and OAS/RNase L. Moreover, the counterpart of the closest animal coronavirus relative of SARS-CoV-2, bat-CoV RaTG13, can also inhibit human PKR and OAS/RNase L antiviral activities. Thus, the importance of our discovery for understanding the COVID-19 pandemic is 2-fold. First, the ability of SARS-CoV-2 N to inhibit innate antiviral activity is likely a factor contributing to the transmissibility and pathogenicity of the virus. Second, the bat relative of SARS-CoV-2 has the capacity to inhibit human innate immunity, which thus likely contributed to the establishment of infection in humans. The findings described in this study are valuable for developing novel antivirals and vaccines. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic . AlthougInnate immunity provides the first line of defense against virus infection. To establish a productive infection, a virus must be capable of evading the host’s innate immunity. During the replication cycle of a virus, the virus produces pathogen-associated molecular patterns (PAMPs), such as double-stranded RNA (dsRNA), which are detected by the pattern recognition receptors (PPRs)  of the host . The intCoronaviruses produce dsRNA during their replication, which can activate antiviral proteins such as PKR and RNase L –11. SARS–13–Vaccinia virus (VACV), the prototype poxvirus, encodes two proteins, E3 and K3, to suppress dsRNA-activated PKR and/or RNase L antiviral pathways. E3, a dsRNA binding protein, inhibits both PKR and RNase L, while K3 (eIF2α homolog) modulates PKR antiviral activity by interfering with the phosphorylation of the PKR substrate eIF2α , 17. PreIn this investigation, we expressed the SARS-CoV-2 N protein in VACVΔE3ΔK3 and demonstrated that the SARS-CoV-2 N protein can restore virus replication in cells by suppressing both PKR and RNase L antiviral activities. Furthermore, we identified the functional domains of the N protein that are essential for suppressing PKR and RNase L. Finally, we found that the N protein of the bat coronavirus (bat-CoV) RaTG13, the closest relative of SARS-CoV-2, is also capable of inhibiting human PKR and RNase L, indicating that the bat-CoV has the evasion measures to counteract human innate immunity.A previous study reported that SARS-CoV-2 infection leads to the activation of PKR and RNase L in A549 cells expressing human ACE2 (A549/huACE2) at 48 h postinfection (hpi). However, virus replication was not affected at either 24 or 48 h postinfection . It is nAs shown in 21–The SARS-CoV-2 N gene driven by a vaccinia virus early/late promoter, mH5 , was insNext, we examined the dsRNA binding capacity of SARS-CoV-2 N. Previously, we examined VACV E3 and poxvirus E3 orthologs for their dsRNA binding using poly(I·C)-conjugated Sepharose beads . Here, wThe VACV E3 protein is a potent inhibitor of PKR and OAS/RNase L antiviral activities. When the gene encoding the E3 protein is deleted, the VACV deletion mutant virus induces PKR phosphorylation at threonine 446 and activates OAS/RNase L, as shown by the degradation of rRNAs. As shown in Coronavirus N proteins have a modular organization consisting of two structural domains, the N-terminal domain (NTD) and the C-terminal domain (CTD), flanked by three intrinsically disordered regions (IDRs), the N-terminal IDR (N/IDR), the linker region (LKR), and the C-terminal IDR (C/IDR) , 30. We The proteins of all of the truncated SARS-CoV-2 N mutants were expressed from VACVΔE3ΔK3-infected A549/dko cells at comparable levels  and B. IThe dsRNA binding capacity and the inhibition of the PKR activation and RNase L activity of the truncated SARS-CoV-2 N mutants were analyzed as described above. As shown in Next, we examined if the LKR and CTD themselves could perform the function of inhibiting PKR and OAS/RNase L. The individual domains  and a dual LKR-CTD (LKR+CTD), which were driven by the vaccinia virus early/late promoter mH5, were inserted into VACVΔE3ΔK3 as described above . AlthougSimilar to the full-length N protein, the LKR+CTD was fully functional in rescuing the replication of VACVΔE3ΔK3 in A549/wt, A549/PKRko, and A549/RNaseLko cells . FurtherThe CTD and the LKR+CTD bound to poly(I·C), while the NTD could not . In A549The closest coronavirus to SARS-CoV-2 is a bat coronavirus (bat-CoV), RaTG13, with 96.2% genome sequence identity . For theVACVΔE3ΔK3 expressing the bat-CoV RaTG13 N protein replicated at a level comparable to that of VACVΔE3ΔK3 expressing SARS-CoV-2 N in A549/PKRko, A549/RNaseLko, and A549/wt cells, while VACVΔE3ΔK3 did not grow . SimilarA previous study reported that SARS-CoV-2 N could bind to PKR and G3BP1 using an ectopic-overexpression assay . Here, wAs shown in Previous studies have shown that SARS-CoV-2 N binds to G3BP1 and that the N/IDR and CTD are required for binding . In our PKR and OAS/RNase L are major components of innate immunity that inhibit virus replication by shutting off viral protein synthesis , 33. OncCoronavirus N is a multifunctional protein , 30. In 13–Coronavirus N is organized into five distinct domains, including three IDRs  and two structural domains (NTD and CTD). Previous studies have shown that the NTD, LKR, and CTD can bind to viral RNA , 30. In 38–It has been reported that SARS-CoV-2 N can physically interact with PKR and G3BP1 to inhibit SG assembly using an ectopic-expression system , 14. We Based on epidemiological and genomic sequence data, it is generally accepted that the origin of SARS-CoV-2 was a zoonotic spillover from an animal source, although the exact animal reservoir for the virus has not been identified , 47. TheIt is known that coronaviruses encode proteins to inhibit dsRNA-activated pathways. Some coronaviruses encode virus-specific proteins to inhibit dsRNA-activated antiviral pathways. For example, the murine coronavirus nonstructural protein ns2 and MERS-CoV ns4b can cleave 2′,5′-oligoadenylate and inhibit the OAS/RNase L pathway , 50. TheThe coronavirus N protein is the most abundant and highly antigenic structural protein . Several58–In this study, we demonstrate that SARS-CoV-2 N has the capacity to inhibit two important innate antiviral pathways, PKR and OAS/RNase L. It is logical that SARS-CoV-2 N plays an important role in the high transmissibility and pathogenicity of SARS-CoV-2, which are two viral determinants of the COVID-19 pandemic. Based on our findings that the LKR and CTD of the N protein are required to inhibit PKR and OAS/RNase L activities, we are investigating the domain/motif of the N protein that is essential only for PKR and OAS/RNase L inhibition but not for virus replication. This will allow us to analyze the immune-modulating function of the N protein in the context of SARS-CoV-2 infection. The findings in this study are valuable for designing novel antivirals targeting the N protein and for modifying the N protein as a protective antigen for the development of N protein-based vaccines.2. The vaccinia virus (VACV) Western Reserve (WR) strain and the E3L and K3L double-deletion mutant (VACVΔE3ΔK3) were described previously , A549 RNase L knockout (A549/RNaseLko), and A549 PKR/RNase L double-knockout (A549/dko) cells were provided by B. Moss  . A549 ceeviously , and SAReviously . The VACMN908947). The modular structure of the SARS-CoV-2 N protein was based on an alignment with a SARS virus: the N-terminal intrinsically disordered region (N/IDR) (amino acids 1 to 44), the N-terminal domain (NTD) (amino acids 45 to 180), the linker region (LKR) (amino acids 181 to 247), the C-terminal domain (CTD) (amino acids 248 to 364), and the C-terminal IDR (C/IDR) (amino acids 365 to 419)  , 30. The to 419) . The N min vitro , as we pCell monolayers in a four-chambered slide (Thermo Fisher) were infected with the virus at a multiplicity of infection (MOI) of 0.5 and incubated at 37°C. At 24 h postinfection (hpi), cells were washed twice with phosphate-buffered saline (PBS) and fixed using a 3% paraformaldehyde–PBS solution for 10 min at room temperature. The fixed cells were washed 3 times with PBS for 3 min and then permeabilized using a 0.2% Triton X-100–PBS solution for 5 min at room temperature, with gentle rotation. Following another 3 washes with PBS (1 min each wash), the cells were incubated with a blocking buffer  at 4°C for 1 h. The cells were then incubated with the primary antibody , diluted 1/200 in blocking buffer, at 4°C for 2 h. The cells were washed 3 times with PBS for 3 min each wash and incubated with a fluorescence-conjugated secondary antibody specific for the primary antibody , diluted 1/1,000 in blocking buffer, at 4°C for 1 h. The cells were washed 3 more times with PBS for 3 min each. The chambers were removed from the slides and rinsed with water. Prolong Diamond antifade mountant with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen) was added, and the slides were covered with a coverslip and incubated at room temperature in the dark overnight. Slides were imaged using a confocal microscope  at a ×40 magnification.Cell monolayers were infected with the viruses listed in the related figure legends at an MOI of 5 and incubated at 37°C for 12 h (VACV) or 24 or 48 h (SARS-CoV-2) after infection. The total RNA was extracted using the RNeasy minikit (Qiagen) according to the manufacturer’s instructions. RNA concentrations were determined using the Qubit 4 system (Thermo Fisher) and diluted to a final concentration of 5 ng/μL. Ten nanograms of the RNA samples was analyzed by RNA electrophoresis using the high-sensitivity RNA ScreenTape system (Agilent) according to the manufacturer’s instructions, and rRNA was visualized using the 2200 TapeStation system (Agilent).g for 10 min, and an aliquot was removed and used as a prepulldown control. The rest of the supernatant was incubated with the poly(I·C)-conjugated Sepharose beads at room temperature for 2 h with end-over-end rotation. The beads were washed 3 times with nondenaturing lysis buffer to remove unbound proteins. Bound proteins were released from the poly(I·C) beads using an SDS-PAGE loading buffer  with boiling at 95°C for 10 min. Samples were analyzed using Western blotting as described previously (Poly(I·C) (Sigma-Aldrich)-conjugated Sepharose beads  were prepared as previously described . BHK21 ceviously .g at 4°C for 5 min. To prepare the cell lysate containing nonphosphorylated or phosphorylated PKR, wild-type A549 cells were mock infected (for nonphosphorylated PKR) or infected with VACVΔE3ΔK3 (for phosphorylated PKR) for 8 h, and the cell lysate was then prepared as described above for the N and mutant cell lysates. Coimmunoprecipitation was performed using the Dynabeads-protein G immunoprecipitation kit (Invitrogen) according to the manufacturer’s instructions. A mouse anti-FLAG antibody (Sigma-Aldrich) was used to bind to SARS-CoV-2 N and its mutants. The final precipitated protein complex was eluted from the Dynabeads-protein G using SDS sample buffer as described above and analyzed using Western blotting as described previously (For the preparation of the SARS-CoV-2 N and mutant protein cell lysates, A549/dko cells were infected with the corresponding viruses for 8 h. Following one wash with PBS, the cell lysate was collected using the nondenaturing lysis buffer described above, and nonsoluble cell debris was removed by centrifugation at 2,000 × eviously .Confluent cell monolayers in a 12-well tissue culture plate were infected with the viruses and collected at the time points indicated in the related figure legends. SDS-PAGE, membrane transfer, antibody blotting, and protein band detection were performed as described previously . Total P


Its evolution can lead to potential zoonotic transmission, seasonal epidemics, and the emergence of drug-resistant mutants. To gain comprehensive insights into the mutational effects and drug resistance profiles of NA, we employed a high-throughput profiling system to quantify the replication capacity of NA mutants at the single-nucleotide level in mouse lung tissues. The fitness of NA mutants is generally correlated with natural mutation occurrence and is constrained by both the requirement to maintain protein stability and NA function. Leveraging this system, we profiled the drug resistance to the three most commonly used neuraminidase inhibitors (NAIs): zanamivir, oseltamivir, and peramivir. In addition to identifying previously reported drug resistance mutations, we validated novel mutants. Notably, we identified an allosteric mutation that confers resistance to all three drugs, which may affect drug binding by interfering with the tetramerization of NA. Moreover, the fitness cost associated with drug-resistant mutations may limit their widespread dissemination. In summary, we provided a parallel characterization of NA’s fitness and drug resistance landscape in an in vivo is essential for comprehending the evolutionary constraints and making informed choices regarding drug selection to combat resistance in clinical settings. In the current study, we established an efficient deep mutational screening system in mouse lung tissues and systematically evaluated the fitness effect and drug resistance to three neuraminidase inhibitors of NA single-nucleotide mutations. The fitness of NA mutants is generally correlated with a natural mutation in the database. The fitness of NA mutants is influenced by biophysical factors such as protein stability, complex formation, and the immune response triggered by viral infection. In addition to confirming previously reported drug-resistant mutations, novel mutations were identified. Interestingly, we identified an allosteric drug-resistance mutation that is not located within the drug-binding pocket but potentially affects drug binding by interfering with NA tetramerization. The dual assessments performed in this study provide a more accurate assessment of the evolutionary potential of drug-resistant mutations and offer guidance for the rational selection of antiviral drugs.NA is a crucial surface antigen and drug target of influenza A virus. A comprehensive understanding of NA’s mutational effect and drug resistance profiles  Influenza viruses represent an ongoing public health threat, causing acute respiratory disease with high morbidity and mortality . The priin vivo context. However, in contrast to in vitro screening, which can accurately control factors such as the number of cells and multiplicity of infection (MOI), in vivo screening encounters a stronger infection bottleneck. Therefore, optimizing the complexity of the library and the infection scheme becomes crucial to ensure reliable profiling results and maintain library complexity in vivo.We and others have integrated the mutagenesis system with high-throughput sequencing to explore the mutational effects of HA in cell culture. These investigations have characterized antibody escape mutations, reconstructed the evolutionary history of antigenic epitopes, and identified potential vaccine targets on HA , 5, 6. HTo date, NA is the primary drug target for influenza virus treatment. Neuraminidase inhibitors (NAIs), such as zanamivir (ZA), oseltamivir (OS), and peramivir (PE), have become widely used antiviral agents for the management and prevention of influenza infections –18. Thin vivo context represents a more biologically relevant condition and provides information on immune-sensitive mutations.In this study, we established a deep mutational profiling system for NA. By introducing a library of single-nucleotide mutants of NA into mouse lung tissues and employing deep sequencing, we quantified the replication fitness of each mutant. To ensure the reliability of our fitness profiling data set, we controlled the complexity of the mutant library, the infection dose, and the timing of sample collection from infected mouse lungs. Compared with screening in cell culture, fitness profiling in the in vivo. We identified and validated several new drug-resistant mutants by comparing the mutational effects on viral replication under both drug selection and no drug selection conditions. Interestingly, we found an allosteric drug-resistant mutation (V205I) outside the drug-binding pocket on the NA tetramerization interface. Finally, we analyzed the global dynamics of NA variants and noted that the frequency of NAI-resistant mutations did not increase significantly over the last decade, probably due to the impact of these mutations on viral replication fitness.Using this system, we characterized the resistance profiles of OS, ZA, and PE in vivo, we established a deep mutational screening system in a mouse model  in mice peaked on days 2 to 4 after infection, so we harvested lung tissues on day 3 to select the virus libraries (10 RF = 0), while a clear separation in the distribution of RF scores was observed between synonymous mutations and nonsense mutations . Amino acid positions that incurred a high fitness cost upon mutation tended to be highly conserved. The correlation between natural occurrence frequency and RF score was also significant at each mutation level (P < 0.0001). However, there were instances where discrepancies emerged between entropy and RF scores.To examine if the fitness of NA mutants correlated with their natural occurrence, we analyzed the sequence diversity of 12,667 human N1 NA sequences (2010–2020) from the Influenza Research Database (IRD). The entropy at each site was generally correlated with the RF score measured in our screening  showed relatively high entropy but were deleterious upon mutation. This discrepancy could be attributed to compensatory mutations in other influenza strains or selection pressures beyond NA activity alone, such as the interplay between HA and NA activity 30. On tTo explore the biophysical basis of mutational effects, we performed protein stability simulations using PyRosetta , 32. At in vivo screening may also reflect the ability of mutants to effectively spread within the lung environment under the selective pressure exerted by the host immune system. To directly demonstrate this, we conducted an additional selection of the same amplified viral library stock in A549 cell culture  treatment . The viral copy number was examined at 24 h post-infection, and the IFN sensitivity was calculated as the fold decrease in viral copy number upon IFN treatment. Notably, one of these mutations, V67G, was significantly more sensitive to IFN selection than WT viruses . Alternatively, if multiple mutations in a single residue displayed a resistance phenotype , they were also considered resistant. A total of 99 mutations were identified as resistant to OS .Comparing the drug-resistant mutations across the three NAIs, we noticed some positions exhibiting resistance to multiple drugs . As the Besides the literature-reported drug-resistant mutations, we aim to validate new mutations that could potentially confer reduced sensitivity to NAIs. We picked 11 nonreported mutations that confer resistance to 2, or all tested NAIs, and constructed them individually. The six mutations with RF scores less than 0.1 in the control condition were too deleterious to obtain sufficient virus for downstream drug-resistance assays . The recFurthermore, we tried to elucidate the potential mechanism of drug resistance of the two new mutations from a structural perspective. Similar to other well-recognized drug-resistant mutations, L134 is located at the intra-hydrophobic core, consisting of eight functional residues . The side chain of the mutation L134M may directly alter the catalytic pocket and consequently reject the drug binding . InteresConsidering the mounting prescription of NAIs for early influenza infections, it is necessary to monitor the circulating NAI-resistant variants at the population level –46. Toin vitro and in vivo settings , we observed that nearly 97% of mutations conferring resistance to the three NAIs exhibited compromised replication capacity , includisettings .In the current study, using a mouse model, we employed a high-throughput genetics platform to map the fitness cost and drug resistance of single-nucleotide mutations of influenza A NA. Our findings revealed a strong correlation between fitness effects and the natural occurrence of mutations, which could be attributed to protein stability and functional constraints. The drug resistance profiling confirmed previously reported mutations and validated new mutations, including one with a potential allosteric regulatory function. Finally, we analyzed the dynamics of recent occurrences of drug-resistant mutations and elucidated their limited global spread by considering their fitness cost in the absence of drug pressure.in vitro systems , which could result in false positives in detecting drug resistance mutations. Finally, we constructed the NA library using the A/WSN/33 (H1N1) virus. Different genetic backgrounds may exhibit varying profiles of drug-resistant mutations, particularly when compensatory mutations are involved .Influenza A/WSN/33 WT and the mutant viral libraries were generated using the eight-plasmid reverse genetics system . HEK-293CAGGTCTCATAAATGAATCCAAACCAGAAAAT (flu6-M1F-BsaI), CAGGTCTCATGGAACCAATTCTTATGCCATTG (flu6-M1B-BsaI), CAGGTCTCAGGGTGGGCTATACACAGCAAAGA (flu6-M2F-BsaI), CAGGTCTCAGCCAGCCCATTCCATCATGACAT (flu6-M2B-BsaI), CAGGTCTCATCGGTTGCTTGGTCAGCAAGTGC (flu6-M3F-BsaI), CAGGTCTCACATTCAACTCTATCGATTTAGTA (flu6-M3B-BsaI), CAGGTCTCATTCAAGATCGAGAAGGGGAAGGT (flu6-M4F-BsaI), CAGGTCTCAATGAAAATCCCTTTACTCCGTTT (flu6-M4B-BsaI), CAGGTCTCAGGCCCAGTGTCTGCTGATGGAGC (flu6-M5F-BsaI), and CAGGTCTCAGTAGCCCCCTGATTAATTCAACC (flu6-M5B-BsaI).Influenza A/WSN/33 NA mutant plasmid libraries were generated as previously described . In brieCACGTCTCATTTAAACTCCTGCTTTCGCTCCC (flu6-V1F-BsmBI), CACGTCTCTTCCAAAGGAGATGTTTTTGTCAT (flu6-V1B-BsmBI), CACGTCTCCACCCACGGATGGGACAAAGAGAT (flu6-V2F-BsmBI),The corresponding vectors for each of the five mutant libraries were generated by PCR using the following primers: CACGTCTCCTGGCTAACAATCGGAATTTCTGG (flu6- V2B-BsmBI),CACGTCTCACCGATTCAAACCTTGAATTGTAC (flu6- V3F-BsmBI), CACGTCTCGAATGCACCTAATTCTCACTACGA (flu6- V3B-BsmBI), CACGTCTCTTGAAAATTTTGTACGAGGCCAGC (flu6- V4F-BsmBI), CACGTCTCTTCATATAAGTATGGCAATGGTGT (flu6- V4B-BsmBI), CACGTCTCGGGCCACAGCTGCCTGTTCCATCT (flu6- V5F-BsmBI), and CACGTCTCGCTACCTGAGGAGGACGCAATCTG (flu6- V5B-BsmBI).The PCR product was digested by DpnI (New England Biolabs) to remove the input plasmid.The amplified segment was gel purified for each small library, BsaI digested, ligated to the BsmBI digested vector, and transformed into MegaX DH10B T1R cells (Life Technologies). As each small library was expected to have ~1,000 single mutations, ~50,000 bacterial colonies were collected to ensure ~15× coverage for each mutant (considering ~33% of the library is single-nucleotide mutations). The plasmid libraries were extracted using PureLink HiPure Plasmid Filter Midiprep Kit (Invitrogen).50) were measured using MDCK cells. To further amplify the mutant viral libraries, ~10 million MDCK cells were infected with the individual mutant virus libraries at an MOI of 0.1 (library coverage ~300-fold). Cells were washed with PBS three times at 2 h post-infection. Viruses were collected 48 h post-infection from the supernatants.To generate the mutant viral library, ~30 million 293T cells were transfected with each plasmid library  and the other 7 plasmids encoding WT viral proteins . Transfections were performed using Hieff TransTM Liposomal Transfection Reagent (Yeasen). The medium was replaced at 12 h post-transfection. Viruses were collected 60 h post-transfection. Viral titers  were lightly anesthetized with inhaled isoflurane and then inoculated intranasally with 1.0 × 105 TCID50 of the NA mutant viral libraries in 50 µL (library coverage ~30-fold). At 6 h post-infection, NAIs were administered  to each mouse group. The control group of mice was left untreated in parallel. The animals were monitored daily for clinical signs of disease, body weight changes, and survival and were administered three NAIs daily for 3 days post-infection. The dosage was the same as that on day 1. Mice that lost over 30% of their initial body weight were euthanized. Then, the lung of each mouse was harvested on day 3 post-infection.We determined the dose-response curve of each drug to ensure that the selection pressure was consistent among the three drugs. We selected a relatively high drug concentration . Groups of 6- to 8-week-old female BALB/c mice (SLACOM)  and ACC ATT GTT CCA ACT CCT TT (reverse).Each mouse lung sample was mixed with 1 mL of TRIzol reagent (Cwbiotech) and homogenized at 4°C. Viral RNA was extracted using TRIzol reagent and reverse transcribed using HiScript II Q RT SuperMix for qPCR (Vazyme). Quantitative real-time PCR was performed using 2× T5 Fast qPCR Mix (SYBR Green I) (Tsingke). For viral copy number, a series of standard samples ranging from 1 × 106 genome copies were used to amplify the mutated segment. The primers used for amplification are as follows: GAGTTTCTGGAGATCCAAACCAGAAAAT (flu6-S1F-BpmI), TCCTTTCTGGAGAATTCTTATGCCATTG (flu6-S1B-BpmI), TCCGTGCTGGAGCTATACACAGCAAAGA (flu6-S2F-BpmI), TGTTAGCTGGAGCATTCCATCATGACAT (flu6- S2B-BpmI), TTGAAT CTGGAG CTTGGTCAGCAAGTGC (flu6- S3F- BpmI), AGGTGC CTGGAG CTCTATCGATTTAGTA (flu6- S3B-BpmI), AAATTT CTGGAG TCGAGAAGGGGAAGGT (flu6- S4F- BpmI), CTTATA CTGGAG TCCCTTTACTCCGTTT (flu6- S4B-BpmI), GCTGTG CTGGAG TGTCTGCTGATGGAGC (flu6- S5F-BpmI), and CTCAGG CTGGAG CCTGATTAATTCAACC (flu6- S5B-BpmI).For each mouse sample, at least 1 × 10The amplified regions were BpmI digested and ligated with the sequencing adaptor, which had a three-nucleotide multiplexing ID to distinguish between samples.Deep sequencing was performed with Illumina sequencing NovaSeq PE250. The three-nucleotide population IDs were used to de-multiplex raw sequencing reads. The same table of the multiplexing index for each sample is provided in Sequencing errors were corrected by filtering out unmatched forward and reverse reads. Mutations were identified by comparing sequencing reads with the WT sequence. The RF score was calculated for each point mutation, and only mutations with frequencies above 0.01% in the plasmid library were reported.whereFor a particular drug, each mutation’s drug resistance score (W) was computed as the proportion of the RF score with drug selection to that without drug selection.t-test, P < 0.001), or multiple mutations in one residue with min W >1.5 for all three replicates.We define a residue as functional for viral replication if more than 50% of its mutations have RF <0.1 and ΔΔG <0. A residue is essential if the frequency of its mutations with RF <0.1 is >80%. Under each drug selection condition, drug-resistant mutations include those with a significantly higher W than synonymous mutations (two-tailed The protein stability of a single mutation on NA (ΔΔG) was predicted by PyRosetta . The PDBAmino acid sequences of the influenza NA protein were downloaded from the IRD. IRD is an open online resource funded by the US NIH/NIAID that facilitates the retrieval, analysis, and visualization of influenza virus data. Shannon entropy was used to quantify the sequence diversity at each amino acid site.50) were measured with MDCK cells. To amplify the mutant viruses further, ~10 million MDCK cells were infected with individual mutant viruses at an MOI of 0.1. Cells were washed with PBS three times at 2 h post-infection. The viruses were collected 48 h post-infection from the supernatants. To confirm the genotype of the mutant viruses, each mutant viral RNA was extracted and reverse-transcribed to cDNA. Each mutant’s sequence was PCR amplified and then subjected to Sanger DNA sequencing. To measure the growth kinetics of mutant viruses with and without NAI selection, ~0.2 million A549 cells were pre-treated with NAIs for 2 h, respectively, and then infected with each mutant virus at an MOI of 0.01 with/without NAIs, and the supernatants were collected at 48 h post-infection.Individual mutant viral plasmids were generated by a PCR-based site-directed mutagenesis strategy. To generate a single mutant virus, ~2 million 293T cells were transfected with 4 µg DNA. Transfections were performed using Hieff TransTM Liposomal Transfection Reagent (Yeasen). The medium was replaced at 12 h post-transfection. Viruses were collected 60 h post-transfection. Viral titers  were lightly anesthetized with inhaled isoflurane and then inoculated intranasally with 1.0 × 105 TCID50 of the mixed viruses in 50 µL. Lung tissues were harvested at day 3 post-infection, viral RNA extracted, reverse transcribed, amplified with the input viral mix, and subjected to Sanger sequencing. Relative peak heights of mutant and WT nucleotides at each position were quantified.We evaluated the relative replication capacity of mutants by mixing each mutant with WT viruses at an equal ratio and performing intranasal infection on mice. Groups of 6- to 8-week-old female BALB/c mice (SLACOM)  (PBL Assay Science) for 20 h and then infected with individual mutants or WT virus at an MOI of 0.01. Cells were washed with PBS three times at 2 h post-infection, and 1,000 U/mL IFNa2 was added back into the culture medium. The viral copy number was examined 24 h post-infection. IFN sensitivity was calculated as the decreased viral copy number upon IFN treatment.6 µM for 50% inhibition of the NA activity (IC50) was calculated from the fitted dose-response curve using GraphPad Prism 8.3.0 software .We measured the NA activity in recombinant viruses using a fluorometric assay with the fluorogenic substrate MUNANA  . BrieflyNA WT and mutant recombinant proteins were produced by the Bac-to-Bac baculovirus expression system. Protein coding sequences were cloned into pFASTBAC-1 vectors with an N-terminal GP67 signal peptide and a C-terminal His-tag. Baculoviruses were produced by sf9 insect cells. Proteins were expressed by High5 insect cells and secreted into the media. The culture medium was collected when the cell viability was ~10%. The secreted proteins were purified by Ni-column and Superdex 200 . Fractions containing target proteins were pooled and concentrated.To test the effects of the mutations on NA proteins, we measured the binding affinities between the drug and purified NA proteins by ITC. The tests were performed at 25°C . The buffer contained 20 mM MES (pH = 6.5) and 200 mM NaCl. The protein concentration in the syringe was around 0.006 mM, while in the reaction cell, it was around 0.125 mM. All titration data were calculated and analyzed by MicroCal ITC-ORIGIN Analysis Software .


Recent developments in artificial nucleic acid and drug delivery systems present possibilities for the symbiotic engineering of therapeutic oligonucleotides, such as antisense oligonucleotides (ASOs) and small interfering ribonucleic acids (siRNAs). Employing these technologies, triplex-forming oligonucleotides (TFOs) or peptide nucleic acids (PNAs) can be applied to the development of symbiotic genome-targeting tools as well as a new class of oligonucleotide drugs, which offer conceptual advantages over antisense as the antigene target generally comprises two gene copies per cell rather than multiple copies of mRNA that are being continually transcribed. Further, genome editing by TFOs or PNAs induces permanent changes in the pathological genes, thus facilitating the complete cure of diseases. Nuclease-based gene-editing tools, such as zinc fingers, CRISPR-Cas9, and TALENs, are being explored for therapeutic applications, although their potential off-target, cytotoxic, and/or immunogenic effects may hinder their in vivo applications. Therefore, this review is aimed at describing the ongoing progress in TFO and PNA technologies, which can be symbiotic genome-targeting tools that will cause a near-future paradigm shift in drug development. The recent advancement of artificial nucleotides, as well as their applications, have enabled the development of nucleic acid drugs, such as antisense oligonucleotides (ASOs) and small interfering ribonucleic acids (siRNAs). Combined with emerging drug delivery system (DDS) technologies, therapeutic oligonucleotides are recently attracting increasing attention as new drug modalities. We recently summarized the physicochemical properties of these artificial modifications (ASOs and siRNAs)  and DDS +), recognizes the guanine (G)·C base pairs to form a C+·G:C triad. On the other hand, A or T recognizes the A:T base pairs to form an A·A:T or T·A:T triad, and G recognizes the G:C base pairs to form a G·G:C triad in the antiparallel triplex.TFOs exhibit a short oligonucleotide sequence, which binds to their target DNA duplex by forming a triple-helix structure on the major groove of the DNA via the Hoogsteen  in the promoter or transcribed regions ,12; theyVarious TFO-treatment mechanisms have been reported. First, TFOs were developed for antigene strategies targeting clinically essential genes, particularly oncogenes, such as c-myc, bcl-2, HER2, and Ets-2 . TFOs caAs mentioned in Recognition of the thymine–adenine base pair in a parallel motifN4-(3-carboxypropyl)deoxycytosine nucleobase (1) for the recognition of a T:A base pair in a parallel motif. They predicted that its constituent carboxylic acid could interact with the 6-amino group in A of the T:A base pair. The ultraviolet (UV)-melting experiments of a 15-mer TFO containing 1 selectively recognized a T:A base pair in dsDNA. The melting temperature (Tm) of this TFO  was 7 °C higher than that of the triplex comprising the G·T:A triad pyrimidine-2(7H)-one as their core structures. They derivatized the 6-position of this core skeleton to introduce additional functionality. They noted that the nucleobase AP (4) was the optimum analog for the C:G recognition in both affinity and selectivity ,,N-methylormation . The seqRecognition of the thymine–adenine base pair in an antiparallel motif7). However, the association constants (Ka) of the triplex formation depended on the neighboring bases, and the adjacent dG base at the 5′ side of the target, T, appeared to be required for stable triplex formation -butyrate ( and 22) . We demoN-7 of purine bases, particularly G, to form a highly stable coordination complex, resulting in intrastrand and interstrand crosslinking in DNAs S,S-cycloS/R)-bm-PNA (37), was developed by Ganesh’s group [S/R)-bm-PNA contains an additional nucleobase at the γ-position of the PNA backbone, which enables bifacial recognition-forming duplexes at the B1 and B2 sides. The thermal stability of the DNA1/Cγ(S/R)-bm-PNA/DNA2 complexes was higher than those of their respective isolated duplexes [38), contains an additional nucleobase at the α-position of the PNA backbone [38, and 37 exhibited similar properties, although the target sequences were restricted to homothymine and homocytosine. These bimodal PNAs can be used to generate novel higher-order assemblies with DNAs and RNAs. For example, they reported a pentameric complex comprising a triplex and two duplexes, DNA1–Cγ(S/R)-bm-PNA/DNA2–Cγ(S/R)-bm-PNA/DNA3 [A novel class of PNAs, bimodal PNA, Cγ-bm-PNA , 37, was’s group ,95,96,97duplexes ,95. The +·G:C triad base pairing. Therefore, the inability to form stable hydrogen bonds with the T:A and C:G base pairs, as well as the necessity of protonating C, limit their in vivo application, including parallel TFOs. Some of the designed base analogs for TFOs can be used for PNA. For example, the base analogs 2 [5 [12) [NP (39), forms more stable hydrogen bonds than the other heterocycles [PNA forms a triplex structure via the Hoogsteen hydrogen-bond-forming T·A:T triad and Cnalogs 2  and 5 [9ogs 2 [5    can be u [5 [12) ,102. Rec[12) [PN , 39, for40) [40 forms stable base pairing through the synergistic effect of improved van der Waals contacts base stacking with hydrogen bond formation. Rozner’s group examined 2-aminopyridine M (41) as a more basic C nucleobase [Thio-pseudoisocytosine , 40 [103ridine M , 41 as acleobase ,105. The42) [43) [As mentioned in 42) . Hudson’42) . Recentl42) [43) .N-7 methylguanine (N7-Me-G), which was designed to cause steric and electrostatic repulsions between the G-clamp. The modified PNAs were used in the detection of the dsDNA target, the RT-RPA amplicon, from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with single nucleotide resolution, discriminating between two SARS-CoV-2 strains. Each strand of modified PNA exhibited fast strand invasion in the physiological condition and formed stable complexes with low equivalents of PNA.G-clamp is a phenoxazine-derived tricyclic C analog that can strongly bind to G through additional interactions, such as π-stacking, the electrostatic attraction of a positively charged amine, and hydrogen bonding at the Hoogsteen face . The int44–47) [44–47 enable the targeting of any sequences, they could be novel gene-targeting tools, and their applications for therapeutic purposes will be investigated subsequently.An investigation of the Janus–Wedge triple PNA helix was pioneered by McLaughlin’s group . Therein44–47) ,113. The21 and 22), which enabled the easy conjugation of psoralen in the N-terminus of PNA and subsequent photo-crosslinking evaluation [21 and 22 were 48% and 45%, respectively, after 30 s of irradiation; the yields were much higher than those of corresponding PNAs prepared using SPB (15%). The applications of these novel Ps–PNAs are being studied in our lab.Glazer and Nielsen’s group developed psoralen-conjugated pcPNAs, demonstrating that they can be used to induce single-base substitutions and deletions within the target site ,115. Thealuation . The yieCrosslinkable furan-derived nucleobases were developed for PNA. The furan generates reactive species immediately after the activation of reactive oxygen species (ROS), which can be exploited for DNA crosslinking. Sequence-specific crosslinking was realized with γ-Lys-modified PNA ,117. RecRecently, Tonelli’s group reported the therapeutic applications of PNAs in targeting the MYCN gene ,120. TheIn 2016, Glazer et al. reported an in vivo correction of a β-thalassemia mutation in mice by miniPEG γ-modified tcPNA (γtcPNA) . An γtcPOur group recently described the physicochemical properties of therapeutic oligonucleotide  and DDS


EGFR) gene. However, rare mutations such as G719X and S768I lack standard anti-EGFR targeted therapies. Understanding the structural differences between wild-type EGFR and these rare mutants is crucial for developing EGFR-targeted drugs. We performed a systematic analysis using molecular dynamics simulations, essential dynamics (ED), molecular mechanics Poisson–Boltzmann surface area, and free energy calculation methods to compare the kinetic properties, molecular motion, and free energy distribution between wild-type EGFR and the rare mutants’ structures G719X-EGFR, S768I-EGFR, and G719X + S768I-EGFR. Our results showed that S768I-EGFR and G719X + S768I-EGFR have higher global and local conformational flexibility and lower thermal and global structural stability than WT-EGFR. ED analysis revealed different molecular motion patterns between S768I-EGFR, G719X + S768I-EGFR, and WT-EGFR. The A-loop and αC-helix, crucial structural elements related to the active state, showed a tendency toward active state development, providing a molecular mechanism explanation for NSCLC caused by EGFR S768I and EGFR G719C + S768I mutations. The present study may be helpful in the development of new EGFR-targeted drugs based on the structure of rare mutations. Our findings may aid in developing new targeted treatments for patients with EGFR S768I and EGFR G719X + S768I mutations.Non-small cell lung cancer (NSCLC) is often driven by mutations in the epidermal growth factor receptor (  Among lpatients . Among outations . In coal therapy .EGFR mutations (19-Del and L858R) while improving progression-free and overall survival rates [EGFR mutations in the treatment of NSCLC. Therefore, developing targeted drugs for rare EGFR mutations has become a crucial focus for researchers to improve patient treatment options.Numerous EGFR tyrosine kinase domain inhibitors (TKIs) and vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) neutralizing antibodies have been developed and approved for the treatment of NSCLC . The FDAal rates . Current791QLMPF795) that connects the N-terminal lobe to the C-terminal lobe; and the C-terminal lobe, which includes the Asp-Phe-Gly (DFG) motif, catalytic loop (HRD837LAARN), and A-loop [The structure and function of EGFR protein have proven useful in developing new targeted drugs. Research has shown that EGFR is a transmembrane receptor tyrosine kinase composed of 1,210 amino acids and has a molecular weight of approximately 134 kDa. The protein-coding gene is located in the human chromosome 7p12-14 region. It consists of 28 exons, which include an extracellular ligand-binding domain that has 621 amino acids (AA) (25–645), an α-helical transmembrane domain that has 23 AA (646–668), a cytoplasmic tyrosine kinase domain that has 273 AA (707–979), and a carboxy-terminal  signaling domain that has 229 AA  [The ATP-binding cleft is at the interface between the N-lobe and C-lobe . The actEGFR mutation have poorer prognoses due to shorter median survival and progression-free survival than other EGFR mutations [EGFR, specifically in the αC-β4 loop. In this location, the hydrophilic serine is replaced by a hydrophobic isoleucine, which has greater steric resistance than the previous serine, resulting in stronger hydrophobic interactions between the αC-helix and the adjacent β9 chain. This enhances the effect and stabilizes the enthalpy of the active conformation of “αC-in.” G719 is in the N-terminal lobe and represents the initial glycine in the P-loop’s widely preserved “GXGXXG” motif. When non-glycine residues  replace G719, it leads to a substantial decrease in the flexibility of the P-loop, which weakens the inhibition of the hydrophobic interaction of αC-helix in the inactive configuration and destabilizes the inactive conformation, thus leading to the active conformation.Patients with S768I utations . S768 isEGFR [EGFR mutations.According to the structure analysis by Du et al. , both G7EGFR . Wang etFurthermore, G719C + S768I accounted for 68.82% of all G719X + S768I mutations . The S76Therefore, this study aims to provide an overview of EGFR mutations in NSCLC, emphasizing their prevalence and the distinction between common and rare mutations while highlighting the significance of targeted therapies over chemotherapy. Our primary research focus was to comprehensively analyze the kinetic and thermodynamic behavior of EGFR mutations, specifically S768I, G719X, and G719X + S768I, in comparison to wild-type EGFR, using molecular dynamics (MD) simulations and free energy mapping. This analysis seeks to elucidate these mutants’ conformational flexibility, kinetic properties, molecular motion, and free energy distribution, shedding light on their role in NSCLC and contributing to developing more effective targeted treatments.EGFR S768I and G719X + S768I mutations. Through MD simulations and the reconstruction of free energy maps, we sought to answer the following key questions:How do the conformational flexibility and kinetic properties of S768I-EGFR, G719X, and G719X + S768I-EGFR differ from those of WT-EGFR, and what implications do these differences have for the activation state of the protein?What are the distinctive patterns of molecular motion exhibited by these mutant EGFR proteins, and how do they contribute to our understanding of their behavior in the context of NSCLC?What insights into the free energy distribution of these EGFR mutants can be gained, and how can these insights inform the development of targeted treatment strategies for NSCLC patients carrying these rare mutations?Various computational models were utilized to understand the kinetic mechanism of EGFR mutations, such as geometric properties of EGFR, binding free energy, hydrogen bond analysis, and stability analysis . These mEGFR S768I, G719X, and G719X + S768I mutation leading to NSCLC and can help in developing new targeted treatment strategies for patients with these rare mutations.In this study, we conducted a systematic computational study to compare the kinetic and thermodynamic behavior of WT-EGFR, G719X-EGFR, S768I-EGFR, and G719X + S768I-EGFR. Our results showed that S768I-EGFR, G719X, and G719X + S768I-EGFR exhibited higher conformational flexibility and a greater tendency to transition to the active state than WT-EGFR. These findings provide a molecular mechanism explanation for the 22.1https://www.rcsb.org) database (PDB code: 2GS7). The missing residues were modeled using MODELER [The structure of the inactive wild-type EGFR was downloaded from the Protein Data Bank  and 1.4 nm (long-range). The solute and solvent temperature were maintained at 300 K using a coupling time constant τt of 0.1 ps . The pre2.3Root mean square deviation (RMSD) and Cα root mean square fluctuation (RMSF) were calculated using the GROMACS tools “gmx rmsd” and “gmx rmsf,” respectively .2.4For ED analysis in this study, we utilized principal component analysis , a widel2.5The free energy landscape (FEL) was used to characterize the thermodynamic and energetic properties of WT-EGFR, G719X-EGFR, S768I-EGFR, and G719X + S768I-EGFR. The GROMACS-20.6 software package was used to perform MD simulations for both systems, and the ED analysis was carried out to obtain the first few eigenvectors. The two-dimensional preparatory subspace formed along eigenvectors 1 and 2 was used as the reaction coordinates to reconstruct the FEL of the two systems. The global free energy minimization region was characterized by different sizes and depths of free energy wells, indicating a rugged and rough free energy surface.33.1To evaluate the stability of the structures of WT-EGFR, G719X-EGFR, S768I-EGFR, and G719X + S768I-EGFR during simulation, we calculated the RMSD values of the backbone atoms for each simulation replica concerning the initial structure . The RMSThese results indicate that G719C-EGFR and G719A-EGFR are easier to achieve equilibrium fluctuations than WT-EGFR. The time required for G719A + S768I-EGFR and WT-EGFR to reach equilibrium fluctuations is similar. G719S-EGFR, G719C + S768I-EGFR, S768I-EGFR, G719D-EGFR, and G719D + S768I-EGFR are harder to achieve equilibrium fluctuations than WT-EGFR. In other words, the structural stability of G719A + S768I-EGFR is similar to that of WT-EGFR, and the structural stability of G719C-EGFR and G719A-EGFR is higher than that of WT-EGFR. Additionally, the structural stability of G719S-EGFR, G719C + S768I-EGFR, S768I-EGFR, G719D-EGFR, and G719D + S768I-EGFR is lower than that of WT-EGFR. Therefore, the lower the structural stability, the higher the ability of the protein to undergo conformational changes.These findings indicate increased structural dynamics in these mutants, which may have important implications for their functional properties. In particular, the elevated conformational flexibility of the A-loop region, associated with the active state of EGFR, suggests that these mutants may have an enhanced capacity to transition from an inactive to an active conformation. This insight is crucial for understanding the potential oncogenic properties of these mutants in NSCLC, where EGFR dysregulation is a vital driver of tumorigenesis. This observation suggests that these mutants may have a heightened propensity to adopt functionally relevant states, including the active conformation. Clinical implications arise as these mutants could drive aggressive NSCLC phenotypes and respond differently to targeted therapies.3.2To ensure that the calculated RMSF values accurately reflect the natural conformational flexibility, we observed Upon careful examination of the RMSF diagram , it was Moreover, our ED analysis revealed that certain mutants experience larger structural fluctuations and conformational changes than WT-EGFR, implying the existence of distinct dynamic behavior that may be exploited in drug design. These mutants could be more susceptible to compounds that selectively stabilize or disrupt specific conformations, opening up new avenues for drug development strategies tailored to individual EGFR mutations. The increased conformational diversity observed in certain mutants opens new avenues for precision medicine. Targeted therapies that specifically exploit the unique structural dynamics of EGFR mutants may provide a more effective treatment strategy for patients. For example, drugs designed to stabilize or disrupt specific conformations associated with mutant EGFR variants could improve treatment outcomes. Our study identifies potential vulnerabilities in these mutants that can be targeted with tailored therapeutic interventions.3.3The ED analysis based on the MD simulation trajectory was used to extract the protein’s most important or largest scale motion mode in the multi-dimensional conformational space. The eigenvectors and eigenvalues of wild-type and mutant EGFR were obtained by analyzing the ED of their respective tandem equilibrium trajectories.2, respectively , was calculated for G719D + S768I-EGFR, S768I-EGFR, G719A + S768I-EGFR, G719D-EGFR, G719C + S768I-EGFR, WT-EGFR, G719S-EGFR, G719C-EGFR, and G719A-EGFR. The TMSF values were 75.507, 61.299, 52.804, 51.522, 49.712, 49.196, 47.750, 47.507, and 43.594 nmectively . These vThe top 30 eigenvalues of the eigenvectors of G719D + S768I-EGFR, S768I-EGFR, G719A + S768I-EGFR, G719D-EGFR, G719C + S768I-EGFR, WT-EGFR, G719S-EGFR, G719C-EGFR, and G719A-EGFR are shown in The ED analysis revealed that the eigenvalues of the first eigenvectors of G719D + S768I-EGFR, S768I-EGFR, G719A + S768I-EGFR, G719D-EGFR, G719C + S768I-EGFR, G719S-EGFR, G719C-EGFR, and G719A-EGFR were significantly higher than those of WT-EGFR, indicating that these mutants experienced more drastic structural fluctuations or conformational changes than WT-EGFR along the first eigenvector. The cumulative contributions of the first two eigenvectors of WT-EGFR, S768I-EGFR, and G719C + S768I-EGFR to TMSF were 35.2, 38.1, and 37.3%, respectively, while the cumulative contributions of the first ten eigenvectors were 78.9, 79.5, and 79.6%, respectively. These results suggest that the essential subspace formed by the first two eigenvectors contains the most significant collective motion and the most dominant conformational states or substrates obtained by MD simulation sampling.Furthermore, the first eigenvectors of G719D + S768I-EGFR, S768I-EGFR, G719A + S768I-EGFR, G719D-EGFR, and G719C + S768I-EGFR had larger eigenvalues than WT-EGFR, indicating that these mutants had larger motion amplitude or conformational freedom degree than WT-EGFR along the first eigenvector. In such a high-dimensional conformational space, only the first ten eigenvectors contributed more than 80% to the TMSF , indicating that our intrinsic kinetic analysis successfully extracted the most dominant movement patterns of EGFR. Overall, compared with WT-EGFR, G719D + S768I-EGFR, S768I-EGFR, G719A + S768I-EGFR, G719D-EGFR, and G719C + S768I-EGFR required a smaller number of eigenvectors to reach the same level of cumulative contribution.The motion pattern of the two EGFRs along the first eigenvector is shown in a glitch diagram . The firMoreover, the movement direction of the αC-helix, another crucial structural element related to EGFR activation, was downward and inward, opposite to the outward rotation of αC-helix “out” conformation. The αC-helix tended to develop toward an “in” conformation trend. These conformational change trends of A-loop and αC-helix provide an essential basis for the transition of EGFR from an inactive to an active conformation.3.4The FEL of WT-EGFR, G719A-EGFR, G719C-EGFR, G719D-EGFR, G719S-EGFR, S768I-EGFR, G719A + S768I-EGFR, G719C + S768I-EGFR, and G719D-EGFR were reconstructed using the first and second eigenvector projections as reaction coordinates . AnalysiCompared with S768I-EGFR, WT-EGFR has two larger free energy wells (≤−21 kJ/mol), implying higher thermal stability. Greater conformational diversity signifies higher conformational freedom or structural variability, allowing S768I-EGFR to sample more conformational states or substrates. However, the increased degree of conformational freedom also leads to decreased thermal and structural stability of S768I-EGFR. Therefore, S768I-EGFR has greater conformational diversity and more complex dynamic behavior than WT-EGFR, with an improved ability to approach the active state.4EGFR disrupts its inactive form, leading to an equilibrium shift toward the active state. This shift enables the receptor to activate independently of ligand binding [EGFR S768I and G719X + S768I mutations affect the structure of EGFR and its relation to NSCLC remains unclear. Understanding the molecular basis of the conformational distribution of the S768I and G719X + S768I mutants of EGFR is vital for in-depth research into the molecular mechanisms of mutation-activated NSCLC and related drug development. In this study, we conducted long-term MD simulations of S768I-EGFR, G719X + S768I-EGFR, and WT-EGFR to investigate the structural and kinetic differences between the rare mutants and wild type.Activating wild-type EGFR requires ligand binding, which induces a conformational change promoting receptor homodimerization or heterodimerization with other EGFR family members. This event leads to the autophosphorylation of tyrosine residues in the EGFR tail and the activation of several signaling pathways . These p binding . The conThe RMSF analysis results of S768I-EGFR, G719X + S768I-EGFR, and WT-EGFR indicated that S768I-EGFR and G719D + S768I-EGFR had greater global and local conformational flexibility than WT-EGFR, suggesting that the latter had more enthalpy or entropy factors that positively contributed to its structural stability, consistent with previous EGFR structural analyses of L858R and T790M mutations . The stuThus, without ligand induction, S768I-EGFR, G719A + S768I-EGFR, G719C + S768I-EGFR, and G719D-EGFR could reach the active state through a conformational selection mechanism. In contrast, due to its weak conformational flexibility and high conformational rigidity, WT-EGFR must be combined with the ligand to promote further conformational change. Otherwise, it could not reach the active state.The ED analysis of S768I-EGFR, G719X + S768I-EGFR, and WT-EGFR revealed that S768I-EGFR and G719X + S768I-EGFR have a greater degree of conformational freedom, confirming the previous findings. A comparison of their large-scale collective molecular motions revealed that S768I-EGFR and G719X + S768I-EGFR have more substructures and larger collective displacement amplitudes. These motion direction and amplitude differences lead to distinct conformational changes, particularly in the A-loop and αC-helix of S768I-EGFR and G719D + S768I-EGFR, which tend to transition to the active conformation. The transition of the A-loop and αC-helix between active and inactive states is critical for EGFR regulation, and mutations associated with cancer dysregulate these conformations. The short α-helix of the A-loop interacts with αC-helix autoinhibition to stabilize the “out” conformation of αC-helix, so the conformational change of the short αC-helix will alter its autoinhibitory interaction with αC-helix, promoting the development of αC-helix toward the “in” conformation. Notably, the S768I and G719X + S768I mutations induce more significant molecular movement in the N-terminus, suggesting that the mutation changes the adjacent conformation around the mutant amino acid, leading to differences in overall and local flexibility between S768I-EGFR, G719X + S768I-EGFR, and WT-EGFR .5Limited research has been conducted on G719X + S768I by MD. However, Liu et al. utilized the MD method to investigate. They discovered that the G719C mutation impacted the binding free energy of EGFR mutants to gefitinib compared to the classical mutation . ChakrabAdditionally, they found that the G719S mutation brings the ligand closer to the hinge region, while the T790M mutation moves the ligand out of the binding pocket . This ma6In conclusion, our study illuminates the dynamic structural characteristics of various EGFR mutants, shedding light on their conformational flexibility and distinct behaviors. Notably, the G719X + S768I mutant, which has been relatively underexplored in MD studies, exhibits remarkable conformational flexibility and structural changes, potentially contributing to its resistance to EGFR-targeted therapies. These findings underscore the importance of precision medicine approaches tailored to specific EGFR mutations. Future research directions should encompass in-depth investigations into the dynamic behaviors of less-studied mutants, developing next-generation inhibitors, and clinical studies to optimize personalized treatment strategies.Building upon the insights derived from this study, several promising avenues for future research and clinical applications emerge. First and foremost, further investigations are warranted to explore the dynamic behaviors and structural characteristics of EGFR mutants, especially the less-studied G719X + S768I variant, using advanced computational methods and experimental validation. Understanding the distinct conformational landscapes of these mutants can facilitate the development of novel targeted therapies that specifically exploit their structural vulnerabilities. Moreover, the findings hint at the potential for rational drug design targeting the altered binding forces between the mutant EGFR and existing EGFR-TKIs. Future research can delve into developing next-generation inhibitors and combination therapies designed to tackle EGFR resistance mechanisms effectively, ultimately improving patient treatment outcomes. Additionally, clinical studies focusing on the efficacy of tailored treatments based on specific EGFR mutation profiles, as highlighted in our research, can provide valuable insights into personalized medicine approaches for NSCLC patients. Overall, the future directions of this study lie in leveraging these structural insights to advance the understanding of EGFR-related cancers and to develop more precise and effective therapeutic strategies.


The cytidine deaminase, Apolipoprotein B mRNA editing enzyme catalytic subunit 3B , is a critical mutation driver that induces genomic instability in cancer by catalyzing cytosine-to-thymine (C-to-T) conversion and promoting replication stress (RS). However, the detailed function of A3B in RS is not fully determined and it is not known whether the mechanism of A3B action can be exploited for cancer therapy. Here, we conducted an immunoprecipitation-mass spectrometry (IP-MS) study and identified A3B to be a novel binding component of R-loops, which are RNA:DNA hybrid structures. Mechanistically, overexpression of A3B exacerbated RS by promoting R-loop formation and altering the distribution of R-loops in the genome. This was rescued by the R-loop gatekeeper, Ribonuclease H1 . In addition, a high level of A3B conferred sensitivity to ATR/Chk1 inhibitors (ATRi/Chk1i) in melanoma cells, which was dependent on R-loop status. Together, our results provide novel insights into the mechanistic link between A3B and R-loops in the promotion of RS in cancer. This will inform the development of markers to predict the response of patients to ATRi/Chk1i. Apolipoprotein B mRNA editing enzyme catalytic subunit 3B  belongs to the cytidine deaminase APOBEC3 family, which can cause the accumulation of cytosine-to-thymine (C-to-T) mutations in DNA and induce genome instability . Thus, AR-loops are intermediates of transcription consisting of a complementary RNA:DNA hybrid plus a displaced DNA strand (ssDNA) . They arn = 67), adjacent normal (n = 5) and benign nevi tissues (n = 14). Concordant with previous reports . Th. Th35]. p < 0.05. Pearson correlation analysis was performed on GEPIA (http://gepia.cancer-pku.cn/).All statistical analyses were performed using GraphPad Prism 7.0 . Descriptive values are presented as mean + standard error of the mean (SEM) unless stated elsewhere. Differences between two groups were analyzed by unpaired Student’s t-test while differences among multiple groups were analyzed by one-way analysis of variance (ANOVA) with post-hoc intergroup comparisons with Turkey’s test. When data did not meet the normal distribution, the Mann–Whitney U-test was performed. Test details were indicated in the figure legends. Results were considered statistically significant when Supplementary Figures and TablesSupplementary Materialchecklist


The avian influenza virus (AIV) PA protein contributes to viral replication and pathogenicity; however, its interaction with innate immunity is not well understood. Here, we report that the H5 subtype AIV PA protein strongly suppresses host antiviral defense by interacting with and degrading a key protein in interferon (IFN) signaling, Janus kinase 1 (JAK1). Specifically, the AIV PA protein catalyzes the K48-linked polyubiquitination and degradation of JAK1 at lysine residue 249. Importantly, the AIV PA protein harboring 32T/550L degrades both avian and mammalian JAK1, while the AIV PA protein with residues 32M/550I degrades avian JAK1 only. Furthermore, the residues 32T/550L in PA protein confer optimum polymerase activity and AIV growth in mammalian cells. Notably, the replication and virulence of the AIV PA T32M/L550I mutant are attenuated in infected mice. Collectively, these data reveal an interference role for H5 subtype AIV PA protein in host innate immunity, which can be targeted for the development of specific and effective anti-influenza therapeutics. The highly pathogenic avian influenza virus (HPAIV) circulates in several countries and causes multiple human infections worldwide. In the present study, we found that two novel residues 32T and 550L in the PA protein of AIV inhibit the IFN-mediated immune response by diminishing JAK1 protein levels and maintaining optimum polymerase activity in mammalian cells. We further revealed that the two amino acids were responsible for the pathogenicity of H5 HPAIV in infected mice. Our study emphasizes the importance of PA protein in the increased pathogenicity of HPAIV and provides important insights for understanding the molecular mechanisms of virus-host interactions, which are critical for developing control strategies for HPAIV. An increasing number of highly pathogenic avian influenza viruses (HPAIV), including the H5 and H7 subtypes, have acquired the ability to cross the species barrier to infect and kill humans, continually posing a serious threat to public health –3. In 19The interferon (IFN) response constitutes an essential part of host defenses that restrict viral infections. IFNs are the primary antiviral cytokines produced by infected cells. The engagement of type I IFNs and their cell surface receptors activates JAK-signal transducer and activator of transcription (STAT) signaling, which promotes the transcription of an extended array of IFN-stimulated genes (ISGs) to exert antiviral activity ,16. The The IAV RNA-dependent RNA polymerase complex, which is composed of PB2, PB1, and PA subunits, is essential for viral genome transcription and replication and determines the evolution of adaptive viral changes to overcome host restrictions . PB2-E62In a recent study, we showed that the IAV PB2 proteins mediate the degradation of human JAK1 to benefit its replication . To furtIt was found that PA-CZ downregulated endogenous JAK1 protein but not IFNAR1, STAT1, or STAT2 in HEK293T cells . To soliThe ubiquitin-proteasome and autophagy-lysosome pathways are two systems that control protein degradation in eukaryotic cells. We found that PA-mediated human JAK1 degradation was mostly restored by treatment with the proteasome inhibitor MG132, but not with the lysosome inhibitor ammonium chloride (NH4Cl) or chloroquine (CQ) . Next, wTo identify the key residues in JAK1 that are targeted by PA for ubiquitination and degradation, we first designed and constructed truncated plasmids . JAK1-WTIFIT1, and TAP1). The qRT-PCR results indicate that the IFIT1 and TAP1 mRNA levels were dramatically decreased in the presence of JAK1 K249R or JAK1 K859/860R overexpression in the shJAK1 cells by comparing with that of JAK1 WT restoration (To further understand the role of JAK1 (K249R) during IAV infection and eliminate the effect of endogenous JAK1 on viral replication, we established a JAK1 shRNA-knockdown (shJAK1) cell line and constructed a mutant JAK1 expression plasmid (ΔJAK1) that contains 7-nucleotide nonsense mutations in the shJAK1 target sequences to restore the JAK1 expression in shJAK1 cells . In the toration , suggestSince a previous study has shown that JAK1 interacts with the IAV PB2 protein , we deteM-CZ or PA-JY  and rCZ-PAJY, respectively). JAK1 co-localized with PA in rCZ-infected cells  . It is nor PA-JY . To examed cells , but thied cells . Notably of JAK1 , indicattination . Similar of JAK1 . These dThe amino acid sequences of JAK1 from human and mouse are conserved, with a similarity of 94.72%, while the similarity between human and chicken JAK1 is only 81.09%. However, the Lysine 249 residue is conserved among human, mouse, and chicken . TherebyIFIT1 (TAP1 (T32M. We then explored the ability of PA to regulate the expression of ISGs. Quantitative polymerase chain reaction (qPCR) analysis further indicated that overexpression of PA-CZ and PAT32M-CZ, but not PAL550I-CZ or PAM-CZ, significantly inhibited IFNβ-triggered transcription of IFIT1 and TAP1 in human A549 cells  by monitoring the GFP expression, suggesting that PA (32T/550L) protein dampened the secretion of antiviral factors induced by SeV /mL at 72 hpi  for the rCZ-PA49 cells . All of t 72 hpi . We furtt 72 hpi . Furtherd 48 hpi .T32M-infected HEK293T cells than that in the rCZ-infected HEK293T cells. However, rCZ-PAL550I infection has significantly lower levels of viral NP mRNA, vRNA, and cRNA than rCZ virus in HEK293T cells . In HEK293T cells, the polymerase activity of the single mutation PA-T32M was increased by approximately 4 fold, whereas a significant reduction in polymerase activity was observed for PA-L550I. However, the double mutation T32M/L550I in the PA protein recovered the polymerase activity, with a 9-fold increase . The vir3T cells . Interesed cells . There wed cells . These fL550I or rCZ-PAM exhibited lower morbidity than those infected with rCZ , a panel of two double mutants, was generated, with PA T32M and PA L550I coupled with PB2 M283I and PB2 K526R. rCZ-(PAM+PB2M) replicated with virus titers significantly lower than those of rCZ-WT and other mutant viruses at 2 and 5 dpi (M+PB2M) was attenuated compared with the rCZ-PAM virus   were higher than those in mice infected with rCZ or rCZ-PAT32M.Moreover, the expression of JAK1 was lower in the lungs of mice infected with rCZ or rCZ-PAAM+PB2M) . We also)  (“X-ORF”) . PA-X plIn summary, we found that the two residues in the PA protein, 32T, and 550L, are critical for mediating mammalian JAK1 degradation and maintaining polymerase activity, which contributes to the virulence of AIV in mammals. Our study provides important insights into the development of rational antiviral and vaccine strategies.Animal experiments were conducted in accordance with the guidelines for experimental animal welfare and ethics. All animal studies followed the protocols of the Jiangsu Province Administrative Committee for Laboratory Animals (permit number: SYXK-SU-2017-0044). All experiments on influenza virus infection were conducted at a biosafety level 3 laboratory and animal facility at Yangzhou University (CNAS BL0015).T32M, rCZ-PAL550I, rCZ-PA, rCZ-PAJY, and rCZ-[PA+PB2]. The rescued viruses were confirmed using sequence analysis, and viruses bearing unwanted mutations were excluded from further analysis.Two HPAI H5N8 viruses, A/goose/eastern China/CZ/2013 (CZ) and A/duck/Eastern China /JY/2014 (JY), were characterized in our previous studies . Recombi2. Cells were inoculated with AIV at the indicated multiplicity of infection (MOI) for virus infection. After adsorption for 1 h at 37°C, the cells were washed with phosphate-buffered saline (PBS) and cultured in DMEM/MEM containing 0.5 μg/mL L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin.Human embryonic kidney (HEK) 293T cells , chicken fibroblast cell line DF-1 cells , and human lung epithelial A549 cells  were cultured in Dulbecco’s modified Eagle’s medium . Madin-Darby canine kidney (MDCK) cells  cells were cultured in minimum essential medium Eagle (MEM). ShJAK1 HEK293T/A549 cells were prepared as previously described , and PA plasmids and treated with MG132. Data are representative of two independent experiments.(TIF)Click here for additional data file.S4 Fig(A) Immunoblots of HEK293T cells transfected with different IAV PA plasmids. (B). Quantification of protein expression on immunoblots (A) and normalized with actin (n  =  3). Data are presented as means ± SD and statistical significance was determined by unpaired two-tailed Student’s t-test. (C) The amino acid differences between the PA protein from different IAVs. (D) Frequencies of different residues at PA 32 and 550 positions in avian and human viruses. All sequences were obtained from GISAID database.(TIF)Click here for additional data file.S5 Fig(A) Amino acid alignment of JAK1 from the different species. The black box highlights amino acid 249 corresponding to human JAK1. (B) Immunoblots of HEK293T cells transfected with PA and chJAK1 plasmids (upper). Densitometry analysis of the ratio of JAK1/Actin on immunoblots from three independent experiments (lower). (C) Luciferase assays in DF-1 cells transfected with Rluc plasmid, and PA-CZ or PA-JY plasmid (n = 3). (D) qPCR analysis of chJAK1 mRNA level in DF-1 cells transfected with PA plasmids from CZ and JY. (n = 3). (E) Co-ip analysis of the interaction of PA with chJAK1 in HEK293T cells. (F) Ni-NTA pull-down analysis of the ubiquitination of chJAK1 in HEK293T cells transfected with chJAK1, HA-Ub, and PA plasmids. WCL, whole-cell lysates. Data are presented as means ± SD and statistical significance was determined by unpaired two-tailed Student’s t-test in B–D. nsP >0.05. Data are representative of three independent experiments.(TIF)Click here for additional data file.S6 Fig(A–C) HEK293T cells or (D–F) DF-1 cells were infected with rAIVs. Levels of NP genes were estimated by quantitative RT-PCR (n = 3). Data are presented as means ± SD and statistical significance was determined by unpaired two-tailed Student’s t-test. nsP >0.05.(TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.