The SARS-CoV-2 pandemic has seriously affected the population in Turkey. Since the beginning, phylogenetic analysis has been necessary to monitor public health measures against COVID-19 disease. In any case, the analysis of spike (S) and nucleocapsid (N) gene mutations was crucial in determining their potential impact on viral spread. We screened S and N regions to detect usual and unusual substitutions, whilst also investigating the clusters among a patient cohort resident in Kahramanmaraş city, in a restricted time span. Sequences were obtained by Sanger methods and genotyped by the PANGO Lineage tool. Amino acid substitutions were annotated comparing newly generated sequences to the NC_045512.2 reference sequence. Clusters were defined using phylogenetic analysis with a 70% cut-off. All sequences were classified as Delta. Eight isolates carried unusual mutations on the S protein, some of them located in the S2 key domain. One isolate displayed the unusual L139S on the N protein, while few isolates carried the T24I and A359S N substitutions able to destabilize the protein. Phylogeny identified nine monophyletic clusters. This study provided additional information about SARS-CoV-2 epidemiology in Turkey, suggesting local transmission of infection in the city by several transmission routes, and highlighting the necessity to improve the power of sequencing worldwide. Coronavirus Disease-19 (COVID-19), caused by the Severe Acute Respiratory Syndrome Virus 2 (SARS-CoV-2), is characterized by clinical manifestations ranging from asymptomatic and mild symptoms to severe disease and death . The couAlongside timely diagnosis, the WHO and European Centre for Disease Prevention and Control (ECDC) advised to detect variant circulation using SARS-CoV-2 whole-genome or spike (S), partial or complete, gene sequencing . Phyloge−3 [Molecular characterization has allowed the identification of genomic substitutions responsible for increased transmissibility and pathogenicity of the SARS-CoV-2 enveloped virus ,14. The −3 . Interes−3 . The SAR−3 . The ana−3 ,17,18. O−3 ,20,21. T−3 . Sahin a−3 ,21. In tThis study was designed to focus on a selected patient cohort resident in Kahramanmaraş city in August 2021. The city is located in southern Turkey and is the capital of Kahramanmaraş Province. The population living in the metropolitan area was 560,000 in 2021 and 583,000 at present. During routine diagnosis by molecular tests, the S region was sequenced, as suggested by the WHO, to identify new SARS-CoV-2 circulating variants, while the N region was analyzed as an important genomic hotspot. Herein, we screened the spike and nucleocapsid whole regions by Sanger sequencing to detect usual and unusual SARS-CoV-2 mutations carried on newly generated sequences. Additionally, we investigated the clusters occurring over a one-month period in the cohort by phylogenetic analysis using concatenate S and N genomic regions. Fifty-eight nasopharyngeal swab samples (collected between 3rd and 24th August 2021) from subjects attending to the Kahramanmaraş Sütçü İmam University Health Practice and Research Hospital Microbiology Laboratory, Turkey, with available clinical data, were included in the analysis. All patients with COVID-19 prediagnosis were confirmed to be SARS-CoV-2-RNA-positive. Viral RNA was detected by a real-time RT-PCR assay, targeting the ORF1ab, nucleocapsid (N), and spike (S) genes . The analytical sensitivity of the Bio-Speedy kit reported by the manufacturer is 98%. The Ct value is inversely related to the viral RNA copy number in clinical samples. Values of 12 ≤ Ct < 20, 21 ≤ Ct ≤ 25, and Ct > 25 were associated with high, medium, and low viral load, respectively. 2, 0.2 mM dNTPs, 0.2 µM forward and reverse primers, and 1U Taq DNA polymerase Solis HOT FIREPol . The S and N genes were amplified and sequenced using primers previously reported [Viral RNA isolation was performed with the HibriGen General RNA Isolation Kit . The purified RNA was reverse-transcribed into complementary DNA (cDNA) using the OneScript Plus cDNA Synthesis Kit . PCR was performed in a 20 μL reaction mix, as follows: 2 mM MgClreported . Sanger ® database [SARS-CoV-2 viral isolates were classified by Pangolin COVID-19 Lineage Assigner v.4.0.6 . Amino adatabase . SARS-CoV-2 genomes of Turkish strains, submitted between 1st and 31st August 2021, were downloaded from the GISAID database. High-coverage Delta (B.1.617.2) VOC sequences without nucleotide gaps and with complete collection dates were included in the analysis. Duplicate sequences identified by the ElimDupes tool were excluded . The refAll patients were positive for both genomic regions by the real-time RT-PCR assay. The median Ct value of 14.96 (ranging from 10.62 to 21.21) was detected for the ORF1ab plus N genomic regions, while a median Ct value of 17.35 (ranging from 13.02 to 23.34) was detected for the S region. Overall, the median Ct value for both regions was less than 25 (ranging from 10.62 to 23.34), identifying a high viral load in all nasopharyngeal swabs. The median age of the 58 patients was 33.9 years (range 0–78) and the percentage of females was 53.4%. Specific COVID-19 symptoms were referred by most of the patients (58.6%). Unfortunately, vaccination status was not available. Demographic and clinical characteristics of the patients at diagnosis are reported in The 58 consensus sequences were all classified as Delta VOC, B.1.617.2 lineage, by the PANGO Lineage tool. Any B.1.617.2 genome was classified as a unique AY sublineage by a genotyping online tool. The S and N regions of the newly identified isolates showed several amino acid mutations. All isolates carried the T19R, G142D (with only one exception coding G), L452R, T478K, D614G, P681R, and D950N spike amino acid substitutions. Notably, some isolates carried one or more unusual mutations, such as A67T, P330L, A372S, T696S, I742N, V915I, A1020T, G1124C, and G1267R. Thirty-six out of fifty-eight (62.1%) isolates showed the T95I mutation. The N proteins of the 58 isolates displayed the usual D63G, R203M, G215C, and D377Y amino acid substitutions, and in one isolate, the L139S unusual mutation. The specific mutational pattern of whole S and N genomic regions for each isolate is reported in To check the prevalence of the unusual L139S mutation, 52 more N sequences of the Delta VOC collected during the same time span were screened, and again only one isolate carried this amino acid substitution. Additionally, we identified two T24I, one A359S, and one P364L N mutations among the fifty-two isolates. The ML phylogenetic tree of the final dataset showed the presence of nine statistically significant monophyletic clusters, including only sequences from Kahramanmaraş city. No clusters among the new isolates and sequences from the rest of Turkey were observed .The nine clusters (from A to I) contained between two to five patients, with a median cluster size of three positive subjects. Thirty out of fifty-eight (51.7%) new isolates did not belong to any cluster, including the two isolates detected in a married couple (#19 and #25 isolates).Cluster A (black) comprised 5/58 isolates, all of them carried the T29A, E156G, and Del157-158 S mutations, while 4/5 carried the T250I, 1/5 (isolate #99) carried the P174S, and 1/5 (isolate #49) carried the V1228L S mutations. The A119S and R319H N substitutions were reported in isolate #49 and isolate #98, respectively. The five sequences belonged to two males and three females, who reported COVID-19 symptoms in 3/5 cases. Cluster B (green) included three  isolates, displaying the E156G and Del157-158 S mutations, from patients  who reported COVID-19 symptoms. Cluster C (turquoise) showed very high bootstrap support (>100) and comprised three isolates carrying the T95I and E1202Q S mutations. In particular, isolate #3 displayed the S E156G and Del157-158 substitutions, while isolate #9 also carried the D1163Y S plus unusual L139S N mutations. The isolate #32 carried the D1163Y S substitution as well. Only the patient infected by isolate #9 reported COVID-19 symptoms, while the patient infected by isolate #3 reported an atypical backache. Cluster D (yellow) included two isolates carrying the T95I, E156G, Del157-158 and I850L S substitutions. Isolate #29 was also characterized by the M153I S mutation. Both isolates belonged to patients with specific symptoms at diagnosis.From Cluster E to Cluster I, all isolates carried the T95I, E156G, and Del157-158 S substitutions. Isolates #7 and #88 of Cluster E (fuchsia) additionally showed E281Q and I850L in the spike region, while isolate #88 also showed the R41Q N mutation. Isolate #120, but not the isolate #127, of Cluster F (dark blue) displayed the N185D S mutation. Inside Cluster G (orange), only isolate #45 was characterized by the I742N S substitution. The isolates #101 and #123, forming Cluster H (light blue) with isolate #97, carried the peculiar G1219C and A67T plus V382L S substitutions, respectively. Cluster I (red) is characterized by S232G and E253D N mutations. In particular, isolate #24 also carried the T696S spike amino acidic substitution. All four patients infected by isolates included in this cluster reported specific COVID-19 symptoms.Despite the highest prevalence of infection being in the 10–19 (14/58) and 30–39 (14/58) age groups, clusters were not significantly associated with age. The prevalence of infection was similar between males and females (46.6% vs 53.4%) in our cohort and in each cluster.This study aimed to evaluate the genetic variability amongst 58 newly generated sequences collected during routine diagnosis within a patient cohort resident in Kahramanmaraş city in August 2021. In addition, cluster analysis among viral strains isolated from patients was performed in a restricted time span (one month) to understand the dynamics of SARS-CoV-2 transmission during the fourth wave in Turkey. At the time of our analysis, the pandemic was characterized worldwide by the Delta VOC . Delta wIn our cohort, diagnosis was performed using the gold standard method, RT-PCR, despite several methods that were developed to detect SARS-CoV-2 RNA in respiratory and saliva specimens . To diag+ position [The genetic variability of SARS-CoV-2 determined the accumulation of mutations over time, influencing transmission, severity of disease, and vaccination efficacy ,35. The position . Since tposition ,37. Withposition . The cluposition . The S protein of all new isolates carried the T19R, G142D (only one exception with G), L452R, T478K, D614G, P681R, and D950N usual substitutions of the Delta VOC. Interestingly, just one isolate did not display the G142D mutation, which interferes with the neutralization of many N-terminal-domain (NTD)-binding monoclonal antibodies (mAbs) . The L45Furthermore, nine unusual mutations were detected from the S protein and one from the N protein. One patient (isolate #24) carried the T696S mutation in the S1/S2 cleavage site. This polybasic cleavage site is strictly related to the ability of SARS-CoV-2 to fuse with cell membranes and to start the infection process . AnotherAll 58 new isolates displayed in the N protein the four  amino acid substitutions peculiar to the Delta VOC . In partSARS-CoV-2 phylogenetic and mutational analyses were, and still are, of paramount importance to slow the diffusion of VOCs in the age of globalization. In Indonesia, Delta was the dominant variant in the second wave (May–August 2021), carrying unique substitutions that differ from those of the first wave . The incIn conclusion, this study provides additional information into SARS-CoV-2 epidemiology in Turkey. The patients, with clinical information available, reported specific symptoms of the Delta VOC. Phylogenetic analysis identified nine clusters characterized by viruses isolated in Kahramanmaraş city. No clusters were intermixed with sequences from different regions of Turkey, validating a local transmission of infection. Sanger sequencing detected the usual mutations of the Delta VOC still maintained in the Omicron lineage and sublineage, such as L452R, T478K, D614G, and P681R mutations, resulting in a fitness advantage. Alternatively, ten unusual mutations were carried by new isolates in a restricted time span (one month), suggesting pressure to select specific S and N mutations, such as T696S in the S1/S2 cleavage site, V915I in HR1, and T24I and A359S, able to destabilize the N protein structure, as reported above. Even if Delta disappeared from the global landscape, the same mutation could be selected by new SARS-CoV-2 VOCs. Novel selected mutation during viral replication could impact on the transmissibility, pathogenicity of infection, and performance of molecular or antigenic diagnostic tests, since both the S and N genomic regions are widely used in clinical practice to diagnose SARS-CoV-2 infection. Despite the analysis being performed on samples collected in August 2021, our molecular investigation highlights the need to improve the power of sequencing worldwide and to monitor viral evolution in different geographic areas.


O-linked N-acetylglucosamine (O-GlcNAc) glycosylation, a prevalent protein post-translational modification (PTM) that occurs intracellularly, has been shown to crosstalk with phosphorylation and ubiquitination. However, it is unclear whether it interplays with other PTMs. Here we studied its relationship with ADP-ribosylation, which involves decorating target proteins with the ADP-ribose moiety. We discovered that the poly(ADP-ribosyl)ation “eraser”, ADP-ribose glycohydrolase (PARG), is O-GlcNAcylated at Ser26, which is in close proximity to its nuclear localization signal. O-GlcNAcylation of PARG promotes nuclear localization and chromatin association. Upon DNA damage, O-GlcNAcylation augments the recruitment of PARG to DNA damage sites and interacting with proliferating cell nuclear antigen (PCNA). In hepatocellular carcinoma (HCC) cells, PARG O-GlcNAcylation enhances the poly(ADP-ribosyl)ation of DNA damage-binding protein 1 (DDB1) and attenuates its auto-ubiquitination, thereby stabilizing DDB1 and allowing it to degrade its downstream targets, such as c-Myc. We further demonstrated that PARG-S26A, the O-GlcNAc-deficient mutant, promoted HCC in mouse xenograft models. Our findings thus reveal that PARG O-GlcNAcylation inhibits HCC, and we propose that O-GlcNAc glycosylation may crosstalk with many other PTMs. O-linked N-acetylglucosamine (O-GlcNAc) glycosylation is a post-translational modification (PTM) that is installed onto the Ser/Thr residues of nucleocytoplasmic proteins to mediate protein-protein interactions, and change protein enzymatic activity, stability or localization , 2. Sinc+)  or poly-ADP-ribosylation (PARylation)  . Writersylation) . Its eraylation) .Both O-GlcNAcylation and PARylation function in chromatin metabolism and stress response, which are critical for DNA repair, cell cycle, and cell death. Both PTMs are enriched in the nucleus and chromatin, with fundamental roles in the DNA damage response , 8. In rPARP inhibitors are intensely investigated and four have been widely used in clinical settings . Synthete.g., c-Myc)  at its N-terminus . During , c-Myc) . Both mR, c-Myc) , suggestHerein we found that PARG is O-GlcNAcylated. Through electron-transfer dissociation (ETD) mass spectrometry (MS), we identified Ser-26 as a major O-GlcNAcylation site. PARG O-GlcNAcylation promotes its nuclear retention and chromatin recruitment during DNA damage. In HCC cells, PARG O-GlcNAcylation promotes DDB1 PARylation, downregulates its auto-ubiquitination and increases its stability, resulting in decreased c-Myc. Our work suggests that PARG O-GlcNAcylation suppresses HCC, revealing a link between O-GlcNAcylation and PARylation.A revealed, endogenous PARG interacts with OGT. Then cells were transfected with HA-OGT and Flag-PARG plasmids, and the cell lysates were IPed with anti-HA (B) or anti-Flag (C) antibodies; the results showed a reciprocal coIP between the two overexpressed proteins. Recombinant GST-OGT proteins were also utilized in GST-pulldown assays (D), and again GST-OGT could pulldown Flag-PARG, indicative of the binding between OGT and PARG. Then we directly tested if PARG is O-GlcNAcylated (E). By supplementing the medium with glucose and Thiamet-G  as previously described (F). When cells were treated with TMG, endogenous PARG was also O-GlcNAcylated (F). Taken together, PARG is O-GlcNAcylated.To examine if PARG is O-GlcNAcylated, we first assessed if PARG associates with OGT. 293T cell lysates were immunoprecipitated (IPed) with anti-PARG antibodies and immunoblotted (IBed) with anti-OGT antibodies. As Figure 1 anti-HA B or antinti-Flag C antibodn assays D, and agAcylated E. By supescribed , we obseescribed F. When cAcylated F. Taken A), close to its NLS. We constructed the S26A mutant accordingly, and the RL2 Western blotting showed ∼50% reduction in the mutant (B and C), suggesting that Ser-26 is a major O-GlcNAc site on PARG; it is possible that PARG harbors other O-GlcNAc sites. Interestingly, Ser-26 is conserved in some mammals , but not in the fly or other model organisms.To identify the O-GlcNAc site on PARG, Flag-PARG was transfected into 293T cells, and the medium was again enriched for O-GlcNAcylation by TMG plus glucose treatment . The celFig. 2Drosophila. Previous investigations have shown that PARG knockout mice show embryonic lethality  and found them to be relatively conserved (Parg (dPargdel) by CRISPR-CAS9. Similar to the recorded allele dParg27.1  human PARG or WT dParg, demonstrating that PARG is functionally conserved from insects to humans. Unfortunately, the human O-GlcNAc-defective PARG-S26A rescues the lethality of Pargdel as WT PARG, indicating that O-GlcNAc of PARG might not be essential for normal fly development. We reason that O-GlcNAcylation of dPARG probably functions under certain conditions such as nutrition stress and/or pathogen invasion. The role of dPARG O-GlcNAcylation still warrants further investigation.We also attempted to examine PARG O-GlcNAcylation in ethality , and PARat 25 °C , suggestonserved . We alsoonserved  . Then we used immunofluorescence microscopy to examine PARG localization . WT PARG localizes to the nucleus, consistent with its role in degrading PARylation in the nucleus , indicating that O-GlcNAcylation promotes nuclear localization of PARG. We also constructed mCherry-PARG-WT and -S26A. When examined under a microscope, mCherry-PARG-S26A also showed more localization in the cytosol .O-GlcNAcylation has been linked with the nuclear shuttling of many proteins , 28, so Fig. 3 nucleus . In starD). We also utilized the TMG + Glucose method to enrich for O-GlcNAcylation and found that chromatin-bound PARG was elevated, but the O-GlcNAc-deficient S26A mutant was not . These findings suggest that O-GlcNAcylation promotes chromatin association of PARG.As PARG has an important role in DNA damage , 29, we Fig. 3C and D prompted us to further examine the role of PARG O-GlcNAcylation in DNA damage. As PARG is recruited to DNA damage sites by PCNA (A). The results showed that S26A downregulated binding with PCNA. PARG catalyzes the dePARylation reaction, so we used the PARP1 protein as the substrate to measure the effect of PARG O-GlcNAcylation. As shown in Figure 4B, PARG-WT efficiently hydrolyzed the PAR chain on PARP1, but the PARG-S26A mutant did not.The results in  by PCNA , 19, we  by PCNA A. The reC). Consistently, Etoposide increased PARG O-GlcNAcylation by about 20% (C). Then we directly measured the recruitment of PARG to DNA damage sites via laser micro-irradiation. mCherry-PARG was used to visualize PARG recruitment. Upon laser micro-irradiation, PARG was accumulated at DNA damage stripes within 10 s, while the intensity of PARG-S26A was decreased to ∼60% (D and E). Taken together, our analysis revealed that O-GlcNAcylation promotes chromatin recruitment of PARG to DNA damage sites, probably via PCNA.We wondered whether PARG O-GlcNAcylation increases during DNA damage, so we used Etoposide to treat the cells and induce DNA damage C. Consisbout 20% C. Then w to ∼60% , D and EA). As DDB1 PARylation counteracts its auto-ubiquitination (B–D). In PARG-S26A transfected cells, DDB1 ubiquitination was upregulated , resulting in decreased protein stability as measured by cycloheximide (CHX) pulse-chase assays (C and D).As recently DDB1 has been shown to be a PARG substrate in HCC , we usedFig. 5tination , we thenE), leading to increased c-Myc protein levels . In sum, we found that increased cytosolic PARG in HCC cells decreased DDB1 PARylation and increased DDB1 auto-ubiquitination. Destabilized DDB1 attenuated c-Myc ubiquitination and elevated c-Myc levels.A downstream target of the DDB1-CUL4 E3 ligase is c-Myc . And PARFig. 5in vivo and thus constructed HCC cells stably expressing PARG-WT and -S26A plasmids (A). These cells were injected into nude mice to perform xenograft assays (B). PARG-S26A significantly increased the tumor size and weight compared to WT (C and D), suggesting that upregulated c-Myc in S26A cells promotes HCC (E).We wondered whether the OGT-PARG-DDB1-c-Myc axis functions plasmids A. These t assays B. PARG-Sed to WT , C and Dotes HCC E.Figure e.g. c-Myc), resulting in HCC tumorigenesis (E).In this report, we examined whether O-GlcNAcylation interacts with PARylation, and revealed that PARG is O-GlcNAcylated. The glycosylation modification ensures the nuclear localization of PARG and is essential for its recruitment to DNA damage sites. Increased cytoplasmic PARG-S26A almost abolishes PARylation of DDB1 in HCC, thus DDB1 auto-ubiquitination is elevated. Hence, downregulated DDB1 stabilizes its substrate , suggesting that O-GlcNAcylation functions positively for PARG-mediated DNA damage repair. In sum, our findings reveal a new layer of regulation of PARG, which could be utilized for targeting PARG to treat HCC.For therapeutic purposes, PARG inhibitors could be exploited for HCC suppression and they could be used synergistically with anti-PD-1 antibodies . DDB1 wamycoplasma contamination for all experiments. PARP1  and a C18 separating capillary column (75 μm × 15 cm) packed in-house with Luna 3 μm C18 ( cm and aMS data were acquired in data-dependent mode with a full MS scan (300–1700 M/z) in FT mode at a resolution of 60,000 followed by ETD MS/MS scans on the 10 most abundant ions with multiple charges in the initial MS scan. Automatic gain control (AGC) targets were 1e6 ions for Orbitrap scans and 5e4 for MS/MS scans. For dynamic exclusion, the following parameters were used: isolation window, 2 m/z; repeat count, one; repeat duration, 25 s; and exclusion duration, 25 s. The ETD activation time was 150 ms. Charge state dependent time and supplemental activation for ETD were enabled.Homo sapiens database (https://www.expasy.org/) . Carbamidomethyl (Cys) was chosen as a static modification, and oxidation (Met) was chosen as a variable modification. Mass tolerance was 10 ppm for precursor ions and 0.6 Da for fragment ions. Maximum missed cleavages were set as 2. Peptide spectral matches (PSM) were validated using the Percolator algorithm, based on q-values at a 1% FDR at both the peptide and protein levels.Data processing was carried out using Thermo Proteome Discoverer 2.4 using a SwissProt Indirect immunofluorescence staining was carried out as described previously . AntibodSCR_003070) (link). The laser microirradiation experiments were repeated three times, with 10 cells per experiment.U2OS cells were grown on a confocal dish and then irradiated with a 365 nm pulsed nitrogen UV laser  . Real-time images were taken every 10 s with a Dragonfly confocal imaging system (Andor). Images were quantitated with ImageJ . After inoculation, tumor volumes were measured from day 5 to day 14. On the 14th day, tumors were dissected, and the tumor volume was calculated according to the following formula: volume = ((4 × 3.14/3) × (L/2) × (W/2) × (D))/2. The tumor weights were measured at necropsy. The mice were obtained from the Animal Research and Resource Center, Yunnan University, with the Certification NO. SCXK(Dian) K2021 to 0001. All animal work procedures were approved by the Animal Care Committee of Yunnan University.A series of pilot studies was first undertaken to test the feasibility of transplanting Huh7 cell lines. Huh7 cells were infected with lentivirus of pHAGE-FLAG-VET, pHAGE-FLAG-PARG and pHAGE-FLAG-S26A, respectively, and then screened by puromycin (10 mg/ml) to obtain stable Huh7 cells for VEC, PARG-WT and PARG-S26A. The transplantation protocol followed published guidelines , and 6-wvia the PRIDE (PXD041119 and 10.6019/PXD041119.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium he PRIDE  partner This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Wolbachia infection on the biological characteristic of fruit fly Drosophila melanogaster. Results showed that high temperature and Wolbachia infection had a two-way interaction effect on hatching rate, developmental durations, emergence rate, body weight and body length of F1, F2 and F3 flies, and the interaction effect also existed on oviposition amount of F3 flies, and on pupation rate of F2 and F3 flies. The study also showed that high temperature stress deceased the Wolbachia vertical transmission efficiency between generations, and both high temperature stress and Wolbachia infection can reduce the survival of D. melanogaster. The results indicated that high temperature stress and Wolbachia infection had negative effects on the growth and development of D. melanogaster.This study explored the interaction effects of high temperature stress and Wolbachia in the host, but only a few papers reported the effect of the interaction between high temperature and Wolbachia on the biological characteristic of the host. Here, we set four treatment Drosophila melanogaster groups: Wolbachia-infected flies in 25 °C (W+M), Wolbachia-infected flies in 31 °C (W+H), Wolbachia-uninfected flies in 25 °C (W-M), Wolbachia-uninfected flies in 31 °C (W-H), and detected the interaction effect of temperature and Wolbachia infection on the biological characteristic of D. melanogaster in F1, F2 and F3 generations. We found that both temperature and Wolbachia infection had significant effects on the development and survival rate of D. melanogaster. High temperature and Wolbachia infection had interaction effect on hatching rate, developmental durations, emergence rate, body weight and body length of F1, F2 and F3 flies, and the interaction effect also existed on oviposition amount of F3 flies, and on pupation rate of F2 and F3 flies. High temperature stress reduced the Wolbachia vertical transmission efficiency between generations. These results indicated that high temperature stress and Wolbachia infection had negative effects on the morphological development of D. melanogaster.It was reported that temperature affects the distribution of  Wolbachia is a kind of Gram-negative endosymbiotic bacteria that is widely distributed in arthropods and nematodes [Wolbachia [Wolbachia spreads within and between host species through vertical transmission and horizontal transmission. Vertical transmission refers to the transmission from oocyte to offspring through reproduction [Wolbachia can occur between different individuals of the same host, as well as between different species of host [Wolbachia on its hosts is one of the reasons why it was widely studied in recent years [Wolbachia infection is associated with a variety of reproductive anomalies in the host, including cytoplasmic incompatibility [Wolbachia also has many other impacts on the host. For example, the Wolbachia strain wMel can change the composition of gut commensal bacteria in Drosophila melanogaster [Wolbachia are less susceptible to mortality induced by a range of RNA viruses [Wolbachia infection significantly decreases developmental duration in a bean beetle Callosobruchus chinensis [Delia radicum [Wolbachia infection in a small spider Hylyphantes graminicola (Linyphiidae) can shorten the developmental duration of the host [Cimex lectularius (common bed bug) had nutritional dependence on their Wolbachia (Wb) symbiont, and the authors verified that Wb can provide B-vitamins for this bed bug through adding B-vitamin to the blood meal of aposymbiotic bedbugs [ematodes . Weinertolbachia . Wolbachoduction ,4. The h of host . Reprodunt years ,7. Wolbatibility ,9, parthtibility , male sttibility , feminiztibility ,13 and ftibility ,15. In anogaster . The fru viruses . Wolbachhinensis , but sig radicum . Su et athe host . Hickin  bedbugs .Wolbachia-infected D. melanogaster also indicated thermal impacts on the cost of carrying a symbiont [Temperature is a key environmental modulator of host–pathogen interactions, which constrains the rate of biological reactions and sets limits to performance and survival ,23. Tempsymbiont .Wolbachia and temperature on hosts. For example, Strunov et al. analyzed the effects of Wolbachia infection, Wolbachia variants, the host’s sex and temperature on developmental life-history traits, lifespan and fecundity of D. melanogaster, and the results indicated that temperature and infection had highly significant effects on fecundity, and significant interactions were found between temperature and infection. They also found significant two-way interactions between temperature and infection on longevity of D. melanogaster and no interactions existed between infection and temperature on juvenile development time [D. melanogaster [Wolbachia infection nor variant, nor interactions with temperature on juvenile development time. To further explore the interaction between temperature and Wolbachia on the development of D. melanogaster, in this study, we set four treatment group flies: Wolbachia-infected flies cultured in 25 °C (W+M), Wolbachia-infected flies in 31 °C (W+H), Wolbachia-uninfected flies in 25 °C (W-M), Wolbachia-uninfected flies in 31 °C (W-H), and detected oviposition amount, hatching rate, developmental duration, pupation rate, emergence rate, adult body weight, adult body length, adult lifespan and survival rate in each group, and we then analyzed the interaction between high temperature stress and Wolbachia infection on D. melanogaster.So far, extensive research was conducted on the interaction between symbiotic bacteria and their hosts, as well as the interaction between temperature and symbiotic bacteria. Only a few papers studied the interaction between ent time . Howevernogaster . By contnogaster  also indD. melanogaster were collected in August 2018 from Shahu park , which were identified based on morphology. Gravid female flies were reared individually on standard treacle–semolina–yeast–agar media in an artificial climate incubator . After each female gravid fly laid eggs, the female adult flies were then used to extract DNA for Wolbachia infection screening and Wolbachia strain diagnosis. DNA of single flies were extracted using DNA extraction kit . Wolbahica infection status of D. melanogaster was detected with Wolbachia-specific primers and conditions as described by Braig et al. (1998) [Wolbachia strain of D. melanogaster were diagnosed through amplifying a variable tandem repeat region of the Wolbachia genome (VNTR-141) [wMel variants, and isofemale lines of wMel D. melanogaster (W+) were established in laboratory. Isofemale lines of Wolbachia-uninfected flies (W-) was subsequently generated by tetracycline treatment following established protocol [Wolbachia-free by PCR using Wolbachia surface protein gene (wsp).. (1998) . WolbachNTR-141) ,34. Thenprotocol  and conf+ and W- flies from the isofemale lines reared in laboratory, and 4 treatment combinations were set: W+ flies cultured in 25 °C (W+M), W+ flies in 31 °C (W+H), W- flies in 25 °C (W-M), W- flies in 31 °C (W-H). In this study, through preliminary experimental testing, the optimal high temperature for the survival of D. melanogaster was 31 °C. Therefore, 31 °C was used as the temperature value for high temperature stress in this experiment.We selected W1 generation of W+M, W+H, W m and W-H populations, respectively, and reared with standard treacle–semolina–yeast–agar media. When the virginal flies were at the 5-day-old stage, five flies each  were selected randomly and were put into a Petri dish with standard treacle–semolina–yeast–agar media and cultured in artificial climate incubator ; After 6 h, the female and male adult flies were removed, and the number of eggs were recorded with a magnifying glass. Then, we calculated the hatching rate of each treatment, respectively, after 24 h. Then, the number of pupae, the number of emergence and the time from egg hatching to emergence were recorded successively. There were 12 biological replicates for each treatment.Virginal fruit flies  were collected individually within 8 h after emergence from F1 generation of W+M population. The same sampling method was applied to the F1 generation of W-M, W+H and W-H populations. Then, the body length and the weight of the flies from four treatments were measured .A total of 20 unmated female flies and 20 unmated male flies were selected within 8 h after emergence from F1 generation of W+M population and reared individually on standard treacle–semolina–yeast–agar media in an artificial climate incubator . The same sampling method was applied to the F1 generation of W m (cultured in 25 ± 0.5 °C), W+H (cultured in 31 ± 0.5 °C) and W-H (cultured in 31 ± 0.5 °C) populations. The number of dead flies from four treatments was recorded, respectively, every 24 h.A total of 30 unmated flies were selected within 8 h after emergence from F1 generation of W+M, W+H, W m and W-H populations were reared individually on standard treacle–semolina–yeast–agar media in an artificial climate incubator . After these gravid female individuals laid eggs, which then developed into adult flies through larvae and pupae stages, we obtained the F2 generation fruit flies. We obtained F3 generation flies of W+M, W+H, W m and W-H populations from gravid female flies of F2 generation. The growth and development characteristics and lifespan of F2 and F3 generation flies of W+M, W+H, W m and W-H populations were also recorded using the same method as those of the F1 generation.Gravid female flies from FD. melanogaster published in the Genebank database. We used the expression of wsp  to detect the density of Wolbachia. The β-actin gene was used as a reference sequence . RT-qPCR was performed according to the method of Livak and Schmittgen [−ΔΔCt method was used to calculate the relative expression of genes [Wolbachia vertical transmission efficiency through relative expression of wsp (Wolbachia surface protein gene) in F1, F2 and F3 generation of W+M and W+H flies. Five unmated W+M flies (within 6 h after emergence) were pooled for a sample for RNA extraction in F1, F2 and F3 generation, and five unmated W+H flies (within 6 h after emergence) were pooled for RNA extraction in each generation. There were three biological replicates per generation.RNA for each treatment was extracted by Trizol Method, and cDNA was obtained by reverse transcription kit . qPCR was conducted by SYBR Green qPCR mix  in real-time fluorescence quantitative PCR instrument . The primers were designed by DNAStar software according to the sequences of actin reference genes of hmittgen . The 2−Δof genes . We deteWolbachia infection. If there was an interaction between the two factors, Tukey HSD was used for multiple comparisons. If there was no interaction between the two factors, Student’s t-test was used for pairwise comparisons. Three-way ANOVA was used to analyze the interaction among temperature, Wolbachia infection and host’s sex. The survival curves were analyzed using the Kaplan–Meier method (log-rank test). SPSS (26.0) and Graph Pad Prism7 were used for data analysis.We first used two-way ANOVA to analyze the interaction between temperature and 1 generation D. melanogaster flies, we found that temperature and Wolbachia infection had no interaction effect on oviposition amount and pupation rate , but there was interaction effect on hatching rate, developmental durations and emergence rate  . Compared with W+M and W-H groups, the hatching rates and emergence rates were significantly lower in W+H group (p < 0.05) , and the oviposition amount of flies in 31 °C was significantly higher than that of the flies in 25 °C under the same Wolbachia infection status   (t-test showed that Wolbachia infection and high temperature had an effect on the oviposition amount and pupation rates of F1 generation D. melanogaster.In F001) see . W m fly < 0.05) B,E, whicduration C, which < 0.001) A. The pu< 0.001) D. An ana2 generation flies . W-M fly group showed the highest hatching rate, pupation rate and emergence rate. Compared with W-M group, the hatching rates, pupation rate and emergence rates were significantly lower in W+M, W-H and W+H groups (p < 0.05). In addition, compared with W-M group, the developmental duration was longer in W+H, W-H and W+M groups (p < 0.05). The oviposition amount of W+ flies was significantly lower compared to the W- flies at the same temperature . The oviposition amount of flies in 31 °C was significantly lower than that of the flies in 25 °C under the same Wolbachia infection status   A.3 generation flies . W-M fly group showed the highest oviposition amount, hatching rate, pupation rate and emergence rate. Compared with W-M group, the oviposition amount, hatching rates, pupation rate and emergence rates were significantly lower in W+M, W-H and W+H groups (p < 0.05). In addition, compared with W-M group, the developmental duration was longer in W+H, W-H and W+M groups (p < 0.05).In Flies see , we founWolbachia infection and temperature on the effect of the body weight in F1, F2 and F3 generation flies . The interaction between Wolbachia infection and sex on the body weight of F1 generation flies was found , and temperature and sex had an interaction effect on the body weight of F2 and F3 generation flies . There was a significant interaction effect among Wolbachia infection, temperature and sex on the weight of F3 generation flies  , while W+H fly group had the lowest body weight in F1, F2 and F3 generation  (We found a significant interaction between 011) See . W-M flye flies) .Wolbachia infection, temperature and sex on the body length of F1, F2 and F3 generation flies  , while W+H fly group had the lowest body length in F1, F2 and F3 generation  (We found a significant interaction effect between 001) See . W-M flye flies) .Wolbachia negative flies, the survival rate of Wolbachia positive flies from F1, F2 and F3 generation at the same temperature were significantly decreased    . Compare< 0.001) . The reswsp of the W+ flies reared at 31 °C from F1 generation to F3 generation decreased gradually , while there was no significant difference of wsp expression among F1, F2 and F3 generations of W+ flies reared at 25 °C   . The resWolbachia are widely distributed in arthropods, and approximately 52% of arthropods were infected with Wolbachia [Wolbachia on host reproduction and fitness [Wolbachia in hosts were also reported widely [Wolbachia and temperature on hosts [+M, W+H, W-M and W-H) to detect the difference of the growth, development, adult lifespan and Wolbachia distribution of D. melanogaster in F1, F2 and F3 generations, and tested the interaction of temperature and Wolbachia infection on the biological characteristics of host. The result showed that temperature stress and Wolbachia infection had interaction on the growth and development of flies. fitness ,38, and d widely . So far,on hosts . In thisWolbachia infection affected the fecundity and development of D. melanogaster. High temperature stress and Wolbachia infection showed an interaction on the fecundity and development of D. melanogaster. Specifically, the short-term high temperature stress (stress in F1 generation) increased the oviposition amount and shortened the pupation rate of D. melanogaster, which seemed to have a positive impact on fecundity of the host. However, long-term interaction between temperature and Wolbachia infection  can reduce the oviposition amount, hatching rate, pupation rate, emergence rate and prolong the development duration, which is a negative effect on the host. Strunov et al. [Wolbachia and temperature interactions on the host in the short term high temperature stress, and they found that the development time, femur lengths and the total number of ovarioles were significantly affected by higher temperature in F1 generation D. melanogaster; moreover, both Wolbachia and temperature had effects on longevity and female fecundity of F1 generation D. melanogaster, and significant two-way interactions between temperature and Wolbachia infection were found on fecundity and longevity of F1 generation. In our study, we not only detected the two-way interactions between temperature and Wolbachia infection on the F1 generation D. melanogaster, but we also tested the interaction between temperature and Wolbachia infection on the F2 and F3 generation. These results indicated that high temperature stress and Wolbachia infection had negative effects on the morphological development of D. melanogaster. This may be a morphological adaptation to extreme temperature. Research showed significant differences between the survival curves of infected flies and uninfected controls at both 25 °C and 29 °C [1, F2 and F3 generation, and we found that both high temperature stress and Wolbachia infection can reduce the survival of D. melanogaster.Our results showed that both high temperature stress (31 °C) and v et al.  studied nd 29 °C . In thisWolbachia-infected hosts prefer cooler temperatures and might be likely to seek out cooler microclimates, which would reduce exposure to higher temperature and lessen the fitness consequences of high temperatures [D. melanogaster exhibits strong circadian and neurally controlled temperature preference behavior, which centers around 24–27 °C [Wolbachia-infected flies prefer a cooler mean temperature than uninfected flies. Therefore, we suspect that Wolbachia-infected flies may be able to reduce the survival pressure caused by heat stress, reduce the impact of high temperature on their growth and development and reduce their own heat generation.Research showed that eratures . D. mela24–27 °C ,42. The 24–27 °C  showed tWolbachia infection had an interaction effect on oviposition amount, hatching rate, developmental durations, pupation rate and emergence rate of F3 generation flies. Compared with W-M group, oviposition amount, hatching rate, pupation rate and emergence rate of F3 generation flies in W+H group significantly decreased, while the developmental durations in W+H group were significantly longer than that in W-M group. These results indicated that Wolbachia infection imposed a cost to fly hosts at 31 °C, which resulted in flies laying fewer eggs compared to the W-M group. Due to limited nutrition obtained from the mother in the egg, it subsequently leads to low hatching rate, pupation rate, emergence rate and longer developmental period. Our results provided evidence that endosymbionts impose a cost on their host under physiological stress.Endosymbionts rely on their hosts for nutrition, and they can impose a cost on their host ,44. Cost


Pomotrelvir is a novel Mpro inhibitor that has recently completed a phase 2 clinical trial. In this report, we demonstrated that pomotrelvir is a potent competitive inhibitor of SARS-CoV-2 Mpro with high selectivity against human proteases. In the enzyme assay, pomotrelvir is also active against Mpro proteins derived from human coronaviruses CoV-229E, CoV-OC43, CoV-HKU1, CoV-NL63, MERS, and SARS-CoV. In cell-based SARS-CoV-2 replicon and SARS-CoV-2 infection assays, pomotrelvir has shown potent inhibitory activity and is broadly active against SARS-CoV-2 clinical isolates including Omicron variants. Many resistance substitutions of the Mpro inhibitor nirmatrelvir confer cross-resistance to pomotrelvir, consistent with the finding from our enzymatic analysis that pomotrelvir and nirmatrelvir compete for the same binding site. In a SARS-CoV-2 infection assay, pomotrelvir is additive when combined with remdesivir or molnupiravir, two nucleoside analogs targeting viral RNA synthesis. In conclusion, our results from the in vitro characterization of pomotrelvir antiviral activity support its further clinical development as an alternative COVID-19 therapeutic option.The unprecedented scale of the COVID-19 pandemic and the rapid evolution of SARS-CoV-2 variants underscore the need for broadly active inhibitors with a high barrier to resistance. The coronavirus main protease (M In addition, the other four human CoVs  are believed to result in 5%–30% of “common colds,” for which therapeutic intervention is generally limited to over-the-counter symptomatic relief (Coronaviruses (CoVs) are enveloped, positive-sense, single-stranded RNA viruses; seven of which are known to infect humans  –7. Whic relief .pro) is essential for viral replication by processing the majority (11 of 16 cleavages) of the viral polyprotein into individual functional proteins. The liberated mature proteins are subsequently assembled into the replication machinery and participate in the transcription of new viral RNA which are packaged into virions to produce infectious CoV progeny virus , in which Cys145 acts as a nucleophile and His41 functions as the acid base in the proteolytic process. Mpro exclusively cleaves polypeptides after a Gln residue, and no known human protease displays the same cleavage specificity, which favors the discovery of Mpro inhibitors with high selectivity and low toxicity to host cells. Therefore, Mpro has been an attractive drug target in the development of SARS-CoV-2 inhibitors  is the only SARS-CoV-2 Mpro inhibitor with NDA approval in the United States for treating high-risk patients . A second Mpro inhibitor (ensitrelvir) has received emergency regulatory approval for the treatment of high-risk COVID-19 patients only in Japan (https://www.fda.gov/media/143189/download). The Mpro inhibitor nirmatrelvir requires co-administration with RTV, which introduces significant drug-drug interaction considerations in high-risk patients who are also on medications metabolized by the cytochrome P450 3A pathway  and phase 2 studies in otherwise healthy COVID-19 patients not at risk of developing serious diseases (NCT05543707). In this report, we describe pomotrelvir potency and mechanism of action, broad activity against SARS-CoV-2 clinical isolates, combination studies with other SARS-CoV-2 inhibitors, and cross-resistance profile compared with nirmatrelvir.Two classes of direct-acting antiviral (DAA) drugs have received worldwide or US regulatory Emergency Use Authorization or New Drug Authorization (NDA) approval to treat SARS-CoV-2 infections in high-risk patients: molnupiravir and remdesivir are inhibitors targeting the viral RNA-dependent RNA polymerase (RdRp) ; in the in Japan . Even thin Japan . The virpro proteins alone and in co-complex with Mpro inhibitors. The co-crystal structure of pomotrelvir bound to SARS-CoV-2 Mpro was solved at a resolution of 2.15 Å (pro inhibitor nirmatrelvir (PF-07321332) in which the nitrile substituent forms a reversible covalent thioimidate adduct with the catalytic Cys145 (pro kinetic assay using highly sensitive microfluidics capillary electrophoresis to detect cleaved peptide substrate. As shown in Ki) was calculated to be 2.7 nM; the alpha factor was 10.4 (>>1), suggestive of the competitive inhibition model (γ) approached ∞ when combinations of both inhibitors (1–8 nM) were present in the assay, demonstrating that pomotrelvir and nirmatrelvir compete for the same active site and their binding to the Mpro enzyme is mutually exclusive.Pomotrelvir  was discf 2.15 Å . A covalon model . An enzyon model  between pro enzymes derived from other human CoVs was evaluated in an endpoint IC50 assay. Pomotrelvir inhibited wild-type (WT) SARS-CoV-2 Mpro with an IC50 of 24 nM. Similar activity (IC50 of 34 nM) was observed with the P132H variant, which is commonly observed in Omicron isolates and has been shown to have no effect on nirmatrelvir inhibition , cathepsin K (Ki = 0.289 µM), cathepsin B (Ki = 1.27 µM), and cathepsin L (Ki = 7.4 µM), respectively. These results demonstrated that pomotrelvir is highly selective against host serine and cysteine proteases.Potential off-target activity of pomotrelvir against host serine and cysteine proteases was evaluated in the microfluidic assay with relevant peptidyl substrates. As described above, pomotrelvir showed a The inhibitory activity of pomotrelvir against SARS-CoV-2 was assessed using three cell-based systems: (i) Pluripotent induced stem cell-derived human lung alveolar type 2 (iPS-AT2) cells were infected with passage 5 of SARS-CoV-2 WA-1 . Viral replication was quantified using a plaque assay to measure reduction in infectious viral titer and a RT-PCR assay to measure reduction in viral RNA copy number. (ii) A549-hACE2 cells overexpressing the human ACE2 receptor for SARS-CoV-2 were infected with SARS-CoV-2 NLuc, generated by inserting the nanoluciferase gene into the SARS-CoV-2 WA1 strain which allows viral replication to be quantified by measuring nanoluciferase activity. (iii) Huh7 cells were transfected with the SARS-CoV-2-EGFP replicon RNA; the viral replication was measured by the expression of the EGFP reporter. As summarized in Additionally, pomotrelvir antiviral activity was assessed against CoV-229E and CoV-OC43. Pomotrelvir was active in the CoV 229E and CoV-OC43 infectious virus CPE assays, with mean EC50 values of 180 nM and 380 nM, respectively . CytotoxThe effect of human serum on pomotrelvir antiviral activity was evaluated in the SARS-CoV-2 NLuc assay. As shown in pro region and the 11 Mpro cleavage sites were well conserved; the only substitution of note was the P132H change, which was observed in all Omicron isolates used in our study (data not shown).The activity of pomotrelvir against clinical isolates was evaluated in A549-AT cells (A549 cells overexpressing ACE2 and TMPRSS2). After infection of A549-AT cells, immunostaining of the viral nucleoprotein was used to monitor infection and determine levels of viral replication. pro inhibitors developed for SARS-CoV-2  compared with the WT protease. The few exceptions were enzymes containing substitutions Y54A, E166A, E166V, L167F, and H172Y (as single mutations or in combination with other substitutions), with catalytic efficiency ~5- to ~10-fold lower than that of the WT enzyme.Nirmatrelvir is one of the first MRS-CoV-2 , and resreported , 17–19In the pomotrelvir inhibition assay, cross-resistance was conferred by many nirmatrelvir resistance substitutions . AnalysiNo resistance (<threefold). Pomotrelvir was fully active against these 10 substitutions: G15S, T21I, L50F, K90R, T135I, H164N, Q189K, D248E, A260V, and T21I + L50F.pro assays with single substitutions A173V and T304I are necessary to fully evaluate the impact of these changes. A173V + T304I conferred 6.7-fold resistance, similar to the more complex combinations of T21I + A173V + T304I (6.1-fold) and T21I + C160F + A173V + V186A + T304I (5.7-fold), demonstrating that these additional amino acid substitutions did not change the resistance level of the double mutant. T21I + L50F + A193P + S301P conferred 4.9-fold resistance. Among the substitutions in this quadruple combination, T21I + L50F was shown to confer no resistance, so the observed reduction in pomotrelvir inhibition was likely due to A193P and/or S301P.Low-level resistance (3- to 10-fold). Found in this group are single substitutions Y54A and S144A. To deconvolute combinations of substitutions, we noticed that while T21I and L50F showed no resistance, double mutants T21I + A173V and L50F + A173V both conferred four to fivefold resistance. Assuming changes at T21 or L50 do not affect distal sites such as A173, we postulate that A173V could confer the same four to fivefold resistance as a single substitution. T21I + T304I, L50F + T304I, and T135I + T304I showed similar levels of resistance (~threefold), suggesting that T304I is likely to confer the same ~threefold resistance as a single substitution. However, the possibility cannot be ruled out that combinations of T21I or L50F with substitutions such as A173V and T304I may act together via yet-unidentified mechanisms; MModerate-level resistance (10- to 50-fold). Found in this group are single substitutions F140A, E166A, and L167F. The double-mutant L50F + E166A showed similar resistance to the single-substitution E166A (36-fold vs 35-fold); the double-mutant T21I + S144A conferred 13-fold resistance, within twofold of that by the single-mutant S144A (6.7-fold). The results showed that combination with T21I or L50F did not significantly change the resistance of single substitutions. T21I + S144A + T304I conferred 23-fold resistance, approximately multiplicative of resistance by S144A (6.7-fold) and T304I (estimated as ~threefold) as single substitutions, suggesting that S144A and T304I each independently contributed to the resistance of the combined mutant. Of the two combinations containing F140L, F140L + A173V conferred 10-fold resistance and L50F + F140L + L167F + T304I conferred 40-fold resistance. Only data on F140A (14-fold resistance) was available, and testing of the single substitutions F140L is required to deconvolute the aforementioned combinations.pro assay. The triple-mutant L50F + E166A + L167F conferred the highest level of resistance (188-fold), which was approximately multiplicative of the fold increase caused by E166A (36-fold) and L167F (13-fold) as single substitutions, suggesting that E166A and L167F each independently contributed to the resistance of the combined mutant.High-level resistance (>50-fold). Found in this group are single substitutions E166V and H172Y. E166V conferred 51-fold resistance, and combination of E166V with T21I or L50F did not change its resistance level. The double-mutant H172Y + P252L conferred 62-fold resistance, similar to that conferred by the single-substitution H172Y (71-fold) suggesting that P252L had no impact on pomotrelvir activity in the Mpro inhibitor GC376 .The activity of the control Mor GC376  was simiTo further assess the effect of nirmatrelvir resistance substitutions on pomotrelvir activity, 12 mutations were selected and cloned into SARS-CoV-2-EGFP replicons. Four of the mutant replicons  generated no or a low number of GFP-positive cells after transfection  and wereCheckerboard assays  were perpro with high selectivity against host proteases; it is broadly active against SARS-CoV-2 clinical isolates, including Omicron variants. Pomotrelvir is also shown to inhibit other human CoVs in enzyme and cell-based infection assays. Combination studies demonstrate additivity between pomotrelvir and two RdRp inhibitors remdesivir and molnupiravir, which is consistent with the fact that the two classes of inhibitor target different viral proteins , supporting the potential combination with remdesivir and molnupiravir in treating SARS-CoV-2 infections. In contrast, the binding exclusivity study has shown that pomotrelvir and nirmatrelvir to Mpro compete for the same binding site, suggesting that co-administration of the two Mpro inhibitors is not likely to offer additional improvement in efficacy.In this study, we have shown that pomotrelvir is a potent competitive inhibitor of SARS-CoV-2 Mpro assay. However, the resistance profiles of pomotrelvir and nirmatrelvir are not identical. For example, substitutions G15S, T135I, H164N, and D248E were reported by Pfizer to confer ~fourfold resistance in the Mpro assay  or with poor replication of mutant SARS-CoV-2 replicons . This observation suggests that there is a delicate balance between selection of resistance to Mpro inhibitors and cost to viral fitness and underlies the potential of developing next-generation Mpro compounds which can overcome resistance to current inhibitors like pomotrelvir and nirmatrelvir.Given the finding that pomotrelvir and nirmatrelvir compete for the same binding site, it is not surprising that many  nirmatrelvir resistance substitutions confer cross-resistance to pomotrelvir. Of particular interest is the substitution E166V; it was detected in nirmatrelvir/ritonavir-treated patients  and confro assay  but no cpro enzyme assay with those from the mutant SARS-CoV-2-EGFP replicon assay, we detected two types of discordance. For the double-mutant T21I + L50F , no resistance was observed in the Mpro assay, but a ~fourfold increase in EC50 and EC90 was seen in the replicon assay. On the contrary, for a series of mutants containing E166A  with low enzyme activity, the IC50 fold increase in the Mpro assay  is greater than the EC50 fold increase in the replicon assay . It is plausible that the Mpro enzyme assay is more sensitive in measuring resistance with poorly active enzymes , whereas the cell-based replicon assay is better equipped to detect changes in susceptibility caused by the global impact on viral replication such as improved polyprotein processing due to a more active Mpro . Further studies with additional Mpro mutants are required to confirm this observation, but it underscores the importance of conducting both enzymatic and cell-based assays in evaluating the impact of Mpro substitutions on SARS-CoV-2 replication and susceptibility to inhibitors.When comparing the results from the mutant Min vitro characterization of pomotrelvir antiviral activity and phase 1 clinical studies . At this stage of the COVID-19 pandemic, with lower viral burden and more rapid symptom resolution due to high levels of underlying population immunity, it has become more challenging to demonstrate the virologic effect and clinical benefit of SARS-CoV-2 therapeutics on the non-high risk population . Other examples of Mpro inhibitors in late-stage development include STI-1558, currently in the phase 3 clinical trial in adult subjects with mild or moderate COVID-19 (NCT05716425). Recently, the topline results of the phase 2 trial of the Mpro inhibitor EDP-235 have been announced . In similar non-high-risk patients, EDP-235 failed to demonstrate the effect on viral RNA decline or infectious viral load in nasal samples, again highlighting the challenge of demonstrating a virologic effect on this patient population. Despite the progress and challenges, as long as COVID-19 remains as a major public health issue, efforts from public and private sectors should continue to develop safe, highly effective, and orally administered agents without drug-drug interactions for treatment of SARS-CoV-2 infections. Furthermore, a pan-coronavirus inhibitor with high barrier to resistance would be of great value to the preparedness of future coronavirus pandemics. We believe the lessons learned from the development of pomotrelvir will contribute to the understanding of SARS-CoV-2 replication and the discovery of novel therapeutics for COVID-19 and other human CoV infections.Results from the  studies  supportePomotrelvir and nirmatrelvir were synthesized by Pardes Biosciences . GC376 was provided by WuXi AppTec . Compounds were dissolved in DMSO and stored at −20°C.A549-hACE2 cells overexpressing the human ACE2 receptor for SARS-CoV-2 were obtained from Dr. Ralph Baric at UNC Chapel Hill  and cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin and streptomycin, and 1× minimal essential amino acids . Pluripotent stem cell-derived human lung alveolar type 2 (AT2) cells were cultured as described previously  and platpro (nsp5) sequence, mutations were designed by SnapGene software obtained from GSL Biotech, LLC , and introduced into the plasmid containing the SARS-CoV-2 WT replicon using the two-step red-mediated recombination method (A passage 3 stock of the SARS-CoV-2 WA-1 isolate MN985325) was obtained from the CDC  and passed twice in Vero E6 cells to generate high-titer passage 5 stock for experiments described in this study. SARS-CoV-2-expressing nanoluciferase (SARS-CoV-2 NLuc) was generated by cloning the seven genomic fragments from the clinical isolate WA1 separately into vector plasmids. The nanoluciferase gene was introduced into the ORF7 , 25. The was obtan method . The cloVmax), Michaelis constant (Km), and inhibition constant (Ki), pomotrelvir was tested in the presence of 3 nM SARS-CoV-2 Mpro  and 2.3–300 μM (1:2 dilution starting at 300 µM) of substrate peptide (FAM-TSAVLQSGFRK-NH2) in assay buffer  at room temperature; cleavage was measured by microfluidic electrophoresis using Caliper’s LabChip 3000 . Initial velocity (v) was derived from progress curves, Vmax, and Km were determined using the Michaelis-Menten model. To determine the inhibition constant Ki, a global fit was performed using the following general mixed inhibition model:The assay was performed by Nanosyn Inc. . To determine maximal velocity  was very close to enzyme concentration (3 nM), Morrison correction was made to account for compound depletion using the following equation:in which [Ki of 2.7 nM. All local curve fittings and global fit were performed by using XLFit software .The final analysis returned a To evaluate the binding exclusivity between pomotrelvir and nirmatrelvir, initial velocity was determined in the presence of pomotrelvir (0–20 nM) and nirmatrelvir (0–8 nM) with 3 nM enzyme and 1 µM peptide substrate in the assay buffer described above. The binding mode was assessed by the Yonetani-Theorell equation:V12 is the initial velocity in the presence of the two test compounds, Vo is the initial velocity in the control sample (without compounds), I1 and I2 are concentrations of the two test compounds, Ki1 and Ki2 are constants of interaction of the two test compounds with Mpro enzyme, and γ is the binding exclusivity coefficient. If γ = 1, the two compounds bind independently of one another to two different binding sites; for compounds that positively affect each other’s binding, γ < 1. If γ has a finite value > 1, the two compounds demonstrate antagonistic binding; a finite but large (>>1 to ∞) value of γ indicates that the two compounds bind to the same site on the enzyme.where Evaluation of pomotrelvir inhibition of host proteases in the kinetic assay was performed by Nanosyn Inc. following protocols established by the facility.pro, codon-optimized cDNA of SARS-CoV-2 Mpro (QIZ13716.1_ORF1a) was cloned into the pET28a vector, expressed in E. coli, and purified. To generate mutant SARS-CoV-2 Mpro proteins, cDNAs containing resistance substitution(s) were synthesized and cloned into the pET28a vector. The reaction buffer consisted of 20 mM of Tris-HCl (pH7.3), 100 mM of NaCl, 1 mM of EDTA, 5 mM of TCEP, and 0.1% BSA. The cleavage of substrate (Dabcyl- KTSAVLQ‖SGFRKM-Edans) obtained from Genscript  was measured at 30°C by a SpectraMax M2e microplate reader . The kinetic parameters  of each mutant protease were determined using the Michaelis-Menten model. The enzyme and substrate concentration and reaction time were selected for each mutant Mpro to ensure that the assay was within the linear range, the signal-to-noise ratio (S/N) was ≥3, and the substrate concentration was one- to twofold greater than Km.The assay was performed by WuXi AppTec . To generate WT SARS-CoV-2 Mpro proteins from other human CoVs were also cloned, expressed in E. coli and purified by WuXi AppTec. The accession numbers and sequences of peptide substrates are shown as follows: CoV-229E_Mpro (AGW80947.1), YGSTLQAGLRKM; CoV-OC43_Mpro (AGT51517.1), KTSAVLQSGFRKM; SARS_Mpro (AAR87511.1), KTSAVLQSGFRKM; MERS_Mpro (ASU89965.1), KTSAVLQSGFRKM; CoV-NL63_Mpro (AGT51378.1), YNSTLQSGLKKM; and CoV-HKU1_Mpro (ARB07597.1), KTSAVLQSGFRKM. The assay conditions were similar to those of SARS-CoV-2 Mpro.Mg for 10 min at 10°C and set up for co-crystallization using the sitting drop method against reservoir containing 24.1% PEG 3350, 0.1 MES pH 7.5 and incubated at 20°C.The study was performed by Schrödinger Inc. . The SARS-CoV-2 Mpro protein provided by WuXi AppTec was diluted to 7 mg/mL and mixed with pomotrelvir at a 2:1 molar ratio in 20 mM Hepes pH 7.5, 50 mM NaCl and 2.5 mM DMSO and incubated for 60 min at 4°C. The sample was then clarified by centrifugation at 16,800 × Co-crystals were harvested and flash frozen in liquid nitrogen directly from the crystallization drop. X-ray diffraction data were collected at beamline BL13-XALOC, ALBA synchrotron, Cerdanyola del Vallès, Barcelona, Spain, using a wavelength of 0.979261 Å. The X-ray diffraction data were reduced using XDS , scaled,GTGCTCATGGATGGCTCTATTA-3′), reverse (5′CGTGCCTACAGTACTCAGMTC-3′) primers, and probe (5′-FAMACCTACCTTGMGGTTCTGTTAGAGTGGT-BHQl-3′). RNA copy numbers were interpolated from a standard curve produced with serial 10-fold dilutions of nsp4 gene RNA run on the same sample plate. Cell viability was determined by the Toxilight Bioassay Kit from Lonza  according to the manufacturer’s instructions. Luminescence was detected using a Synergy Hl Plate Reader .AT2 cells were infected with SARS-CoV-2 WA-1 at a multiplicity of infection (MOI) of 0.004 plaque-forming units (PFU)/cell in AT2 medium  for l hrA549-hACE2 cells were infected with SARS-CoV-2 NLuc at an MOI of 0.025. After incubation for 45 min, inoculum was removed, and cells were treated with titrations of pomotrelvir for 48 hr in growth media containing 10% FBS supplemented with 0, 5, 10, or 20% human serum . Viral replication was quantified by measuring nanoluciferase activity using the Nano-Glo Luciferase Assay System  according to the manufacturer’s specifications. Cell viability was determined by CellTiter-Glo Assay (Promega) according to the manufacturer’s instructions. Luminescence was detected using a Synergy H1 Plate Reader (BioTek).in vitro transcribed replicon RNA and seeded at 4,000 cells/well in 384-well plates containing serially diluted compounds and then cultured at 37°C for 1 d. The replication of SARS-CoV-2 replicon was measured by the expression of the EGFP reporter gene. Fluorescence intensity was determined using Acumen Cellista , and the antiviral activity of compounds was calculated based on the reduction of number of GFP-positive cells. The acceptable threshold of GFP signal was >30 GFP-positive cells/well. Cell viability was measured with CellTiter-Glo following manufacturer’s instructions.The assay was performed by WuXi AppTec. Huh7 cells were transfected with The assay was performed by Retrovirox Inc. . A549 cells overexpressing ACE2 and TMPRSS2 (Retrovirox Inc.) were plated in DMEM with 10% FBS for 24 hr followed by addition and incubation of serial dilutions of test compounds (in DMED with 2% FBS) for 1 hr at 37°C. Cultures were then infected with SARS-CoV-2 resuspended in DMEM with 2% FBS at MOI of 0.005 TCID50/cell. Cell culture media with the virus inoculum was not removed after virus adsorption. After 48 hr of incubation, the extent of infection was monitored by immunostaining using a monoclonal antibody against the SARS-CoV-2 nucleocapsid (NP) followed by incubation with horseradish peroxidase conjugated polyclonal antibodies against human IgG (HRP-donkey anti-human IgG). The expression of the viral antigen (NP) in infected cells were quantitated using a colorimetric readout (absorbance at 492 nm). Cell viability was determined with the CellTiter 96 AQueous One Solution cell proliferation assay (MTS) from Promega. The clinical isolates used in the study are listed as follows:MEX-BC2/2020, representative of the B.1 lineage, carrying the D614G mutation (GISAID database ID: EPI_ISL_747242), MEX-BC12/2021, representative of the B.1.1.7 lineage, referred as the “Alpha” variant of concern (GISAID database ID: EPI_ISL_2455241), MEX-BC10/2021, representative of the B.1.429 lineage referred as the “Epsilon” variant (GISAID database ID: EPI_ISL_2455244), MEX-BC15/2021, representative of the B.1.617.2 lineage, referred as the “Delta” variant of concern (GISAID database ID: EPI_ISL_2249247), MEX-BC18/2021, representative of the B.1.617.2 + AY.20 lineage, also referred to as the “AY.20 Delta” variant (GISAID database ID: EPI_ISL_9149176), MEX-BC16/2021, representative of the B.1.621 lineage, referred to as the “Mu” variant (GISAID database ID: EPI_ISL_6944671), RVX-01/2022, representative of the BA.1.1 sub-lineage of the Omicron variant of concern (GISAID database ID: EPI_ISL_10954105), USA/RVX05/2022, Omicron BA.2 sub-lineage (EPI_ISL_12470781), USA/RVX07/2022, representative of BA.2.12.1 sub-lineage of the Omicron variant of concern (EPI_ISL_13351626), MEX/BC28/2022, Omicron BA.4.1 sub-lineage (EPI_ISL_14319831), USA/RVX13/2022, Omicron BA.5.2.1 sub-lineage (EPI_ISL_14319830).In the CoV-229E assay, compounds diluted in assay medium  were added to MRC5 cells , which were then infected with 200 TCID50 per well of CoV-229E virus. The resulting cultures were incubated at 35°C and 5% CO2 for an additional 3 d, at which time the virus infection in the virus control  displayed significant CPE. In the CoV-OC43 assay, compounds diluted in cell culture medium were added to Huh7 cells , which were then infected with 100 TCID50 per well of CoV-OC43 virus. The resulting cultures were incubated at 33°C and 5% CO2 for an additional 7 days, at which time the virus infection in the virus control displayed significant CPE. The CPE was measured by CellTiter-Glo following the manufacturer’s manual.IC50, EC50, EC90, and CC50 values were calculated using the nonlinear regression model of log(inhibitor) vs response-variable slope (four parameters) by Graphpad Prism  or by Dotmatics software . IC50 and EC50 were defined as the drug concentration achieving the response halfway between the fitted bottom and top (maximum) inhibition%; EC90 was defined as the drug concentration achieving 90% inhibition between the fitted bottom and top (maximum) inhibition%. To calculate fold change, the activity  of each mutant was divided by the activity of the corresponding WT from the same experiment.https://synergyfinder.org/#!/) to calculate ZIP synergy scores A549-hACE2 cells were infected with SARS-CoV-2 NLuc at an MOI of 0.025 PFU/cell and incubated for 48 hr with 0.0013–1 μM pomotrelvir in combination with 0.02–2.5 μM remdesivir (RDV) or 0.0013–3 μM of pomotrelvir in combination with 0.04–2.5 μM NHC. Viral replication was quantified by measuring nanoluciferase activity using the Nano-Glo Luciferase Assay System (Promega). To carry out synergy analysis, the nanoluciferase signal expressed from SARS-CoV-2 NLuc infection treated with pomotrelvir × RDV or pomotrelvir × NHC was normalized to signals from cells treated with DMSO alone and percent inhibition was calculated. The resulting data sets served as input in SynergyFinderPlus software (y scores . When thLess than −10, the interaction between two drugs is likely to be antagonistic.From −10 to 10, the interaction between two drugs is likely to be additive.Larger than 10, the interaction between two drugs is likely to be synergistic.


Background: A fluid-attenuated inversion recovery (FLAIR) method eliminates the cerebrospinal fluid (CSF) signal, enhancing white matter lesion detection by enhancing the contrast between the lesion and CSF. Three-dimensional (3D) volume acquisition has the advantage of multiplanar reformation of contiguous slices yielding improved signal-to-noise ratios (SNRs) and contrast-to-noise ratios (CNRs). To our knowledge, there are only three studies comparing 3D- and 2D-FLAIR sequences with respect to multiple sclerosis (MS) lesions at 3 tesla.Aims and objectives: This study aimed to determine the efficiency of 3D-FLAIR in the detection of lesions of multiple sclerosis in terms of spatial and contrast resolutions in comparison with 2D-FLAIR sequences.Methodology: A total of 75 patients with MS undergoing magnetic resonance imaging (MRI) brain at the Department of Radiology, Krishna Institute of Medical Sciences (KIMS), Secunderabad, Telangana, India. This is an observational comparative study. Independent-samples t-tests were performed in the present study to compare the number of lesions detected. The measured CNR and SNR values were subjected to Mann-Whitney U test.Results: As a result of the 3D-FLAIR, more lesions were found as compared to 2D-FLAIR (p = 0.001). There was a greater CNRs for 3D-FLAIR images than for 2D-FLAIR images (p = 0.001). Lesions, CSF, white matter, and gray matter showed significantly higher SNRs with 3D-FLAIR (p = 0.001).Conclusion: 3D-FLAIR has exhibited greater sensitivity in detecting lesions associated with MS when contrasted with the 2D-FLAIR sequence. Significantly more lesions and higher SNRs and CNRs were detected with 3D-FLAIR in contrast to 2D-FLAIR. 3D-FLAIR may be considered the sequence of choice for MS imaging in the future. Magnetic resonance imaging (MRI) plays a chief role in the diagnostic workup of multiple sclerosis (MS) ,2. HenceInitially, 3D-FLAIR sequences were primarily conducted using a multislab approach, which led to the emergence of Venetian blind artifacts when the resulting images were remodeled . The sinFurthermore, almost no CSF flow artifacts were seen with the 3D-FLAIR sequence in brain imaging . In addiThis is an observational comparative study, and an informed consent form was obtained from all the included participants. The Institutional Human Ethics Committee (IHEC) of KIMSHEALTH and Dr. P. Raja Gopal, Director of Medical Education, issued approval . Seventy-five patients  with clinically definite MS undergoing MRI brain at the Department of Radiology, Krishna Institute of Medical Sciences (KIMS), Secunderabad, Telangana, India, were included in this study. Inclusion and exclusion criteriaAll the patients with clinically diagnosed MS undergoing MRI brain imaging at the department of radiology were included in the study.Patients with concomitant neurological disease in addition to MS; patients with a history of claustrophobia; patients with cardiac pacemakers, prosthetic heart valves, cochlear implants, or any metallic implants in the body; and patients who did not consent to be a part of the study were excluded from the study.MR image acquisitionA 16-channel phased-array HR SENSE NV 16 coil was used on a 3T MRI system . We have obtained an additional sequence (3D-FLAIR) along with routine MRI sequences (including 2D-FLAIR) for MS. 3D-FLAIR refers to single-slab 3D fast spin echo (FSE) T2 FLAIR, which is an advanced MRI sequence. Very long spin-echo trains are created by utilizing variable flip-angle refocusing radiofrequency (RF) pulses. As a result, this approach gives a slower signal decay when compared to traditional FSE sequences that employ a continuous flip angle of 180° (13). In addition, employing refocusing RF pulses with flip angles substantially below 180° can result in a reduction in the specific absorption rate (SAR).The 2D-FLAIR sequence was acquired in an axial plan, while the 3D-FLAIR sequence was obtained in the sagittal plane, which was then reformatted into axial slices. The width of slices of 2D-FLAIR and 3D-FLAIR was 5 mm and 0.9 mm, respectively. Imaging parameters used for the 2D- and 3D-FLAIR sequences were as follows: (a) repetition time msec/echo time msec, 10000/125; inversion time msec, 2800 and matrix, 300x205 for the 2D-FLAIR sequence (the remaining parameters are as follows: 5 mm section thickness with a 2 mm intersection gap and 230x230x142 mm field of view); (b) repetition time msec/echo time msec, 4800/315; inversion time msec, 1650 and matrix, 252x249 for the 3D-FLAIR sequence (the other variables were as follows: 0.9 mm section thickness with no intersection gap and 250x250x168 mm field of view).Image analysisOnly axial slices were used for the evaluation. Two neuroradiologists, each possessing a minimum of five years of experience in the field, assessed the lesions and reviewed the sequences in a randomized manner. Lesions were calculated and assigned to the following brain regions: periventricular (PV), non-periventricular/juxtacortical (NPV & JC), and infratentorial (IT) Figure . The anaStatistical analysisIn this study, we used IBM SPSS Statistics for Windows, version 17  to perform all statistical calculations. The total number of lesions identified on two sequences was compared by using an independent sample t-test. The CNRs and SNRs measured on the two sequences were calculated using a Mann-Whitney U test.Table When the 3D-FLAIR and 2D-FLAIR sequences were compared, the single-slab 3D-FLAIR sequence was dominant over the 2D-FLAIR sequence regarding the evaluation of the number of lesions and higher SNRs and CNRs. To our knowledge, there are only three studies comparing 3D- and 2D-FLAIR sequences regarding MS lesions at 3T MRI. 2D-FLAIR has a greater potential for lesion detection than traditional 2D spin echo sequences, as demonstrated in the literature ,14-18. MThe subtype of the 3D FSE T2 FLAIR sequence is the 3D-FLAIR sequence. Utilizing modulated flip-angle refocusing RF pulses enables the generation of extended echo trains, primarily because they yield a more gradual signal decay owing to their high output. In this way, more echoes are generated without causing extensive blurring and artifacts . In addi3D-FLAIR shows more lesions when 2D-FLAIR and 3D-FLAIR sequences are compared. This was due to the identification of small lesions that remained undetected on 2D-FLAIR images Figure -7. Moreo3D sequences offer advantages in terms of signal strength and contrast properties, even when utilizing small voxels ,24,25. OIn the present investigation, three reference tissues  showed significantly higher CNR values in 3D-FLAIR images  than in 2D-FLAIR images . It is plausible that improvements in MRI techniques will influence the diagnostic criteria for MS. The latest additions to the McDonald criteria, incorporating the MR criteria, have relied on imaging studies conducted at a magnetic field strength of 1.5T. The combination of 3T MRI with 3D-FLAIR sequence, as shown in our study, can be valuable in diagnosing MS.A single-slab 3D-FLAIR sequence identifies a greater number of lesions as compared to a 2D-FLAIR sequence. The upgraded presentation of 3D-FLAIR sequences can be recognized to the presence of a substantial quantity of thin, contiguous slices, high spatial resolution, and remarkable increase in the SNRs and CNRs provided by the technique. The effectiveness of 3D sequences in MS diagnosis is still under research and their potential. However, the confirmation of this will require larger-scale studies. The study indicates that 3D-FLAIR is superior to the conventional 2D-FLAIR with respect to MS imaging. 3D-FLAIR can take over 2D-FLAIR in MS imaging in the future. Diagnosis of MS at the initial phase can be expected by using high-field MRI combined with 3D sequences.


The Pneumoviridae family of viruses includes human metapneumovirus (HMPV) and respiratory syncytial virus (RSV). The closely related Paramyxoviridae family includes parainfluenza viruses (PIVs). These three viral pathogens cause acute respiratory tract infections with substantial disease burden in the young, the elderly, and the immune-compromised. While promising subunit vaccines are being developed with prefusion-stabilized forms of the fusion glycoproteins (Fs) of RSV and PIVs, for which neutralizing titers elicited by the prefusion (pre-F) conformation of F are much higher than for the postfusion (post-F) conformation, with HMPV, pre-F and post-F immunogens described thus far elicit similar neutralizing responses, and it has been unclear which conformation, pre-F or post-F, would be the most effective HMPV F-vaccine immunogen. Here, we investigate the impact of further stabilizing HMPV F in the pre-F state. We replaced the furin-cleavage site with a flexible linker, creating a single chain F that yielded increased amounts of pre-F stabilized trimers, enabling the generation and assessment of F trimers stabilized by multiple disulfide bonds. Introduced prolines could increase both expression yields and antigenic recognition by the pre-F specific antibody, MPE8. The cryo-EM structure of a triple disulfide-stabilized pre-F trimer with the variable region of antibody MPE8 at 3.25-Å resolution confirmed the formation of designed disulfides and provided structural details on the MPE8 interface. Immunogenicity assessments in naïve mice showed the triple disulfide-stabilized pre-F trimer could elicit high titer neutralization, >10-fold higher than elicited by post-F. Immunogenicity assessments in pre-exposed rhesus macaques showed the triple disulfide-stabilized pre-F could recall high neutralizing titers after a single immunization, with little discrimination in the recall response between pre-F and post-F immunogens. However, the triple disulfide-stabilized pre-F adsorbed HMPV-directed responses from commercially available pooled human immunoglobulin more fully than post-F. Collectively, these results suggest single-chain triple disulfide-stabilized pre-F trimers to be promising HMPV-vaccine antigens. Immune responses to the fusion (F) glycoprotein trimer of human metapneumovirus (HMPV) could form the basis of an effective vaccine. However, the F glycoprotein assumes two conformations or shapes, prefusion (pre-F) and postfusion (post-F), and the shape of the most effective HMPV-vaccine immunogen has been unclear. Here, we use structure-based design to create a single chain version of F, which we stabilized in the pre-F shape with three additional disulfide bonds and assessed structurally, antigenically, and immunogenically. Structurally, we confirmed by cryo-electron microscopy that the three designed disulfides formed. Antigenically, the single-chain triple disulfide-stabilized F trimer was well recognized by the pre-F specific antibody, MPE8. And immunogenically, the single-chain triple disulfide-stabilized F trimer elicited high titer neutralizing responses, both in naïve mice and in pre-exposed rhesus macaques. Overall, our results suggest a highly stabilized pre-F trimer, such as the single-chain triple disulfide-stabilized F trimer that we described here, may be a highly effective HMPV-vaccine immunogen. Pneumoviridae family of viruses includes respiratory syncytial virus (RSV) and human metapenumovirus (HMPV). The Paramyxoviridae family includes several important human pathogens, such as mumps virus, measles virus, and parainfluenza viruses (PIVs)  was codon optimized, synthesized and subcloned into VRC vector VRC8400. The His and Strep tags were appended to the C terminus with TEV protease cleave site. Antibodies were expressed in Expi293F cells via transient transfection with ExpiFectamine 293 (Thermo Fisher). Supernatants were harvested 6 days post-transfection, and purified using Nickel Sepharose Excel Resin (Cytiva), followed by SEC on a HiLoad Superdex 200 pg 16/600 column (Cytiva).2. The total dose was fractionated for 2.5 s over 50 raw frames.Samples for cryo-EM grid preparation were produced by first mixing v3B_D12_D454C-V458C with MPE8 scFv at a molar ration 1:1.2 (monomeric F:scFv), purifying the complex by size exclusion chromatography and concentrating to 1 mg/ml. Purified complex was then diluted to 0.6 mg/ml in PBS and adjusted to have a final concentration of 0.005% (w/v) n-Dodecyl β-D-maltoside (DDM) to prevent preferred orientation and aggregation during vitrification. Cryo-EM grids were prepared by applying 3 μL of sample to a freshly glow discharged carbon-coated copper grid (CF 1.2/1.3 300 mesh). The sample was vitrified in liquid ethane using a Vitrobot Mark IV with a wait time of 30 s, a blot time of 3 s, and a blot force of zero. Cryo-EM data were collected using Leginon software  installeCryo-EM data process workflow for the HMPV-scFv MPE8 complex, including motion correction, CTF estimation, particle picking and extraction, 2D classification, ab initio reconstruction, homogeneous refinement, heterogeneous refinement, non-uniform refinement, and local resolution estimation, were carried out with C3 symmetry in cryoSPARC 3.3 . Using 1All mouse experiments were reviewed and approved by the Animal Care and Use Committee of the Vaccine Research Center, National Institutes of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) and were housed and cared for in accordance with local, state, federal and institute policies within an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International accredited facility at the NIH. To assess the effectiveness of recombinant HMPV F trimer designs at eliciting neutralizing antibodies, groups of 10 CB6F1/J mice were immunized twice at weeks 0 and 3 intramuscularly via a needle syringe combination in the caudal thighs with 50 μg of recombinant HMPV F glycoprotein trimer designs combined with 10 μg Poly I:C and week 5 sera were assessed for autologous HMPV virus neutralization in vitro. Neutralizing antibody titers were determined using HMPV plaque-reduction neutralization assays.The nonhuman Primate (NHP) Animal Study Proposal was reviewed and approved by the Animal Care and Use Committee of the Vaccine Research Center, NIAID, NIH and all animals were housed and cared for in accordance with local, state, federal and institute policies in an AAALAC International accredited facility at the NIH. Female and male Indian origin rhesus macaques with body weights between 2–9 kg were used for immunization studies. For each immunization, 1 ml of 100 μg immunogen mixed with 20% of Adjuplex, an adjuvant based on lecithin and carbomer homopolymer ,34, in PThe neutralization assays were done using live HMPV based on a published method . BrieflyAbsorption assays were conducted using a fortéBio Octet Red384 instrument. In brief, Flebogamma  was titrated with either prefusion or postfusion immunogen after the removal of the purification 6xHis tag. A series of 2-fold dilutions of prefusion immunogen   for posiIntroduction of the cysteines D454C and V458C to v3B_Δ12 increased both expression as well as affinity to MPE8 . IntroduProline substitution is another widely used strategy to stabilize conformations of type 1 fusion machines –39, and Thermal stability analysis by nano differential scanning fluorimetry verified the thermostability of the HMPV F variants with additional disulfides or proline mutations . To evalWhile SDS-PAGE confirmed the formation of interprotomer disulfide bonds, the formation of intraprotomer disulfides was unclear, and we therefore sought to obtain structural information. Attempts to obtain the cryogenic electron-microscopy (cryo-EM) structure of v3B_Δ12_D454C-V458C with the MPE8 antigen-binding fragment (Fab) failed because of Fab-Fab interactions that caused the formation of ordered arrays on cryo-EM grids. Substitution of the MPE8 Fab with the single-chain variable fragment (scFv) lead to dispersed particles, with reconstruction density at 3.25 Å resolution Figs .Clear electron density between Cys140-Cys147 and between Cys454-Cys458 confirmed the formation of these intraprotomer disulfide bonds . The rec2 of epitope surface that spanned over two neighboring protomers with ~75% of the surface provided by the DI and DIII domains of the major binding protomer and ~25% of the surface from the DII domain of the neighboring protomer  as well as these same variants with additional 3P mutations were assessed for their ability to elicit HMPV-neutralizing responses in naïve mice. We immunized CB6F1/J mice with 10 μg doses of each of the HMPV F glycoprotein variants combined with 50 μg polyinosinic-polycytidylic acid (poly-I:C) adjuvant at weeks 0 and 3 and measured the functional activity of week 5 sera in a plaque reduction neutralization assay (PRNT) using the homologous HMPV B2 strain. The optimized single chain variants  elicited especially high neutralizing titers, averaging over 11,000, 20-fold higher than elicited by post-F and 1.5-fold higher than those elicited by v3B_Δ12 . NotablyIn addition to vaccination of infants, who are naïve to HMPV, other target vaccine populations include the elderly, who have likely been previously exposed to HMPV. To assess the ability of the v3B_Δ12_D454C-V458C trimer to recall neutralization, we chose to assess in rhesus macaques, who are known to be infected by HMPV ,43. We aWe observed responses to post-F to also be high, suggesting that the recall response may be less discriminating, in terms of its being triggered by a specific immunogen. We note in this context that immunization with RSV F DS-Cav1 does not induce HMPV neutralizing titers in naïve mice but can induce HMPV-neutralizing responses in pre-exposed adults as reported by Phung and colleagues .Adsorption analysis provides another means to assess the v3B_Δ12_D454C-V458C immunogen. In this type of analysis, antigens are assessed for their ability to adsorb antibodies in serum as well as for their ability to detect what remains after adsorption. Previous analysis with a first-generation single proline-stabilized HMPV F trimer of human Flebogamma, pooled human globulin used for treatment of primary immunodeficiency, found this first-generation HMPV F trimer to deplete about the same titers as post-F of HMPV . Here, wDetecting with pre-F after depletion with either pre-F or post-F, however, we observed substantial depletion at two-to-three orders of magnitude lower amounts of pre-F than with post-F for RSV and PIV3, but for HMPV, only one order of magnitude lower. These observations suggest there to be substantially more postfusion F responses that cross-react with pre-F from HMPV than with pre-F from RSV or PIV3. Detecting with post-F, we observed both pre-F and post-F to deplete similarly with RSV and PIV3, while with HMPV, more titers remained after pre-F adsorption, indicating higher levels of post-F-specific responses against HMPV than for RSV and PIV3. Overall, these adsorption results indicate substantially more post-F responses for HMPV than RSV and PIV3; the triple disulfide-stabilized pre-F v3B_Δ12_D454C-V458C, nonetheless, adsorbed HMPV-directed responses more fully than post-F.With structure-based design, it is critical to know the appropriate target shape to fix or to stabilize. Initial immunogenicity results with the HMPV F revealed the immunogenicity of first-generation pre-F and post-F  as well A recent publication suggests processed pre-F HMPV trimers with a Pro mutation to be superior to uncleaved trimer in terms of thermal stability (62.4°C versus 55.5°C), shedding light on the significance of the F2-F1 linker between HMPV subunits to pre-F thermal stability . RelevanIn naïve mice, we observed elicited titers from disulfide-stabilized variants of pre-F to be significantly higher than those elicited by post-F . RecentlIn phase III trials of recently approved RSV vaccines, a single immunization of prefusion-stabilized F glycoprotein prevents RSV-associated lower respiratory tract illness in adults –49. SimiThe triple-disulfide stabilized pre-F immunogen, v3B_Δ12_D454C-V458C, appears to be a promising vaccine immunogen. It will be critical, however, to assess this immunogen in appropriate target populations. Almost all humans are infected with HMPV by the age of five –52, suggS1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 FigA) Nano Differential Scanning Fluorimetry (NanoDSF) and (B) Differential scanning calorimetry (DSC). (C) Summary of melting temperature of HMPV F variants.The melting temperature of variants was determined by ((TIF)Click here for additional data file.S3 FigA) and variants with disulfide bonds were shown in (B). (C) SDS-PAGE and antigenic analysis of disulfide and 3P stabilized HMPV F variants after incubation at 37°C for the indicated time.Variants with proline substitution were shown in panel ((TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(A) Representative micrograph of HMPV F in complex with scFv MPE8. (B) Representative 2D class averages are shown. (C) The gold-standard Fourier shell correlation resulted in a resolution of 3.25 Å for the overall map using non-uniform refinement with C3 symmetry (left panel); the orientations of all particles used in the final refinement are shown as a heatmap (right panel). (D) The local resolution of the final overall map is shown contoured at 0.448 (5.6s). Resolution estimation was generated through cryoSPARC using an FSC cutoff of 0.5. (E) Representative density is shown for the interface between CDR H3 and HMPV F. The contour level is 1.5σ.(TIF)Click here for additional data file.S7 FigA) Correlation of antigenicity and immunogenicity of HMPV F variants. (B) Comparison of titers elicited by single chain prefusion stabilized with and without 3P mutations.((TIF)Click here for additional data file.S8 FigA) Pre-exposed titers were measured using pre-bleeding by ELISA against HMPV F prefusion (HMPV-F v3B D12_D454C-V458C) and postfusion(postF) immunogens. Neutralization titer were measured by plaque reduction neutralization tests (PRNT) using HMPV subtype A and B strain. 10 NHPs highlighted as color red were selected for HMPV F immunization. (B) Measured anti-F ELISA titer is corelated with neutralization titers. (C) 10 selected NHPs were assigned to prefusion group (blue) and postfusion group (green); ELISA IgG responses with area under curve (AUC) and endpoint titers of each group was shown. Pre-exposed titers against RSV were measured by ELISA with IgG endpoint titers against RSV F DS-Cav1 (right panel).((TIF)Click here for additional data file.S9 Fig(A) Anti-HMPV prefusion and postfusion IgG responses of NHP sera were measured by ELISA against HMPV F prefusion or postfusion immunogens. Ratios of prefusion vs postfusion were in right panel. (B) Anti-HMPV prefusion and postfusion responses of NHP sera were probed by Octet. (C) Three mouse study groups were selected, and anti-HMPV prefusion and postfusion responses were probed by Octet.(TIF)Click here for additional data file.