P<0.05). SNPs in the microRNA (miRNA) binding region of the TNP2 gene 3′-UTR may have contributed to the phenotypic differences. The phenotypic differences are caused by the altered expression of the miRNAs and their targets. Bioinformatics analysis predicted that the g.1536 C>T site in the TNP2 3′-UTR is located in the bta-miR-154 binding region. The quantitative real-time polymerase chain reaction results showed that the TNP2 mRNA relative expression in bulls with the CT and CC genotypes was significantly higher than those with the TT genotype (P<0.05) in the g.1536 C>T site. The luciferase assay also indicated that bta-miR-154 directly targets TNP2 in a murine Leydig cell tumor cell line. The SNP g.1536 C>T in the TNP2 3′-UTR, which altered the binding of TNP2 with bta-miR-154, was found to be associated with the semen quality traits of Chinese Holstein bulls.Transition protein 2 (TNP2) participates in removing nucleohistones and the initial condensation of spermatid nucleus during spermiogenesis. This study investigated the relationship between the variants of the bovine TNP2 gene and the semen quality traits of Chinese Holstein bulls. We detected three single nucleotide polymorphisms (SNPs) of the TNP2 gene in 392 Chinese Holstein bulls, namely, g.269 G>A (exon 1), g.480 C>T (intron 1), and g.1536 C>T (3′-UTR). Association analysis showed that the semen quality traits of the Chinese Holstein bulls was significantly affected by the three SNPs. The bulls with the haplotypic combinations H6H4, H6H6, and H6H8 had higher initial semen motility than those with the H7H8 and H8H4 haplotypic combinations ( The round spermatids of mammals undergo complex morphologic, physiologic, and biochemical modifications that result in the formation of mature spermatozoa. Major restructuring of the somatic chromatin occurs in elongating and condensing spermatids. Nucleohistones are first replaced by a group of arginine- and lysine-rich proteins called transition proteins (TNPs). The TNPs are then replaced by protamines At the transcriptional and the posttranscriptional levels, the haploid number during the differentiation phase of spermatogenesis in mammals is a striking system of developmental regulation of gene expression. Numerous new mRNAs are synthesized in early haploid cells into round spermatids. During the transcriptional inactivation of chromatin in late haploid cells, the round spermatids change into elongated spermatids miRNAs are small non-coding endogenous RNA molecules (18/nt to 24 nt). miRNAs mainly bind to partially complementary sequences in the 3′-untranslated region (UTR) to inhibit the target mRNA translation into protein or accelerate mRNA degradation The present study attempts to contribute in overcoming disturbances by identifying potentially expressed candidate genes and studying the association in male gametes and in reproductive tissues in bulls. This study also focuses on the association of TNP2 gene with the semen quality traits in Chinese Holstein bulls and the effects of testicular miRNA on TNP2 gene expression.The association of SNPs with semen quality traits  was analyzed using 392 Chinese Holstein bulls from three bull stations . All of collected semen samples were collected by company workers with permission from the animal owners. This study was approved by the Chinese Ministry of Agriculture Bureau of Animal Husbandry and Veterinary and the Ministry of Agriculture Dairy Cattle Frozen Semen Quality Supervision Testing Center.The semen quality traits of each ejaculate sample were determined under light microscopy according to the guidelines provided by the World Health Organization. The sperm samples were labeled with the date and the age of the bulls. The semen quality traits of each bull were repeatedly measured from 2008 to 2012. Data on 39125 ejaculate samples from the 392 bulls were used, with 4 to 379 ejaculate samples from each bull. The semen quality traits were averaged for each bull using the mean function of the SAS Software. Genomic DNA was extracted from the sperm samples using standard protocols. The samples were treated with mercaptoethanol and digested with proteinase K, followed by salt extraction and isopropanol precipitation. DNA content was spectrophotometrically estimated and the mixture was diluted to 50 ng/µL. All DNA samples were stored at −20°C for subsequent analysis.2, 0.2 mmol/L dNTPs, 0.5 µmol/L of each primer, and 50 ng DNA in a total volume of 25 µL. After an initial denaturation for 5 min at 95°C, PCR was performed with 35 cycles of denaturation at 95°C for 30 s; annealing at 54°C to 62°C (Primers for detecting polymorphism and amplifying the bull TNP2 gene (GenBank accession no. NC_007326.4)  were des to 62°C  for 30 sHpaII and HindIII. The g.269 G>A polymorphism was genotyped via the CRS-PCR method with one of the primers containing a nucleotide mismatch. The CRS-PCR also enabled the use of the restriction enzyme HpaII for discriminating sequence variations HpaII at 37°C for 30 min. The digested products were detected via electrophoresis in 10% PAGE (29 acrylamide∶1 bisacrylamide) in 1× TBE buffer and constant voltage (110 V) for 3.5 h at room temperature. The digested products were then stained with 0.1% silver nitrate The three SNPs of the TNP2 gene  were genotyped according to the sequencing result using PCR Restriction Fragment Length Polymorphism and Created Restriction Site PCR (CRS-PCR). The PCR products were cleaved using http://www.targetscan.org/vert_50/), MicroInspector (http://bio2server.bioinfo.uni-plovdiv.bg/microinspector/), and RNA22 microRNA target detection (http://cbcsrv.watson.ibm.com/rna22.html) We performed MTar searches of the putative miRNA-binding sites of the 3′ UTRs from segments of perfect Watson-Crick complementarity to bases 2–8 of the miRNA numbered from the 5′ end RNA was extracted from the bull testes to compare the differential expression of bta-miR-154 between adult [2.5 years old (post-sex maturation)] and fetal bulls [3 days old (pre-sex maturation)]. An miScript Reverse Transcription Kit (Qiagen) was used for cDNA synthesis to detect miRNA expression, and an miScript SYBR Green PCR Kit (Qiagen) with a bta-miR-154 specific primer was used to detect mature miRNA detection. Bta-let-7g was used as the internal control. Relative gene expression was determined using a previously described method 5′-AAGCCACGCCTGCCACCACT-3′; R:5′-TCCCTCCAAGTTCTTTCTGTTC-3′; product size = 247 bp). The primers of housekeeping internal control β-actin gene were designed according to reference Samples were collected from the testicular tissues of nine bulls from a commercial slaughter farm. Total RNA was extracted from the testicular tissues samples using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. The relative expression of TNP2 mRNA in the testicular tissues of bulls with different genotypes in the SNP (g.1536 C>T) locus was determined via quantitative real-time PCR (qPCR) using the primer . Cloning was performed and used to transform Trans5α cells (Invitrogen). Cloning was also plated on agar containing 100 mg/mL ampicillin and incubated overnight at 37°C. Positive colonies were cultured in 10 mL of lysogeny broth medium (Fisher BioReagents) containing 100 mg/mL ampicillin and incubated overnight at 37°C. Plasmids were isolated using a Plasmid Miniprep Kit (Biomiga) according to the manufacturer's instructions. The plasmid DNA was quantified using a NanoDrop ND-1000. The constructs were sequenced to ensure proper orientation and authenticity in the vector.The TNP2 3′-UTR was amplified using a pair of primers with the following sequences 2 and subcultured every other day. All reagents were from GIBCO unless otherwise specified.A murine Leydig cell tumor cell line (MLTC-1) was purchased from the Shanghai Life Science Institute, Chinese Academy of Sciences Cell Resource Center. MLTC-1 was maintained in RPMI 1640 supplemented with 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate, 2 mM L-glutamine, 500 U/L penicillin, 500 mg/L streptomycin, and 10% (v/v) fetal bovine serum (FBS) gcggaacuugaagauagguuauccguguagccuucgcuuuacuugugacgaaucauacacgguucaccuauuuuuuaguaccaa) and the scrambled control miRNA (CmiR0001-MR04) were purchased from GeneCopoeia.CCS-bta-154-MR04 . The plasmids were cotransfected with 40 ng of β-gal for normalization. All experiments were performed with a negative control plasmid (pMIR-REPORT™). About 48 h after transfection, the cells were washed once with phosphate-buffered saline before being photographed. The cells were also lysed once with 65 µL of reporter lysis buffer per well (Promega). The cell lysates from the transfected cells were prepared and assayed according to the manufacturer's instructions (Promega) for both firefly luciferase and β-gal absorbance assays http://analysis.bio-x.cn) was used to analyze the pairwise linkage disequilibrium and haplotypic frequencies Yijk is the observed value of each semen quality trait; μ is the overall mean; iH is the fixed effect of genotypic or haplotypic combinations; jP is the fixed effect of age  2–3 years; (2) 4–5 years; (3) 6–10 years); kM is the effect of farm; ijke is the random residual error. Values with P<0.05 and P<0.01 were considered significant. Multiple comparisons were performed using Duncan's method.The SHEsis software .Three SNPs were revealed from 40 randomly selected samples, namely, g.269 G>A (exon 1), g.480 C>T (intron 1), and g.1536 C>T (3′-UTR). Direct sequencing and comparisons with the reference sequence (NC_007326.4) were used to select the 40 samples . The SNPHpaII to discriminate sequence variations. HpaII digestion of the PCR products of the TNP2 g.269 G>A locus produced the following: 135-, 33-, and 27-bp fragments for genotype GG; 135-, 60-, 33-, and 27-bp fragments for genotype GA; and 135- and 60-bp fragments for genotype AA. HpaII digestion of the PCR products of TNP2 g.480 C>T locus produced the following: 350-bp fragments for genotype TT; 350-, 237-, and 113-bp fragments for genotype TC; and 237- and 113-bp fragments for genotype CC. HindIII digestion of the PCR products of TNP2 g.1536 C>T locus produced the following: 432- and 103-bp fragments for genotype TT; 535-, 432-, and 103-bp fragments for genotype TC; and 535-bp fragments for genotype CC , and the other genetic indices of the three SNPs of the bovine TNP2 gene. Alleles G, C, and T were the dominant alleles at positions g.269 G>A, g.480 C>T, and g.1536 C>T in the bulls, respectively. At locus g.480 C>T, the frequencies of genotype CC were higher than those of the genotype TT. The locus possessed low genetic diversity (P<0.25). The Chinese Holstein bulls possessed intermediate genetic diversity at the g.269 G>A and g.1536 C>T loci (0.25<P<0.50), which suggests intermediate genetic diversity. The results of the χ2 test showed that all SNPs were inconsistent with the Hardy–Weinberg equilibrium (ilibrium . The selP<0.05). Our results show that cows with genotype TT at locus g.480 C>T had lower deformity rates (P<0.05) than those with genotypes CC and CT. Bulls with genotype CT at locus g.1536 C>T had higher ejaculate volumes and post-thaw cryopreserved sperm motility than those with genotype CC (P<0.05).We analyzed the association of the three SNPs with semen quality traits . We inclACC), H2 (ACT), H3 (ATC), H4 (ATT), H5 (GCC), H6 (GCT), H7 (GTC), and H8 (GTT). The estimated haplotypic frequencies were 1.9%, 4.6%, 0.7%, 1.8%, 25.2%, 47.3%, 4.9%, and 13.6%, respectively. H6 had the highest haplotypic frequency, whereas H3 had the lowest. The correlation among haplotypic combinations and semen quality traits was analyzed (AACCCT/4), H2H2 (AACCTT/3), H1H3 (AACTCC/1), H4H4 (AATTTT/2), H5H4 (AGCTCT/3), and H7H4 (AGTTCT/4) was less than 5, which is not statistically significant. The association between the haplotypic combinations and the semen quality traits of Chinese Holstein bulls was not analyzed.The SNPs g.269 G>A, g.480 C>T, and g.1536 C>T were used for haplotype reconstruction. The haplotypes were H1  among the different haplotypic combinations (three SNPs) (P<0.05) than those with H6H4, H6H6, and H6H8 (P<0.05). The bulls with the haplotypic combination H1H4 had significantly higher sperm densities than those with H5H2 and H6H4 (P<0.05). The bulls with haplotypic combinations H1H4 and H7H8 showed significantly lower deformity rates than those with H6H6 and H6H8 (P<0.05).Statistical analysis showed that initial sperm motility, sperm density, and deformity rate significantly differed (ee SNPs) . No signWe identified miRNAs that can potentially regulate TNP2 expression with one SNP present in the miRNA seed sequence. To identify the miRNA, we computationally predicted which miRNAs might contribute to TNP2 regulation. We used different algorithms to reduce the false positive rate of the target prediction results. We selected miRNA target pairs with context score of 0.10 or above from the possible putative sites. The miRNA target pairs were predicted to bind with the same target site by all the three software. The miRNAs that were then predicted by at least 2 of the algorithms to bind were considered candidates for further study. We integrated the results from the three prediction software programs. The prediction results showed that the SNP g.1536 C>T of the 3′-UTRs, which alters the binding to the bta-miR-154 seed sequence, may regulate TNP2 gene expression. Only bta-miR-154 had a high likelihood for targeting TNP2 3′-UTR with g.1536 C>T-T . TNP2 biHuang et al. (2011) discovered the novel miRNA miR-154 in cows through Solexa sequencing. They detected the differential expression between testicular and ovarian tissues P<0.05; The TNP2 mRNA levels in the testicles of bulls with different genotypes were determined to investigate the effect of SNP g.1536 C>T on TNP2 expression. We found that the expression of TNP2 mRNA with genotypes CT and CC was significantly higher than that with genotype TT P<0.05; . The higP>0.05). By contrast, increasing the miR-154 concentrations in the g.1536 C>T-TT samples dose-dependently repressed the luciferase activity (P<0.05). A bovine TNP2 3′-UTR fragment containing either the wild-type or the mutant miR-154-binding sequence was subcloned downstream of the luciferase expression vector to confirm that miR-154 targets TNP2. The reporter containing the g.1536 C>T-CC TNP2 3′-UTR did not significant influence miR-154 expression and the luciferase activity No regardless of the concentration  that comprise the vast majority of mammalian splice junctions. A previous study demonstrated that TNP2 mutations affect sperm chromatin structure and reduce fertility in mice Association analysis showed that the three SNPs markers g.269 G>A, g.480 C>T, and g.1536 C>T are significantly correlated with sperm quality traits excluding the environment and peculiarities in the AI stations. We identified SNP g.269 G>A as the possible causative polymorphism locus because it changes codon 62 of TNP2 from Arg to His. TNP2 is a 14 kDa protein with distinct structural domains The results also revealed a TNP2 SNP at position g.480 C>T in the intron that was significantly correlated with the sperm quality traits. Although introns do not code for proteins, they have important regulatory roles in gene expression and regulation Bulls with genotype CT at locus g.1536 C>T (3′-UTR) had higher ejaculate volumes and post-thaw cryopreserved sperm motility than those with genotype CC. Furthermore, the expression of TNP2 mRNA with genotypes CT and CC was significantly higher than those with genotype TT at locus g.1536 C>T (3′-UTR). Finally, bta-miR-154 exerted its inhibitory effects through the direct binding of the TNP2 3′-UTR with g.1536 C>T-T, whereas TNP2 binding was not observed in the g.1536 C>T-C SNP mutation. Our findings suggest that SNPs influence TNP2 gene expression by modulating the binding affinities of the miRNA and the target mRNA. The SNP may thus be responsible for the differences in semen quality of bulls. Polymorphisms and mutations in the corresponding sequences, such as the machinery, miRNA precursors, and target sites, are likely to significantly contribute to phenotypic variations such as disease susceptibility In summary, the g.1536 C>T in TNP2 is a functional candidate SNP that interacts with the miRNA target site. The g.1536 C>T in TNP2 has strong biological relevance and is likely involved in spermatogenesis. This research is promising because it shows an effective means of marker-assisted selection for semen quality and fertility of mammals. However, larger sample sets are still needed to confirm the association and for determining the possible effects of the variant on semen quality.


Translation is a regulated process and is pivotal to proper cell growth and homeostasis. All retroviruses rely on the host translational machinery for viral protein synthesis and thus may be susceptible to its perturbation in response to stress, co-infection, and/or cell cycle arrest. HIV-1 infection arrests the cell cycle in the G2/M phase, potentially disrupting the regulation of host cell translation. In this study, we present evidence that HIV-1 infection downregulates translation in lymphocytes, attributable to the cell cycle arrest induced by the HIV-1 accessory protein Vpr. The molecular basis of the translation suppression is reduced accumulation of the active form of the translation initiation factor 4E (eIF4E). However, synthesis of viral structural proteins is sustained despite the general suppression of protein production. HIV-1 mRNA translation is sustained due to the distinct composition of the HIV-1 ribonucleoprotein complexes. RNA-coimmunoprecipitation assays determined that the HIV-1 unspliced and singly spliced transcripts are predominantly associated with nuclear cap binding protein 80 (CBP80) in contrast to completely-spliced viral and cellular mRNAs that are associated with eIF4E. The active translation of the nuclear cap binding complex (CBC)-bound viral mRNAs is demonstrated by ribosomal RNA profile analyses. Thus, our findings have uncovered that the maintenance of CBC association is a novel mechanism used by HIV-1 to bypass downregulation of eIF4E activity and sustain viral protein synthesis. We speculate that a subset of CBP80-bound cellular mRNAs contribute to recovery from significant cellular stress, including human retrovirus infection. Retroviruses are intracellular parasites that utilize the host translation machinery to catalyze viral protein synthesis. The activity of the translation machinery fluctuates during cell cycle progression and is reduced in the G2/M phase. HIV-1 infection causes the cells to arrest in the G2/M phase, which has the potential to alter the activity of the translation machinery. Herein several lines of evidence demonstrated that lymphocyte mRNA translation is suppressed by the action of HIV-1 accessory protein Vpr. The molecular basis of translation suppression is reduced activity of the rate-limiting translation intitation factor, eIF4E. However, synthesis of the viral structural proteins is sustained and is due to the difference in composition of the viral and cellular mRNA-ribonucleoprotein complexes. Both cellular and completely spliced viral mRNAs are predominantly associated with the cytoplasmic cap binding protein, eIF4E. In contrast, unspliced HIV-1 mRNAs are predominantly associated with the components of the nuclear cap binding complex (CBC). The retention of CBC on the viral mRNAs provides a mechanism to sustain viral protein synthesis. This newly characterized interface of the virus-host-protein synthesis machinery is likely a cellular adaptation used to enable synthesis of proteins that reengage the cell cycle and facilitate recovery from stress. Regulation of translation is a fundamental property of cell physiology. In dividing cells, mRNA translation is robust in the G1 growth phase, diminishes during the S phase, and is downregulated during the G2/M phase The mRNP is initially formed upon addition of 7-methylguanosine to the 5′ end of the RNA, triggering binding of the nuclear cap binding complex  Picornaviridae  Downregulation of the eIF4E activity is a strategic tool for cells to rapidly respond to stress or alterations in cell cycle progression In contrast to most host mRNAs, expression of the HIV-1 mRNA encoding the viral structural proteins is dependent upon an alternate pathway for the export of the corresponding mRNAs. While bulk mRNA export uses NXF1 as the export mediator, transport of HIV-1 unspliced and singly spliced RNAs to the cytoplasm involves use of the rRNA and tRNA export factor, CRM1 In addition to the interaction of the HIV-1 RNA with the host gene expression machinery, the HIV-1 protein products interact with cellular pathways to induce alterations in cell function NL4-3 was obtained from AIDS Reagent Reference Program. pHRvprIRESgfp, pHRvpr(R80A)IRESgfp, pHRvpr(Q65R)IRESgfp, pHRvpr(R80A/Q65R)IRESgfp and pNL4-3-VprX were obtained from Vicente Planelles (University of Utah) NL4-3ΔVif were digested with NheI-PflMI and religated to generate ΔVifVprX. HIV-1HXB2R-RI− was obtained from Eric Cohen  5′-CGCGGATCCGAAGTAGTGTGTGCCCGTCT-3′) and gagSDR (5′-CCCAAGCTTCCCTGCTTGCCCATACTATA-3). All plasmid preparations were confirmed by DNA sequence analysis.HIV-1NL4-3 molecular clone and cell-free supernatants were collected and used to infect CEMx174 lymphocytes. Typically one million CEMx174 cells were incubated with 8×106 pg of cell-free Gag for 2 h to generate HIV-1 producer cells. Coculture infections combined producer and naive CEMx174 cells at 1∶10 ratio and intracellular Gag production was evaluated by flow cytometry beginning at 8 h for indicated intervals. At 32 and 46 h post-infection, the cocultures were treated with nocodazole  for 16 h and cyclohexamide  for 2 h, respectively. HEK293 cells were treated with 1 µM nocadozole for 24 hours. HEK293 cells (2×105) were transfected for 48 h in 6-well plates with 1 µg of indicated plasmid, FuGene 6 (Roche) following manufacturer's protocol.CEMx174 lymphocytes were grown in RPMI with 10% fetal bovine serum and 1% antibiotic/antimycotic. HEK293 cells were grown in DMEM with 10% fetal bovine serum and 1% antibiotic/antimycotic. HEK293 cells were transfected with the indicated HIV-17 cells were treated with 200 µM cycloheximide for 15 min at 37°C, washed in cold PBS containing 200 µM cycloheximide, collected by centrifugation at 1540 rpm for 4 min, and lysed in 450 µl gradient buffer . For EDTA treatment, 30 mM EDTA was added to the PBS and gradient buffer. Lysates were layered on the linear sucrose gradients (15–47.5%) and centrifuged at 36,000 rpm for 2.25 h at 4°C in SW41 rotor (Beckman). Gradients were fractionated and A254 absorbance was measured using an ISCO system. Alternative fractions were collected for RNA or protein isolation. For RNA isolation, fractions were treated with an equivalent volume of 100% ethanol and tRNA (1 µg). For protein isolation, fractions were equilibrated in 10% TCA. Samples were incubated at −80°C overnight and centrifuged at 16,000 g for 20 min, and extracted with Trizol (Invitrogen) by the manufacturer's protocol.Ribosomal profiles were prepared as described previously 7) were collected and resuspended in 110 mM KOAc, 25 mM K-HEPES (pH 7.5), 2.5 mM Mg(OAc)2, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 200 µM CHX, 50 U/mL RNasin (Promega), 150 µg/mL digitonin (Sigma) g for 10 min at 4°C. The supernatant containing soluble cytosolic RNPs was collected. The pellet containing membrane-associated RNPs was rinsed twice with ice-cold cytosol buffer lacking detergent and extracted in 400 mM KOAc, 25 mM K-HEPES (pH 7.5), 15 mM Mg(OAc)2, 1 mM DTT, 1 mM PMSF, 200 µM cycloheximide, 50 U/mL RNasin (Promega), 20 mg/mL digitonin. The suspension was incubated on ice for 30 min, centrifuged at 10,000 g for 10 min at 4°C, and the supernatant was collected. Aliquots of the preparations were reserved for immunoblotting.To fractionate cytosolic RNPs and membrane-associated RNPs, CEMx174 cells  for 30 min on ice followed by flow analysis. ModFit generated the cell cycle profiles. GFP expression was analyzed on a FACS Aria cell sorter.Intracellular staining of Gag with FITC conjugated anti-Gag p24 antibody  was used to monitor the progress of viral infections. Cells were fixed and permeabilized with Cytofix/Cytoperm kit (BD Biosciences) according to manufacturer's protocol. Cell cycle analyses were performed using propidium iodide (PI) staining. Briefly, 5×1035S]-cysteine/methionine  (50 µCi/ml). After incubation for 1 hour, the cells were rinsed with PBS and lysed in 50 mM Tris pH 8.0, 0.1% SDS, 1% Triton-X, 150 mM NaCl, 1% deoxycholic acid, 2 mM PMSF. Cell lysates were equilibrated to 10% TCA with 50 µg bovine serum albumin and 0.01% sodium azide and incubated on ice for 30 min. Samples were applied to glass fiber filters, washed twice with ice cold 10% TCA and 100% ethanol, respectively. The [35S]-cysteine/methionine-labeled proteins were measured by scintillation or visualized by SDS PAGE. Gag or Actin proteins were collected by IP using 1 µl of Gag antibody 35S]-cysteine/methionine-labeled proteins were detected by Phosphorimager analysis as described previously Cell cultures were rinsed and incubated for 30 min in RPMI supplemented with 10% dialyzed FBS that lacked methionine and cysteine, followed by addition of -cysteine/methionine for one hour, washed and incubated for indicated intervals after the addition of complete medium. Equivalent volumes of cells were subjected to immunoprecipitation and proteins were resolved by SDS-PAGE and phosphorimager analysis is presented. Positions of Gag p55, p37 and p24 are indicated.(TIF)Click here for additional data file.Figure S2CBP80 and eIF4E co-sediment with polysomes in cells transfected with subgenomic HIV-1 env plasmid. (A) HEK 293 cells were transfected with pgTat and pCMVRev for 48 hour, cytoplasmic extracts were harvested and incubated with or without EDTA, and applied to 15–47.5% linear sucrose density gradients. Representative A254 ribosomal RNA profiles are indicated. (B) Lysates from every other fraction were immunoblotted with CBP80 and eIF4E antibodies. (C) RNase protection assays of RNA isolated from every other fraction. Designations indicate the unspliced (US) reporter transcript; spliced (S) env RNA; and cellular actin RNA.(TIF)Click here for additional data file.Figure S3Unspliced pgTat mRNA coprecipitates with CBP80 and the spliced env transcript coprecipitates with eIF4E. (A) HEK 293 cells were transfected with pgTat and pCMVRev for 48 hour. Equivalent cytoplasmic lysates were immunoprecipitated with the isotype control (IgG), eIF4E or CBP80 antibodies. (B) Graphical representation of pgTat indicating the position of the RPA probe used (top). RNase protection assays of RNA isolated from the immunoprecipitations. Designations are unspliced (US) pgTat transcript and spliced (S) env transcript.(TIF)Click here for additional data file.


Glucose-6-phosphate dehydrogenase (G6PD) deficiency is common in populations living in malaria endemic areas. G6PD genotype and phenotype were determined for malaria patients enrolled in the chlorproguanil-dapsone-artesunate (CDA) phase III clinical trial programme.Plasmodium falciparum malaria, and haemoglobin ≥ 70 g/L or haematocrit ≥ 25%, were recruited into two clinical trials conducted in six African countries . G6PD genotype of the three most common African forms, G6PD*B, G6PD*A (A376G), and G6PD*A- , were determined and used for frequency estimation. G6PD phenotype was assessed qualitatively using the NADPH fluorescence test. Exploratory analyses investigated the effect of G6PD status on baseline haemoglobin concentration, temperature, asexual parasitaemia and anti-malarial efficacy after treatment with CDA 2/2.5/4 mg/kg or chlorproguanil-dapsone 2/2.5 mg/kg (both given once daily for three days) or six-dose artemether-lumefantrine.Study participants, aged > 1 year, with microscopically confirmed uncomplicated Of 2264 malaria patients enrolled, 2045 had G6PD genotype available and comprised the primary analysis population . G6PD deficiency prevalence was 9.0% , 13.3% (273/2045) of patients were heterozygous females, 77.7% (1588/2045) were G6PD normal. All deficient G6PD*A- genotypes were A376G/G202A. G6PD phenotype was available for 64.5% (1319/2045) of patients: 10.2% (134/1319) were G6PD deficient, 9.6% (127/1319) intermediate, and 80.2% (1058/1319) normal. Phenotype test specificity in detecting hemizygous males was 70.7% (70/99) and 48.0% (12/25) for homozygous females. Logistic regression found no significant effect of G6PD genotype on adjusted mean baseline haemoglobin (p = 0.154), adjusted mean baseline temperature (p = 0.9617), or adjusted log mean baseline parasitaemia (p = 0.365). There was no effect of G6PD genotype (p = 0.490) or phenotype (p = 0.391) on the rate of malaria recrudescence, or reinfection .G6PD deficiency is common in African patients with malaria and until a reliable and simple G6PD test is available, the use of 8-aminoquinolines will remain problematic. G6PD status did not impact baseline haemoglobin, parasitaemia or temperature or the outcomes of anti-malarial therapy.NCT00344006 and NCT00371735.Clinicaltrials.gov:  G6PD gene is highly polymorphic with almost 400 reported variants, conferring varying levels of enzyme activity [G6PD is X-linked, and so deficient variants are expressed more commonly in males than in females. Worldwide, an estimated 400 million people are G6PD deficient with the distribution corresponding to areas in which malaria is, or has been, prevalent. This has led to speculation that malaria has been the selection pressure that has favoured the maintenance of this potentially deleterious trait. There is some evidence that hemizygous males may be protected against severe malaria [Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme in the pentose phosphate pathway (PPP) that plays an important role in protecting cells from oxidative damage by producing NADPH and reduced glutathione. In the erythrocyte, which lacks a nucleus, mitochondria and other organelles, PPP is the only biochemical pathway for generating reducing capacity ,2. The G malaria ,3-5.G6PD variants worldwide, in sub-Saharan Africa three variants occur with polymorphic frequencies (> 0.1%); G6PD*B, G6PD*A and G6PD*A-. G6PD*B is the wild type and the most common variant in Africa and worldwide. G6PD*A has a single A→G substitution at nucleotide number 376, though is a normal variant with about 90% of the G6PD*B enzyme activity [Although there are over 400 activity .Individuals with G6PD*A- are normally asymptomatic . HoweverThis study reports the frequency of G6PD genotypes and phenotypes in malaria patients who participated in two Phase III clinical trials of chlorproguanil-dapsone-artesunate (CDA) ,11. The The clinical data from the CDA trials have been published separately and those reports include more comprehensive descriptions of the conduct of the individual trials ,11.This study was conducted in accordance with Good Clinical Practices, applicable regulatory requirements, and the Declaration of Helsinki. Approval was obtained from each participating centre's ethics committee or institutional review board and the WHO Special Programme for Research and Training in Tropical Diseases (TDR). An Independent Data Monitoring Committee (IDMC) was convened and planned to conduct two interim safety analyses. The IDMC appointed an independent end-point reviewer (blinded to treatment assignment). Written or oral witnessed informed consent was required from patients or their parent/guardian plus assent from patients who were ≥ 12-18 years old.This study used genotyping and phenotyping to determine the frequency of G6PD deficiency in malaria patients included in the CDA Phase III clinical trial programme in Africa. Exploratory analyses investigated whether baseline haemoglobin concentration, temperature and asexual parasitaemia were influenced by G6PD genotype. The effect of G6PD status on anti-malarial efficacy was also examined.Patients were screened for inclusion into two clinical trials. Study 005 compared CDA versus artemether-lumefantrine (AL) in malaria patients aged > 1- < 14 years of age and was conducted between June 2006 and August 2007 at 11 centres in five African countries: Bobo-Dioulasso, Burkina Faso; Kintampo, Ghana; Eldoret, Kilifi and Pingilikani, Kenya; Ibadan, Enugu, Jos and Calabar, Nigeria; and Bagamoyo and Kiwangwa, Tanzania .Plasmodium falciparum malaria ; fever at enrollment or history of fever; weight ≥ 7.5 kg; haemoglobin ≥ 70 g/L or haematocrit ≥ 25%; willingness to comply with study procedures.Across the two trials, eligible subjects included males and females who met the following inclusion criteria: acute uncomplicated microscopically verified P. falciparum malaria; known G6PD deficiency, methaemoglobin reductase deficiency, haemoglobin M/E (i.e. increased risk of methaemoglobinaemia), or porphyria; neonatal hyperbilirubinaemia; received potentially haemolytic concomitant medication; concomitant infection ; any underlying disease that could compromise malaria diagnosis or the anti-malarial efficacy assessment; known hypersensitivity/allergy to study treatments or biguanides, sulphones, sulphonamides, or artemisinin derivatives; malnutrition; received recent anti-malarial therapy that may affect the efficacy evaluation or any investigational drug within 30 days or five half-lives (whichever longer); previously participated in the study. A negative pregnancy test was required from women of child-bearing age; breastfeeding mothers were excluded.Subjects were excluded if they had: severe/complicated During screening, all patients provided a full medical history and underwent a comprehensive clinical examination. Drug treatments were CDA 2/2.5/4 mg/kg, CPG-DDS 2/2.5 mg/kg  given once daily for three days  or six-dose AL . Randomization was 2:1 CDA versus the comparator. Details of patient assessments are given in the clinical reports of these trials ,11. PatiG6PD genotype and phenotype were determined by laboratory staff blinded to treatment. Clinical investigators were blinded to drug treatment and G6PD status was not available to clinical investigators until after the trial was complete.msp-1, msp-2 and glurp [Asexual parasite and gametocyte counts were determined by examination of duplicate Giemsa-stained thick blood slides (10 μL thumb prick) using WHO (2003) methods . Thick bnd glurp ,16.G6PD gene containing G6PD*B (wild type), the common African mutation G6PD*A (A376G) and the other deficient variant G6PD*A- . Amplicons were subsequently analysed by restriction fragment length polymorphism (RFLP) and genotypes scored after agarose gel electrophoresis. DNA was extracted using standard methods. PCR was performed using 0.75 U/μL Taq Polymerase, 1.5 mM MgCl2, 200 μM dNTP (mix 10 mM), 2.5 μM of each primer, 10% PCR buffer, and 2.0 μL of DNA. All restriction endonucleases were sourced from New England Biolabs  and used at optimal temperature and digestion period. Genomic DNA was first amplified using primers for A376G, 5'-CCCAGGCCACCCCAGAGGAGA-3' (forward) and 5'-CGGCCCCGGACACGCTCATAG-3' (reverse). Thermocycling was performed at 94°C for 10 min, then 30 cycles at 94°C for 45 sec, 58°C  for 45 sec, and 72°C for 45 sec, with a final extension at 72°C for 7 min. Amplification products were recovered and PCR fragments incubated at 37°C for 16 h with FokI for identification of the A376G mutation (G6PD*A). All samples positive for A376G were then subjected to PCR amplification using: G202A, 5'-CCACCACTGCCCCTGTGACCT-3' (forward) and 5'-GGCCCTGACACCACCCACCTT-3' (reverse) with annealing at 65°C; G542T, 5'-AGGAGATGTGGTTGGACATCCGG-3' (forward) and 5'-ACTCCCCGAAGAGGGGT-3' (reverse) with annealing at 67°C; G680T, 5'-ACATGTGGCCCCTGCACCAC-3' (forward) and 5'-GTGACTGGCTCTGCCACCCTG-3' (reverse) with annealing at 69°C; and T968C 5'-TCCCTGCACCCCAACTCAAC-3' (forward) and 5'-CCAGTTCTGCCTTGCTGGGC-3' (reverse) with annealing at 65°C. Amplicons were then incubated at 37°C for 16 h with NlaIII for identification of the G202A mutation, AccIII for G542T, BstNI for G680T, and NciI for the T968C mutation. Quality control was maintained by comparing results from the two testing laboratories with discrepant results reanalysed.G6PD genotype analysis was performed at two laboratories: Walter Reed Project/Kenya Medical Research Institute, Kisumu, Kenya and Shoklo Malaria Research Unit, Mae Sot, Thailand. After patient randomization, but before the first dose of study therapy, a 10 μL blood sample was collected (thumb prick) onto pre-printed filter paper. The G6PD genotyping protocol was based on published methods -19. SpecPre-dose venous blood samples (2 mL) were collected for G6PD phenotyping, refrigerated to 4°C and transported within seven days to a central laboratory  for analysis using a commercial NADPH fluorescence test . This was a qualitative test designed to distinguish grossly G6PD-deficient samples from those with intermediate/normal enzyme activity. The reaction mixture containing glucose-6-phosphate+NADP (not fluorescent) and spots were made on the filter paper at the beginning (zero-time) and 5, 10, and 20 min after blood incubation with reagent mixture. The spots were visually inspected under long-wavelength (320-420 nm) ultraviolet light. The observed rate of appearance of bright fluorescence is proportional to the blood G6PDH activity; normal samples fluoresce brightly, whereas deficient samples show little or no fluorescence [Day-28 parasite recrudescence and reinfection rates, differentiated using PCR genotyping as described above, were used to evaluate whether G6PD genotype or phenotype had an effect on anti-malarial efficacy.Descriptive statistics were used to report demographic and clinical characteristics for patients with G6PD genotype. G6PD genotype and phenotype was presented by country and centre.A-, A and B) and compared with observed values using Chi-squared.All statistical analyses were performed post-hoc. There was no formal sample size calculation for the analyses reported in this paper. Sample size was calculated for clinical endpoints as reported previously ,11. AnalA logistic model was developed to investigate the effect of G6PD genotype on the following baseline variables: haemoglobin concentration, temperature, and asexual parasite count.The effect of G6PD genotype was evaluated using three categories: normal; heterozygous; or deficient . The effect of G6PD genotype on baseline haemoglobin concentration was modelled with terms for age category , weight, centre, sex, temperature and parasitaemia category (four quartiles). The model for the effect of G6PD genotype on temperature had terms for age category, weight, centre, sex, haemoglobin concentration and parasitaemia category. To investigate the effect of G6PD genotype on parasitaemia, parasite densities were initially log transformed with model terms for age category, weight, centre, sex, haemoglobin concentration and temperature.A logistic model was developed to investigate the effect of G6PD genotype  on anti-malarial efficacy (recrudescence and reinfection rates) with terms for: anti-malarial treatment, study , age category, weight, centre, sex, and parasitaemia category. The same model was used to investigate the effect of G6PD phenotype  on anti-malarial efficacy.The models were used to calculate adjusted mean values for the variable under investigation and a p value calculated to test any significant effect of model terms using analysis of variance. G6PD genotype was further analysed to compare G6PD normal with heterozygous and G6PD normal with G6PD deficient; 95% confidence intervals were calculated and p values generated using analysis of variance.A summary of the patient population that was screened, randomized and analysed for G6PD status is shown in Figure Table G6PD phenotype results are shown in Table Baseline demographic and clinical characteristics of the study participants are summarized in Table Figures Table Reinfection with falciparum malaria occurred in 608/2045 (29.7%) patients overall in the study. Based on the raw data , unusual but clinically important variants can be missed . PhenotyA, B, AA or AB), 4.3% (25/575) and 3.1% (14/446), respectively, were found to be phenotypically deficient. It is possible that these individuals might have had G6PD mutations at loci other than those examined.As shown in Table In the context of this trial, using only phenotype data would have significantly undermined interpretation of the drug-induced G6PD-related haemolytic effect of CDA as about a third of G6PD*A- patients would be expected to have had a clinically important haemolysis ,11. BecaAcross all patients, phenotype correlated reasonably well with the occurrence of significant hemolysis. Of the 878 patients with phenotype data, significant hemolysis occurred in 21.1% (19/90) of those patients who were phenotypically deficient, in 5.6% (5/90) of those who were intermediate and in 1.1% (8/698) of those with normal phenotype. Of the 41/124 (33.0%) G6PD*A- patients treated with CDA who had clinically important haemolysis, 23 had phenotype data available. Of these, 17/23 (73.9%) were phenotypically deficient, 4/23 (17.4%) had intermediate phenotype and 2/23 (8.7%) had normal phenotype.The study design excluded patients with haemoglobin concentrations ≤ 70 g/L and this consideration may have affected the study results. There were no independent effects of G6PD genotype on baseline haemoglobin, temperature, or malaria parasitaemia in the study population Table , 3 and 4P. falciparum clearance after treatment with artemisinin-based combination therapy [As shown in Table  therapy . However therapy .There are limitations in performing post-hoc statistical analysis on the effect of G6PD status on baseline variables and efficacy as was done in this study. Logistic modelling also has limitations; inclusion of the variables for adjustment is somewhat subjective and can never be comprehensive. Therefore, the conclusions that can be drawn from this study should be viewed as exploratory and require verification in prospective studies.The G6PD*A- deficient genotype was relatively common among the diverse African populations included in this study. This represents a significant risk for adverse haemolytic events after treatment with drug therapies that have the potential to induce oxidative stress. G6PD genotyping and phenotyping should therefore be a requisite in clinical trials evaluating the safety and efficacy of such drugs. The G6PD prevalence data presented here are relevant for the design of such anti-malarial clinical trials. Lastly, there were no clinical differences between G6PD-deficient and -normal patients with malaria enrolled into the CDA clinical trials.Nick Carter and Allan Pamba are employees of GlaxoSmithKline. Stephan Duparc is an employee of the Medicines for Malaria Venture.All authors contributed to the study design. Genotype testing and scoring was performed in JW's laboratory. NC conducted the statistical analysis. All authors contributed to the preparation of the manuscript and approved the final version.


There is an error in the legend of Figure 1. The correct legend is:Figure 1. Purification of H6-tagged MHC class II complexes. A) Preparative purification of DRB1*04:01 refolded with the Influenza A peptide HA307–318(H6) (YKYVKQNTLKLATHHHHHH) on IMAC column. A shallow gradient up to 50mM Imidazole in PBS (20% buffer B) eluted peak 1 containing alpha and beta chains without associated peptides, whereas peak 2, containing mature MHC II complexes, eluted by raising the concentration to 250mM imidazole in PBS (100% buffer B). B) SDS-PAGE analysis of eluted fractions, the alpha chain travels as the upper band. C) Functional LOCI assay on load, run through, and peak fractions. Notice the absence of peptide-loaded MHC class II molecules in the run through (RT) and in peak 1. D) Assessing the level of biotinylation of a DRB1*11:01 chain by incubation with increasing amounts of avidin followed by SDS-PAGE analysis.


The BC04 ca virus exhibited α2,3-SA and α2,6-SA dual receptor binding preference while the NL03 ca virus preferentially bound to α2,3-SA. Substitution of the Q226 and G228 (Q-G) by the L226 and S228 (L-S) residues in the HA improved binding to α2,6-SA for NL03 ca. The vaccine viruses with L-S retained the attenuation phenotype. NL03 L-S ca replicated more efficiently than the original NL03 ca virus in the upper respiratory tract of ferrets, and induced higher levels of humoral and cellular immune responses. Prior vaccination with seasonal LAIV reduced H7-specific antibody responses, but did not reduce the H7N7 vaccine mediated protection against a heterologous H7N3 BC04 wt virus infection in ferrets. In addition, the H7N3 and H7N7 vaccine immunized ferret sera cross reacted with the newly emerged H7N9 virus. These data, in combination with the safety data from previously conducted Phase 1 studies, suggest that these vaccines may have a role in responding to the threat posed by the H7N9 virus.Live attenuated influenza vaccine (LAIV) candidates of the H7 subtype, A/Netherlands/219/03 (H7N7, NL03  Influenza A viruses cause yearly seasonal influenza epidemics and occasional influenza pandemics when a novel influenza virus with an antigenically shifted hemagglutinin (HA) emerges in humans resulting in widespread infection, high morbidity and high mortality . Avian ica) and H7N3  LAIVs were generated and evaluated in pre-clinical studies and phase I clinical trials; the H7N3 vaccine candidate was found to be more immunogenic than the H7N7 vaccine [Vaccination is the most effective method to prevent influenza infection. Live attenuated influenza vaccines (LAIV)  have the vaccine ,14,15.As influenza viruses that have caused past pandemics had receptor binding preference for α2,6-linked sialic acid , it is generally believed that avian viruses need to acquire binding specificity to these human cell receptors to establish efficient human-to-human transmission. Most avian viruses, however, preferentially bind to α2,3-SA receptors. Candidate vaccine viruses that contain the HA and NA protein from an avian influenza virus thus may not replicate efficiently in the mammalian upper respiratory tract and may be less immunogenic as a result. Recently, we demonstrated that the H2N2 (A/Ann Arbor/6/60) and H6N1  LAIV viruses with dual receptor binding to both α2,3-SA and α2,6-SA, as specified by L226 and S228 residues in the HA gene, grew efficiently in eggs and induced higher antibody levels in ferrets than those with Q226 and G228 residues that resulted in preferential binding to α2,3-SA . The H7 ca with the multibasic cleavage site removed and H7N3 A/chicken/BC/CN-6/04 (BC04 Q-G) ca vaccine viruses were generated previously [ca) was also generated by reverse genetics using the HA (accession# 439507) and NA (accession# 439509) sequences from the EpiFlu database of the Global Initiative on Sharing All Influenza Data (GISAID). Wild type A/chicken/BC/CN-6/04 is a low pathogenic virus and is handled at the BLS2 facility. The HA mutations were introduced into the live attenuated H7N7 or H7N3 vaccine viruses by reverse genetics and sequences were verified. All viruses were propagated in the allantoic cavity of 10- to 11-day-old embryonated chicken eggs (Charles River SPAFAS). Virus titers were determined in Madin-Darby Canine Kidney (MDCK) cells and expressed as fluorescent focus units (FFU) per ml.Influenza H7N7 A/Netherlands/219/03 (NL03 Q-G) eviously ,14. H7N9Carpenteria, CA). The reactions were detected by TMB  and the absorbance at 450 nm was determined using a SpectraMax plate reader (Molecular Devices). Receptor binding preference of recombinant viruses was also confirmed by hemagglutination of sialic acid-specific red blood cells (RBC) by the method described previously [Receptor binding preference of the vaccine viruses was examined by the glycan binding assay . Two-foleviously ,19.10FFU of virus per 0.2 ml dose. At 3 days post inoculation, lungs and nasal turbinates (NT) were harvested, viral titers in the respiratory tissues were determined in eggs and expressed as 50% egg infectious dose (EID50).All animal study protocols were approved by MedImmune’s Institutional Animal Care and Use Committee (IACUC) and performed in an AAALAC certified facility under protocol number ACF-081-007, 017 and 029. Eight to ten weeks-old male and female ferrets (n = 3-5/group) from Simonsen Laboratories  were used in the studies. To examine the attenuation phenotype, ferrets were inoculated intranasally with 7.0 log10FFU as described above and blood samples were obtained on days 8 and 14 post-vaccination. Hemagglutination inhibition (HI) and microneutralization (MN) assays were performed [ca virus as antigen.For immunogenicity and protection from wild-type challenge studies, ferrets were inoculated with 7.0 logerformed ,21. The erformed ,23 usingca, A/Wisconsin/67/2005 (H3N2) ca and B/Malaysia/2506/2004 ca at 7.0 log10FFU per strain in 0.2 ml. Four weeks later, they were inoculated with 0.2 ml of 7.0 log10FFU of NL03 L-S or PBS. The blood samples were obtained on days 8 and day 14 after the second dose, and serum antibody and T cell responses in PBMC were determined. On day 28 post-dose 2, the ferrets were challenged with wild type (wt) A/chicken/BC/CN-6/04 (BC04 wt) at a dose of 7.0 log10FFU per 0.5 ml and sacrificed three days post challenge to determine viral titers in the NT and lungs.The effect of seasonal vaccination on H7N7 vaccine induced immune responses and protection was also examined. Ferrets were first intranasally inoculated with PBS or LAIV containing A/New Caledonia/20/1999 (H1N1) ca and H7N3 BC04 ca vaccine viruses with HA Q226L and G228S substitution were named as NL03 L-S or BC04 L-S. The viruses with the L-S change grew equally well in eggs, reaching titer of ~8.5 log10FFU/ml. By glycan binding assay, the original BC04 and NL03 ca vaccine viruses with Q226 and G228 (Q-G) bound to α2,3-glycan  . BC04 Q-ca vaccine viruses is defined by preferential replication in the upper respiratory tract compared to the lower respiratory tract of ferrets. As shown in ca viruses replicated efficiently in the NT at levels of >5 log10EID50/g tissue. NL03 Q-G replicated poorly in the NT (1.8 log10 EID50/g) but the NL03 L-S replicated well in the NT reaching a titer of 5.0 log10EID50/g. Thus, acquisition of virus receptor binding to α2,6-SA improved NL03 ca virus replication in the upper respiratory tract of ferrets.The attenuation phenotype of the ca, NL03 ca and NL03 L-S were compared by measuring serum HI antibody titers from immunized ferrets after a single dose of vaccine  was 4-fold lower than the NL03 L-S titer (81). NL03 L-S immunized ferret sera also cross reacted with the heterologous BC04 ca although the titers were again ~3-4-fold lower. Microneutralization (MN) assay against different NL03 ca viruses was also performed . Consistent with the HI data, the NL03 L-S variant induced a higher level of neutralizing antibody against the homologous virus (GMT 202) than the NL03 Q-G (GMT 63), and the antisera cross reacted well with the heterologous BC04 ca virus (GMT 101). These results indicate that vaccine viruses with better replication efficiency in the upper airway induced higher serum antibody responses in ferrets and that the 226 and 228 residues at the receptor binding site did not significantly alter viral antigenicity.The immunogenicity of BC04  vaccine . BC04 caerformed . BC04 caH7N3 BC04 and H7N7 NL03 immunized ferret sera were further evaluated for their cross reactivity against the newly emerged H7N9 A/Anhui/1/2013 strain (AH13). The BC04 Q-G HI and neutralizing Ab GMT against the AH13 strain (51 and 101) was approximately 3 to 4-fold lower than the titer seen for the homologous strain (256 and 320). The NL03 Q-G and NL03 L-S immunized ferret sera cross reacted to H7N9 AH13 at a level of approximately 3-fold lower than what was seen for the homologous strain by both HI and microneutralization assays .Evaluation of vaccine mediated cellular immune responses focused on the comparison of NL03 Q-G and L-S as BC04 L-S did not have a significant impact on viral receptor binding preference  or immunca virus. The animals that previously received seasonal LAIV had reduced antibody response to the H7N7 vaccine compared to the naïve ferrets, exhibiting approximately 3-fold reductions in HI and MN antibody titers  was used as the challenge virus as studies conducted with the highly pathogenic NL03 wt virus required BSL3 facilities not available at MedImmune. The BC04 wt virus replicated to a titer of approximately 7.0 log10EID50/g in both the NT and lung tissues of the PBS immunized ferrets. Seasonal LAIV did not inhibit challenge virus replication in NT, but reduced challenge virus titers in the lung by approximately 50-fold. The animals that received LAIV followed by the H7N7 NL03 vaccine were protected against BC04 wt replication in the respiratory tract at similar levels compared to the animals immunized with NL03 ca only. The challenge virus titers in the NT were reduced by approximately 100-fold and no virus were detected in the lungs of the ferrets that were vaccinated with a single dose of NL03 L-S regardless of their LAIV priming status. These data indicated that previous seasonal LAIV immunization did not diminish H7N7 vaccine induced protection in ferrets.The H7N7 NL03 L-S immunized ferrets, primed with or without LAIV, were examined for protection against heterologous BC04 nfection . A heterca vaccine in preclinical and clinical studies [ca vaccine.The HA-mediated viral entry through cell surface sialyated glycans is important for viral replication, virulence and transmission of influenza viruses . Live at studies ,14,15. Hca virus. The North America lineage H7N3 BC04 strain with Q-G, also replicated well in the respiratory tract of ferrets and humans [The HA residues 226 and 228 are located in the 220 loop of the HA receptor binding site  and theid humans . The H7Nd humans . Bindingd humans . Thus, id humans ; this maca. Previous infection with one subtype of influenza viruses may induce heterosubtypic immunity against infection with an antigenically unrelated subtype [wt virus titer in the lungs (ca virus replication in the upper airway, all the H7N7 NL03 L-S ca vaccinated ferrets were completely protected from the replication of the heterologous H7N3 BC04 wt challenge virus challenge in the lung. The H7N3 BC04 and H7N7 NL03 vaccines are antigenically distinct, exhibiting approximately 3-fold difference in serum antibody cross reactivity. Similarly, the newly emerged H7N9 AH13 virus and the H7N7 NL03 L-S or H7N3 BC04 had approximately 3-fold difference in serum antibody cross reactivity. Based on the cross protection data obtained from the H7N7 NL03 L-S against BC04 wt virus challenge, it is predicted that the existing H7N3 or H7N7 ca vaccines would provide some degree of protection against H7N9 virus. As master virus seeds for these vaccines have already been produced and the safety profile demonstrated in the previous small phase 1 studies, manufacturing with the exiting vaccine virus will save at least 3 months in vaccine product delivery.Consistent with the replication of vaccine viruses in ferret NT, the NL03 L-S variant induced higher antigen-specific antibody and T cell responses than the original NL03  subtype . In thishe lungs  due to tca virus. The H7N9 AH13 vaccine virus elicited neutralizing antibody titer of 320 against the homologous virus. Due to the lack of the BSL3 facility, we were not able to conduct the wt H7N9 AH13 virus challenge study for this report. Should the H7N9 virus become a pandemic threat, the H7N9 ca virus can now be manufactured to respond to the pandemic.We recently conducted the ferret immunogenicity study for the H7N9 AH13


SF162P4 infected macaque for sensitivity to neutralizing monoclonal antibodies (MAbs) B12, 2G12, 4E10 and 2F5. The early Env, isolated from plasma at day 56 after infection, was the most sensitive and the late Env, from day 670, was the most resistant to MAbs. We identified four PNGS in these Envs that accumulated over time at positions 130, 139, 160 and 397. We determined that removal of these PNGS significantly increased neutralization sensitivity to 2G12, and conversely, we identified mutations by in silico analyses that contributed resistance to 2G12 neutralization. In order to expand our understanding of these PNGS, we analyzed Envs from clade B HIV-infected human subjects and identified additional glycan and amino acid changes that could affect neutralization by 2G12 in a context-dependent manner. Taken together, these in vitro and in silico analyses of clade B Envs revealed that 2G12 resistance is achieved by previously unrecognized PNGS substitutions in a context-dependent manner and by subject-specific pathways.HIV-1 Envelope (Env) protein is the sole target of neutralizing antibodies (NAbs) that arise during infection to neutralize autologous variants. Under this immune pressure, HIV escape variants are continuously selected and over the course of infection Env becomes more neutralization resistant. Many common alterations are known to affect sensitivity to NAbs, including residues encoding potential N-linked glycosylation sites (PNGS). Knowledge of Env motifs associated with neutralization resistance is valuable for the design of an effective Env-based vaccine so we characterized Envs isolated longitudinally from a SHIV The HIV-1 envelope glycoprotein complex (Env) is the sole target of neutralizing antibodies (NAbs) and it evolves rapidly to escape this immune pressure. NAbs arise during infection to neutralize autologous variants and a limited number of HIV patients develop broad neutralizing antibodies . There iSF162P4-infected macaque (A141) with a moderate cross-NAb response, better educated the immune system to elicit a cross-reactive response . The presence of the mutation of interest was verified by DNA sequencing using Prism dye terminator kits  on an Applied Biosystems 3730XL genetic analyzer. Nucleotide alignments were manually edited using the Sequencher software program .5 cells/well in 6-well plates in medium . The following day, 293T cells in DMEM were transfected with 1 µg total DNA/well at a 20:1 ratio of backbone to Env plasmids in the presence of 10.5 µg polyethylenimine  per well. Cells were incubated for 48 hours at 37°C. Virus was harvested by centrifugation of the supernatant  and stored at -80°C until use. The pseudovirus was titrated on Tzm-bl cells. Dilutions of the pseudovirus were used to infect TZM-bl cells in the presence of 7.5 µg/ml DEAE-dextran . Two days later, infection was measured by luciferase expression and 200 50% tissue culture infective doses (TCID50) (determined by the Reed-Muench formula) were added to each neutralization assay.Pseudoviruses were produced with the pSG3ΔEnv DNA plasmid encoding the HIV backbone and a plasmid encoding the envelope of interest. Briefly, 293T cells were plated at 5x1050 of virus were added to serial dilutions of monoclonal antibody and incubated in a total volume of 150 µl medium  for 1 h at 37°C before addition of 100 µL of Tzm-bl cells resuspended in medium at 1 x 105 cells/mL in the presence of 7.5 µg/mL DEAE-dextran. Forty-eight hours later, 150 µL of medium was removed from each well and cells were lysed for 2 min directly in the neutralization plate, using 100 µL of Bright-Glo luciferase assay substrate , and immediately analyzed for luciferase activity on a luminometer . All values were calculated with respect to virus only wells [ minus ] divided by .Monoclonal antibodies IgG1 b12 , 2G12, 2F5 and 4E10  were assessed for neutralization of the pseudoviruses bearing the envelope of interest in a TZM-bl neutralization assay as previously described . BrieflyPseudovirus lysates of A141 Envs  and the different PNGS mutants  as well as the Env-deficient pseudovirus (pSG3ΔEnv) were generated as described  and assehttp://swissmodel.expasy.org/) [A three-dimensional model of monomeric gp120 was built based on the structure of the HIV-1 JRFL gp120 core protein complexed with CD4 and the X5 antibody (PDB code 2B4C)  by homolsy.org/) –30 and tsy.org/) . The figsy.org/) .50 between the neutralization of the pseudovirus/PNGS mutant of interest in a TZM-bl cell assay. For each monoclonal antibody, we randomly selected about 50,000 permutations from possible 1.82509x1014 distinct ways to assign 21 observations into 7 equal groups. One-way analysis of variance (ANOVA) was performed on each 50,000 permutation samples followed by 12 contrast comparisons. The permutation p-value for each comparison was obtained from more than 50,000 statistical hypothesis testing.Permutation test was used to investigate the difference in ICSF162P4-infected macaque that developed moderate breadth [Neutralization sensitivity to well-defined MAbs b12, 2G12, 2F5 and 4E10 was used to characterize different A141 Env variants . b12 rec breadth : d56 B3, breadth , these rP = 0.0003 with Δ(N139/N397), P = 0.0014 with Δ(N130/N397) and P = 0.0001 with Δ(N130/N139/N160/N397)). In addition the 2G12 IC50 obtained for the mutants was not statistically different from the IC50 obtained for the model clone  versus d56 B3, P = 0.55, Δ(N130/N397) versus d417 D15 and P = 0.72, Δ(N139/N397) versus d487 A6-1, respectively) thus suggesting that only these PNGS affected 2G12 neutralization and not the d670 A8-9 backbone. Therefore, we identified previously unrecognized PNGS substitutions outside the 2G12 epitope [Given the neutralization data obtained with the different MAbs, especially 2G12 , we inve epitope ,39 that P = 0.0036 between Δ(N130/N139/N160/N397) and d56 B3, P = 0.0006 between Δ(N130/N397) and d417 D15 and P = 0.0003 between Δ(N139/N397) and d487 A6-1, respectively). In addition, Δ(N130/N397) displayed an intermediate neutralization phenotype by b12 (P = 0.0003 compared to d417 D15) thereby showing that other factors outside the studied PNGS also affect neutralization sensitivity. Overall, our results suggest that the elimination of four specific PNGS in gp120 can alter the conformation and/or accessibility of antibody epitopes on both gp120 and gp41.Next, we compared the neutralization of the PNGS mutants by other MAbs . The neuTo identify additional features of these Envs affecting neutralization sensitivity, we assessed the possible participation of specific amino acids besides those involved in PNGS. A comparison of the four A141 Env sequences identified eight amino acid (AA) positions which were mutated in the late variants but not in the early one . These pin silico analyses of clade B Envs from five Amsterdam Cohort human subjects [To further characterize the amino acid changes responsible for 2G12 resistance, we performed subjects ,24, inclin silico analysis of the amino acid sequence revealed additional positions where mutations occurred between the 2G12 sensitive and 2G12 resistant clones in the ACH18839 Envelopes , 139 (V1), 160 (V2) and 397 (V4) that affect 2G12 neutralization and suggest that eliminating these specific PNGS modified Env conformation and/or access to gp120 and gp41. Similar to our observations with the late variant d670 A8-9, Nakowitsch identified 2G12-resistant viruses  that harOur analysis of A141 Envelopes also shows that the late variants accumulate amino acid changes that could alter Env conformation due to different charges or that can directly affect neutralization resistance. Negative charge changes were introduced in V1 AA 141, 151), V2 (AA 160) and C3 AA 348, 354), which render the sequences more acidic. It has been proposed that this evolution in V1V2 may affect Env conformation in the vicinity of the CD4 binding site . Interes, V2 (AA , which rFinally, our data show that the mutations and combinations that conferred 2G12 resistance are subject-specific. One caveat is that our findings result from examination of a single animal and we speculate that other PNGS may be discovered in additional SHIV-infected macaques. Indeed, different combinations of PNGS and amino acid changes from A141 were associated with 2G12 resistance in Envelopes from clade B HIV-infected subjects ,24. We hIn conclusion, our results and those of others ,22,56 de


We found that (1) star-shaped pGG analogs exhibit HA-inhibition activity by interacting with the conserved structural elements of the receptor binding domain (RBD); (2) HA inhibition depends on the number of galloyl substituents in a pGG analog; the best number is four; and when PGG binds with two HA trimers at their conserved receptor binding domains , PGG acts as a molecular glue by aggregating viral particles so as to prevent viral entry into host cells  with HA). pGGs are also effective on a broad-spectrum influenza A subtypes ; this suggests that pGG analogs can be applied to most influenza A subtypes as a prophylactic against influenza viral infections.Hemagglutinin (HA) is essential for Influenza A virus infection, but its diversity of subtypes presents an obstacle to developing broad-spectrum HA inhibitors. In this study, we investigated the molecular mechanisms by which poly-galloyl glucose (pGG) analogs inhibit influenza hemagglutinin (HA)  It is the causative agent of annual seasonal influenza epidemics and occasional global pandemics; such epidemics are a leading cause of morbidity and mortality worldwide Hemagglutinin (HA), a trimeric spike glycoprotein of the influenza virus, is responsible for binding to sialylated glycan receptors on the surface of host cells, thus inducing viral entry via endocytosis and followed by membrane fusion between the viral envelope and the endosome in vitro and in vivo by blocking HA-mediated virus attachment and cell entry. The most protective neutralizing antibodies are mainly directed against the globular head and interfere with HA-receptor docking e.g., amantadine and rimantadine) and neuraminidase inhibitors  Since HA is the major surface antigen of the influenza virus, anti-HA antibodies efficiently neutralize influenza viruses We previously reported that penta-galloyl-glucose (PGG), a poly-galloyl-glucose (pGG) analog, demonstrated anti-influenza virus activity by directly interacting with the HA protein. The cytotoxicity of PGG was evaluated on MDCK cells (EC50∶31.48±4.60 μM). No significant cytotoxicity was observed at the active concentration of PGG (EC50∶2.51±0.31 μM) Phyllanthus emblica Linn with a purity >98% (32). EA, EGCG, and tannic acid were obtained from the Guangdong small molecule tangible library (GSMTL) Gallic acid (GA) was purchased from Sigma-Aldrich. TGG and PGG were isolated from The assay was performed by using chicken red blood cells (RBCs) with standard procedures. Each compound solution (25 μl), after a two-fold serial dilution in PBS, was mixed with an equal volume of influenza virus solution. The mixture was incubated in a 96-well plate for 1 hour at 4°C. Then, it was mixed with 50 μl of 1% chicken RBC suspension and incubated for 30 minutes at room temperature.The influenza viruses (A/WSN/33(H1N1), A/PR/8/34(H1N1), and A/HK/8/68(H3N2)) were pre-incubated with test samples  diluted in MEM on ice for 1 hour. The mixture was then serially diluted and added to MDCK cells in 6-well plates and incubated for 1 hour at 37°C. After washing twice with PBS (−), cells were overlaid with MEM containing 0.8% (w/v) low melting agarose, 0.1% (w/v) BSA, 1% (v/v) vitamins, and 0.03% (w/v) glutamine. After 3 days of incubation, cells were fixed with ethanol: acetic acid (v/v = 1∶1) for 1 hour at room temperature. After removal of the overlaying agarose gel, the cells were stained with 2.5% (w/v) Amino Black 10B. The plaques were counted by visual examination. Means and standard deviations were calculated from three independent experiments.5PFU) was incubated with either pGGs (10 μM) or DMSO  on ice for one hour. The MDCK cells were then inoculated with the viral mixtures at 37°C for one hour. Then the cells were fixed and stained (for immunofluorescence) at two hours post-infection and analyzed via fluorescence microscopy. The cells were co-stained with anti-nucleoprotein antibody and Hoechst 33342.The influenza virus A/WSN/33, A/California/04/2009 (H1N1), A/Netherlands/219/03 (H7N7), and A/Anhui/1/2005 (H5N1)) with the same electrophoresis mobility shift were immobilized via the amine-coupling method on the vertical channel, while poly-galloyl-glucose analogs were injected, as analytes, through the horizontal channel. According to the manufacturer’s instructions, a mixture of N-hydroxysulfosuccinimide  and 1-ethyl-3- (3-dimethylamino-propyl) carbodiimide hydrochloride  in water was injected (flow rate 25 μl/min) to activate the alginate polymer modified surface (GLH chip). After chip activation, four channels were coupled with the four HA subtypes (which were dissolved in 10 μM sodium acetate buffer pH 4.0), while one reference channel was capped with ethanolamine . After the coupling step, the remaining surface of the coupled channel of the chip was deactivated with 1 M ethanolamine (pH 8.5). After extensive washing with running buffer, the chip was ready for binding studies. For analytes, the typical injection time was 300 s, at a rate of 25 μl/min. After association of analytes with ligands, running buffer was injected (for ≥300 s at 25 μl/min) to dissociate the bonded analyte. A regenerated chip surface is needed to get the surface plasmon resonance signal back to the baseline response level; this is accomplished via a buffer and sodium hydroxide solution. The RU was measured by subtracting the reference signal of the protein unlabeled channel from the double reference signal of the vehicle channel. The maximal RU is specified as Rmax; Rmax indicates the maximal binding capacity of an immobilized protein. Then kinetic constants of each sensorgram  were calculated by fitting the binding model (Langmuir) to sensorgram curves using ProteOn Manager Software. Immobilization buffers were 10 mM sodium acetate buffer (pH 4.0). The running buffer was PBS buffer  plus 0.1% DMSO.This experiment was performed with the ProteOn XPR36 Protein Interaction Array System (BIO-RAD) using a GLH sensor chip. The chip contains a 6×6 perpendicularly intersecting channel array. Four HA subtypes (6 PFU) was mixed 1∶1 with either PGG, DMSO or TritonX-100 , diluted in PBS, and incubated on ice for 30 min. The final concentrations of PGG, DMSO or TritonX-100 were 25 μM, 0.1% (v/v), and 0.5% (v/v), respectively. After incubation, each mixture was added onto a 200-mesh and was removed 2 minutes later. Samples were then stained with 2% phosphotungstic acid for 20 seconds and subjected to detection using the JEM-1400 Transmission Electron Microscope .For the TEM study, purified influenza virus A/WSN/33 (106 PFU) was pre-incubated with either a tested compound or DMSO  diluted in PBS for 1 hour on ice, and allowed to deposit on a coverslip; the final concentrations of each compound or DMSO mixture were 50 μM and 0.1% (v/v), respectively. After washing, the deposited viruses were fixed with 2.5% glutaraldehyde for 15 minutes, and then dried with nitrogen gas and detected via the BioScope Catalyst Atomic Force Microscope .Purified A/WSN/33 . The initial structures of HAs were derived from known crystal structures, with some necessary modifications  that accounted for the differences between the sequences of bioassay strains and crystal structures (see the Supplementary 2) whereas solvent molecules were not restrained. This step contained 2000 cycles of steepest descent minimization and 2000 cycles of conjugated gradient minimization. Then, all heavy atoms were restrained with constraints of 10 kcal/(mol·Å2) during the minimization steps, which consisted of 2500 cycles of steepest descent minimization and 2500 cycles of conjugated gradient minimization. For the final step, 5000 cycles of steepest descent minimization and 5000 cycles of conjugated gradient minimization were carried out without restraint. After minimizations, the whole system was first heated from 0 K to 300 K in 50 ps using Langevin dynamics at a constant volume and then equilibrated for 400 ps at a constant pressure of 1 atm. A weak constraint of 10 kcal/(mol·Å2) was used to restrain all the heavy atoms in the receptor-ligand complexes during the heating and steps. Finally, periodic boundary dynamics simulations were carried out for the whole system with NPT conditions . In the production phase, the complex of pGG and HA monomer was simulated for 10 ns; and the complex of pGG and HA dimer was simulated for 100 ns. The MM/PBSA method All MD simulations were performed using AMBER 11 This assay was carried out to verify the agglutination effect of PGG on HA. Recombinant influenza A HA proteins were incubated on ice with excessive concentrations of either PGG or GA for 30 min. After incubation, the samples were mixed with loading buffer ) and loaded into sample wells of nondenaturing 4%–15% Mini-PROTEAN® TGX™ Precast Gel (Bio-Rad). 0.002% Coomassie Blue G-250(w/v) was added to the Tris-Glycine cathode buffer. Electrophoresis was performed in the native PAGE gel at a constant current of 15 mA for 20 min for the stacking gel, and at 25 mA for 90 min for the separating gel (with ice-cooling running buffer). After electrophoresis on the native PAGE, the gels were then stained with the Silver Stain Kit (Thermo Scientific). HA (H7N7) and HA (H5N1), due to severe self-aggregation, can hardly migrate into the stacking gel even in the absence of PGG. Thus, 2% SDS was added in the sample loading buffer to improve the solubility of HA. As for HA (H1N1/PR8) and HA (H1N1/09), the self-aggregation issue is less severe and tolerable for electrophoresis.) to study the relationship between HA inhibitory activities and pGG analog structures.PGG exhibits an anti-viral activity against influenza A viruses by inhibiting virus-induced hemagglutination and viral entry 50  and shown in Hemagglutination inhibition assays reveal that GA cannot inhibit virus-induced hemagglutination; other pGG analogs, however, can significantly inhibit virus-induced hemagglutination in a dose-dependent manner Fig. 1B.A plaque reduction assay was performed to determine the effects of GA, TGG and PGG on viral infectivity. As shown in a (association constant) and Kd (dissociation constant) were derived from the sensorgram. The Ka and Kd are depicted in D) of pGG analogs  for hemagglutinin range from 10 nM to 1 μM. This proves that pGG analogs directly interact with hemagglutinin molecules. No significant interactions with HAs were detected for GA.Surface plasmon resonance (SPR) experiments were conducted to inspect the association or dissociation of pGG analogs and hemagglutinin molecules. After immobilizing purified recombinant monomeric hemagglutinin derived from IAV subtypes (A/Puerto Rico/8/1934 (H1N1), A/California/04/2009 (H1N1), A/Netherlands/219/03 (H7N7), and A/Anhui/1/2005 (H5N1)) on the chip, we analyzed the interactions of pGG analogs and hemagglutinin molecules by flowing pGG analogs through biosensor channels. The ligand-receptor interactions were measured by the sensorgram, which was plotted as response units (RUs) versus time (Supplementary Information). The KNative polyacrylamide gel electrophoreses (PAGE) experiments demonstrate that the concentration of HA monomer or oligomer decreases while the concentration of the incubated PGG increases. Moreover, the concentration of HA polymer increases while the concentration of the incubated PGG increases. The phenomena were also observed in the PGG and other HA subtypes (A/Puerto Rico/8/1934 (H1N1), A/California/04/2009 (H1N1), A/Netherlands/219/03 (H7N7), and A/Anhui/1/2005 (H5N1) PAGE experiments  with additional 50 ns MD simulations. As shown in Fig. S5, model A, in which a PGG molecule aggregates two trans-oriented hemagglutinin trimers, has a significantly lower binding energy than the ones of other models (Table S2). Actually, PGG can aggregate two, three, and more hemagglutinin trimers, which further form hemagglutinin oligomers, and eventually hemagglutinin polymers (Three The process of PGG aggregating the flu-virus particles is proposed in Atomic force microscopy (AFM) experiments were performed to confirm that pGG analogs agglutinate viral particles through binding hemagglutinin. The AFM images of virus, as shown in Transmission electron microscopy (TEM) experiments Fig. 5g,The results of both AFM and TEM experiments suggest that pGG analogs agglutinate influenza virus particles without significantly disrupting the viral membrane structures.A competition binding assay was performed to determine whether PGG directly binds the hemagglutinin receptor binding domain (RBD). Sialic acid (SA), the substrate of hemagglutinin, was used as a competitive agent with PGG in the assay. SA was incubated with the hemagglutinin derived from influenza A/California/04/2009(H1N1) prior to PGG addition. The grayscale in a lane of To study the binding modes of pGG analogs on hemagglutinin, molecular docking, molecular dynamics (MD) simulations, and binding affinity calculations were performed on six receptor-ligand complexes: H1N1/PR8 and PGG, H1N1/PR8 and TGG, H1N1/PR8 and EGCG, H1N1/PR8 and EA, H1N1/WSN and PGG, and H3N2/HK and PGG. The calculated binding energy results are listed in Figs. S3a∼S3d in the supplementary material. The receptor binding domain (RBD) has three conserved regions . PGG, TGG, and EGCG interact with all three conserved regions through hydrogen bonds (H-bonds) and π-stacking. However, EA only interacts with the two conserved regions (loop 220 and 190-α-helix) through hydrogen binding. These binding modes are consistent with the experimental binding affinities .Energy favorable binding modes of the pGG analogs and H1N1/PR8 hemagglutinin complexes are presented in Fig. S3a), and interacts with loop 130 through a σ-π stacking at ALAs 137, 138; H-bonds at the backbone atoms interact with loop 220 at GLN 226 and ARG 224; PGG also interacts with the 190-α-helix through H-bonds with GLU 190. Similarly, TGG interacts with loop 130 through H-bonds at backbone atoms and σ-π stacking at ALA 137 and VAL 134; TGG also interacts with loop 190 with H-bonds at GLU 190, and interacts with loop 220 through GLN 226 and ALA 227 . EGCG also interacts with the three conserved regions through H-bonds with GLU 190 and via σ-π stacking with LEU 194 (190-α-helix); EGCG also interacts via a hydrogen bond at GLN 226 (loop 220), and via backbone atoms forming H-bonds with VAL 135 (loop 130) . EA is small and ridged, and has only two galloyl groups. It cannot interact with the three binding areas of HA at the same time. Therefore, EA has a weaker binding affinity and hemagglutination inhibition . These in silico results are consistent with the in vitro hemagglutination inhibition assay data.The MD simulation results suggest that the activities of pGG analogs depend on the number of galloyl substituents. Increasing the number of galloyl substituents will increase the inhibition. When the number of galloyl substituents reaches four, the inhibitory activity is close to the maximum. PGG has five galloyl substituents , because PGG has a lower electrostatic binding energy (ΔEele) with H3. The solvent-accessible surface area . HA has two subtype groups, H1 belongs to group 1, and H3 belongs to group 2. Compared with H1, H3’s binding pocket is deeper, and has greater solvent accessible surface area (SASA), which makes it more difficult for PGG to bind. H3 binding pocket is also bumpier (at loop 130 and loop 220) than the ones of H1, therefore, PGG binds to H3 in different way , we observed that PGG has a stronger binding affinity with H1, and a weaker binding affinity with H3  to identify the residues that make major contributions to binding affinities . Based upon the alignment result, a phylogenetic tree was generated , which can be selectively bound with pGG analogs , A/Puerto Rico/8/1934(H1N1), A/California/07/2009(H1N1), A/HK/8/1968(H3N2), A/Vietnam/1203/2004(H5N1), A/HZ/1/2013(H7N9), and A/Netherlands/219/03(H7N7)) were aligned at the RBD region using the ClustalW algorithm enerated Fig. 7i.Sequence alignment analyses also demonstrate that the RBDs of 1934(H1N1/PR8), 1933(H1N1/WSN), H3N2, H5N1, H7N7, and H7N9 are highly conserved at residue 190 with glutamic acid . And pGG analogs have strong binding at this site through the carboxyl group of residue 190. On the other hand, pGG analogs are flexible enough to adjust themselves for possible minor mutations at residue 190. This is also supported by our SPR experiments, as shown in S4, pGG analogs can interact with the 130-loop mainly through backbone H-bonds and σ-π stacking without being disrupted by side chain changes.The key residues at the 130-loop of RBD are basically conserved. There are a few mutations, and these involve mainly smaller sized residues . Again, pGG analogs can easily adjust themselves for binding at this domain with minor receptor changes. As shown in The key residues at the 220-loop of RBD are well conserved with minor mutations. Residue 226 is glutamine for most of the subtypes (H1 and H5). pGG analogs bind with residue 226 via H-bonds. In case of H3N2, pGG analogs bind with residues 225 and 227 via Q226L.Therefore, we propose that pGG analogs inhibit a number of hemagglutinin homologs due to they can bind at the RBDs that are conserved, and the RBDs are selective to the pGG scaffold.homo sapiensThe recognition of HA for its receptor is highly dependent on the structural features of HA conserved domains (despite the hyper-variability of the residues surrounding the HA receptor binding site). For HAs from the H1 virus, residues 190 and 225 at the RBD are the determinants of receptor binding specificity. The H1 HAs possessing E190 and G225 preferentially bind to α2–3 receptors in avian species; D190 and G225 bind to both α2–3 and α2–6 receptors in swine species, and D190 and D225 effectively bind to α2–6 receptors in Polyphenols can inhibit influenza viruses by interacting with the viral HA protein. The inhibition leads to virucidal activity by damaging the virions’ structural integrity; polyphenols aggregate virions to prevent the viruses from adsorbing to cells In this study, our results for the first time demonstrate that pGG analogs bind with HA homologs  at the highly conserved structural elements of the RBD of HA virus, through H-bond and σ-π stacking interactions. The RBD is responsible for the virus to recognize host cells. We have revealed that pGG analogs bind to HA resulting aggregating the flu viral particles without disrupting the viral envelope, and stop viral infectivity and entry.i.e., GA, a single galloyl “finger”), can only be self-docked into a HA binding pocket, but cannot grip two HA trimers. Therefore, the star-shaped tertiary structural feature is the key to the aggregating mechanism of pGG’s HA inhibition. Recent NMR studies on protein-PGG interaction also demonstrated that PGG binding involves both hydrophobic interactions and H-bonds Our AFM experiments proved that pGG analogs cause virion aggregations by clumping influenza viral particles. Consequently, influenza viral entry is blocked. The mechanism of action is different from the many sialic acid-containing agents that have been developed as HA inhibitors in vivo. Meanwhile, PGG exhibits anti-allergy activity by down-regulating mast cell surface FcεRI expression both in vitro and in vivo It has been reported that PGG can also enhance phagocytosis on dendritic cells; phagocytosis can play a pivotal role in initiating and controlling the T cell-dependent immune response Figure S1RMSD values of the top ten scoring poses obtained from docking results of MOE, CDOCKER, Ligandfit, Surflex and FlexX.(TIF)Click here for additional data file.Figure S2MD quality analyses by (a) RMSD values of receptor backbone, ligand and pocket residues are in black, red and green lines. (b) B-factor values of HA before/after MD are in black/red lines. Due to space limitation, only one system’s result is shown to be representative.(TIF)Click here for additional data file.Figure S3Representative binding modes of the most popular MD conformations from the trajectories of poly-galloyl-glucose analogs docked with HA/PR8. The secondary structure of the proteins is in grey, PGG and its analogues are colored in yellow: (a) PGG (b) TGG (c) EGCG (d) EA, pocket residues within 5 Å distance from the ligand are colored in cyan. Hydrogen bonds are depicted in green dash lines.(TIF)Click here for additional data file.Figure S4The most popular binding modes derived from MD simulations for PGG docked with (a) H1/WSN, and (b) H3/HK. The secondary structure of the proteins is in grey, PGG is in yellow sticks, receptor residues within 5 Å distance from the ligand are represented in cyan sticks. Hydrogen bonds are depicted in green dash lines. The three HA proteins, H1/PR8(cyan), H1/WSN(magenta), H3/HK(green) were superimposed to compare the secondary structure variations upon PGG binding. (c) Time series of solvent-accessible surface area (SASA) of H1/PR8, H1/WSN and H3N2 pocket residues upon PGG binding, (d) side view of the HA pocket after superimposing the three HA proteins.(TIF)Click here for additional data file.Figure S5Three proposed models of PGG gluing hemagglutinin oligomers. HAs were represented by magenta and cyan ribbons. The red sphere marked the PGG binding site. The three models were built by creating the symmetric projection of the original HA and adding some rotation. (a) Trans-oriented model, created by centro symmetric projection (b) Cis-oriented model, created by mirror symmetric projection (c) Orthogonal-oriented model, created by 90° rotation of mirror symmetric projection model.(TIF)Click here for additional data file.Figure S6Correlation plot of predicted and experimental binding energy. The experimental binding energy is obtained by converting experimental KD values using formula ΔGbinding = RTlnK.(TIF)Click here for additional data file.Figure S7Binding energy decomposition results for another three systems. (a) PGG & H1N1/2009, (b) PGG & H5N1, (c) PGG & H7N9.(TIF)Click here for additional data file.Table S1Sequence identity between the HA strains for bioassay and the available HA 3D structures.(DOC)Click here for additional data file.Table S2Binding affinity calculation results for each conformation and each model.(DOC)Click here for additional data file.Table S3Various energy contributions to the binding energy for each complex system of pGG analogs vs. mono HA.(DOC)Click here for additional data file.Text S1Detailed methods for virus purification, redocking evaluation, BLAST, and binding energy calculations.(DOC)Click here for additional data file.