甲型流感病毒PB1-PA蛋白作用位点的发现与分析

 基于共进化理论,探究了甲型流感病毒PB1蛋白与PA蛋白上具有共同进化可能性的保守九聚片段 (C9MP)。结构信息显示PB1蛋白的第1-15位氨基酸与PA蛋白的第239-716位氨基酸具有相互作用域;对该区域变异分布的分析发现,PA蛋白第670位氨基酸Q所在的C9MP与PB1蛋白的第9位氨基酸F、第12位氨基酸V和第13位氨基酸P所在的C9MP在PB1-MP1相互作用面上具有最低的共进化值。结合DSSP程序的分析表明,由PA蛋白第670位氨基酸Q与PB1蛋白的第9位氨基酸F、第12位氨基酸V与第13位氨基酸P构成的区域可能成为潜在的相互作用位点。     

Single amino acid substitutions in influenza haemagglutinin change receptor binding specificity

The haemagglutinin (HA) glycoproteins of influenza virus membranes are responsible for binding viruses to cells by interacting with membrane receptor molecules which contain sialic acid (for review see ref. 1). This interaction is known to vary in detailed specificity for different influenza viruses (see, for example, refs 2-4) and we have attempted to identify the sialic acid binding site of the haemagglutinin by comparing the amino acid sequences of haemagglutinins with different binding specificities. We present here evidence that haemagglutinins which differ in recognizing either NeuAc alpha 2 leads to 3Gal- or NeuAc alpha 2 leads to 6Gal- linkages in glycoproteins also differ at amino acid 226 of HA1. This residue is located in a pocket on the distal tip of the molecule, an area previously proposed from considerations of the three-dimensional structure of the haemagglutinin to be involved in receptor binding.     

Structure of the δ-opioid receptor bound to naltrindole

The opioid receptor family comprises three members, the μ-, δ- and κ-opioid receptors, which respond to classical opioid alkaloids such as morphine and heroin as well as to endogenous peptide ligands like endorphins. They belong to the G-protein-coupled receptor (GPCR) superfamily, and are excellent therapeutic targets for pain control. The δ-opioid receptor (δ-OR) has a role in analgesia, as well as in other neurological functions that remain poorly understood. The structures of the μ-OR and κ-OR have recently been solved. Here we report the crystal structure of the mouse δ-OR, bound to the subtype-selective antagonist naltrindole. Together with the structures of the μ-OR and κ-OR, the δ-OR structure provides insights into conserved elements of opioid ligand recognition while also revealing structural features associated with ligand-subtype selectivity. The binding pocket of opioid receptors can be divided into two distinct regions. Whereas the lower part of this pocket is highly conserved among opioid receptors, the upper part contains divergent residues that confer subtype selectivity. This provides a structural explanation and validation for the 'message-address' model of opioid receptor pharmacology, in which distinct 'message' (efficacy) and 'address' (selectivity) determinants are contained within a single ligand. Comparison of the address region of the δ-OR with other GPCRs reveals that this structural organization may be a more general phenomenon, extending to other GPCR families as well.     

Crystal structures of oseltamivir-resistant influenza virus neuraminidase mutants

The potential impact of pandemic influenza makes effective measures to limit the spread and morbidity of virus infection a public health priority. Antiviral drugs are seen as essential requirements for control of initial influenza outbreaks caused by a new virus, and in pre-pandemic plans there is a heavy reliance on drug stockpiles. The principal target for these drugs is a virus surface glycoprotein, neuraminidase, which facilitates the release of nascent virus and thus the spread of infection. Oseltamivir (Tamiflu) and zanamivir (Relenza) are two currently used neuraminidase inhibitors that were developed using knowledge of the enzyme structure. It has been proposed that the closer such inhibitors resemble the natural substrate, the less likely they are to select drug-resistant mutant viruses that retain viability. However, there have been reports of drug-resistant mutant selection in vitro and from infected humans. We report here the enzymatic properties and crystal structures of neuraminidase mutants from H5N1-infected patients that explain the molecular basis of resistance. Our results show that these mutants are resistant to oseltamivir but still strongly inhibited by zanamivir owing to an altered hydrophobic pocket in the active site of the enzyme required for oseltamivir binding. Together with recent reports of the viability and pathogenesis of H5N1 (ref. 7) and H1N1 (ref. 8) viruses with neuraminidases carrying these mutations, our results indicate that it would be prudent for pandemic stockpiles of oseltamivir to be augmented by additional antiviral drugs, including zanamivir.     

The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA

Influenza A viruses pose a serious threat to world public health, particularly the currently circulating avian H5N1 viruses. The influenza viral nucleoprotein forms the protein scaffold of the helical genomic ribonucleoprotein complexes, and has a critical role in viral RNA replication. Here we report a 3.2 A crystal structure of this nucleoprotein, the overall shape of which resembles a crescent with a head and a body domain, with a protein fold different compared with that of the rhabdovirus nucleoprotein. Oligomerization of the influenza virus nucleoprotein is mediated by a flexible tail loop that is inserted inside a neighbouring molecule. This flexibility in the tail loop enables the nucleoprotein to form loose polymers as well as rigid helices, both of which are important for nucleoprotein functions. Single residue mutations in the tail loop result in the complete loss of nucleoprotein oligomerization. An RNA-binding groove, which is found between the head and body domains at the exterior of the nucleoprotein oligomer, is lined with highly conserved basic residues widely distributed in the primary sequence. The nucleoprotein structure shows that only one of two proposed nuclear localization signals are accessible, and suggests that the body domain of nucleoprotein contains the binding site for the viral polymerase. Our results identify the tail loop binding pocket as a potential target for antiviral development.     

Epidermal growth factor receptor occupancy inhibits vaccinia virus infection

Vaccinia virus encodes VGF, an early protein of relative molecular mass 19,000 (19K) which, from amino-acid residues 45 to 85, is homologous in 19 residues to epidermal growth factor (EGF), and transforming growth factor-alpha (TGF-alpha). The conserved sequence includes a region of high homology (6 out of 10 amino acids) from residues 71 to 80, corresponding to the third disulphide loop of both EGF and TGF-alpha. This region has recently been shown to contain a binding region of TGF-alpha for the EGF receptor, and this raises the question of whether vaccinia virus utilizes the EGF receptor in order to bind to and infect cells. We now show that occupancy of the EGF receptor inhibits vaccinia virus infection. Inhibition is observed in a dose-dependent fashion by pre-treatment with either EGF or synthetic decapeptide antagonists of EGF's mitogenic activity which correspond to the sequence of the third disulphide loop of VGF or TGF-alpha. The relative ability of the peptides to inhibit vaccinia virus infection parallels their binding affinity to the EGF receptor.     

百合无症病毒衣壳蛋白基因克隆和蛋白分析

根据已报道的LSV CP基因序列合成两条寡聚核苷酸引物,模板为感染LSV的百合叶片的总RNA,通过反转录-聚合酶链式反应(RT-PCR)扩增出大小为876bp的LSV CP基因,经测序后,对该基因编码区全长序列及相应的氨基酸序列用生物信息学软件系统进行序列分析及结构功能预测.结果表明:该基因由876个核苷酸组成,编码291个氨基酸;与GeneBank公布的其他LSV分离物的基因序列同源性为93.4%～99.0%,氨基酸同源性为84.8%～99.5%;它含有一个卷曲螺旋结构和多个磷酸化位点,平均疏水值为-0.432;含有Carlaviruses完整的衣壳蛋白保守结构域,二级结构以α-螺旋和无规则卷曲为主.     

Characterization of the 1918 influenza virus polymerase genes

The influenza A viral heterotrimeric polymerase complex (PA, PB1, PB2) is known to be involved in many aspects of viral replication and to interact with host factors, thereby having a role in host specificity. The polymerase protein sequences from the 1918 human influenza virus differ from avian consensus sequences at only a small number of amino acids, consistent with the hypothesis that they were derived from an avian source shortly before the pandemic. However, when compared to avian sequences, the nucleotide sequences of the 1918 polymerase genes have more synonymous differences than expected, suggesting evolutionary distance from known avian strains. Here we present sequence and phylogenetic analyses of the complete genome of the 1918 influenza virus, and propose that the 1918 virus was not a reassortant virus (like those of the 1957 and 1968 pandemics), but more likely an entirely avian-like virus that adapted to humans. These data support prior phylogenetic studies suggesting that the 1918 virus was derived from an avian source. A total of ten amino acid changes in the polymerase proteins consistently differentiate the 1918 and subsequent human influenza virus sequences from avian virus sequences. Notably, a number of the same changes have been found in recently circulating, highly pathogenic H5N1 viruses that have caused illness and death in humans and are feared to be the precursors of a new influenza pandemic. The sequence changes identified here may be important in the adaptation of influenza viruses to humans.     

Evidence for host-cell selection of influenza virus antigenic variants

Extensive antigenic variability and a capricious epidemiology are characteristics of influenza A and B viruses of man. The haemagglutinin (HA) undergoes frequent and progressive antigenic drift as a result of selection, under immunological pressure, of viruses possessing alterations in the amino acid sequences at specific sites in the molecule. Here we present evidence for an additional selection mechanism for antigenic variants of influenza virus that depends on differing host cell tropisms of virus subpopulations. These studies were initiated after earlier observations of the occurrence of a marked degree of antigenic variation during passage of laboratory strains of influenza virus in eggs and cell cultures (J.C.J., in preparation). We have now shown that cultivation of influenza B viruses in eggs selects subpopulations which are antigenically distinct from virus from the same source grown in mammalian cell cultures. As antigenic characterization of influenza virus strains for epidemiological purposes and for the preparation of influenza vaccines conventionally relies on the cultivation of virus in eggs, our findings may have important practical implications for vaccine design and efficacy.     

Structure of a beta1-adrenergic G-protein-coupled receptor

G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and many are important drug targets. Here we report the 2.7 A resolution crystal structure of a beta(1)-adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey (Meleagris gallopavo) receptor was selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane alpha-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilized by two disulphide bonds and a sodium ion. Binding of cyanopindolol to the beta(1)-adrenergic receptor and binding of carazolol to the beta(2)-adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the beta(2)-adrenergic receptor, directly interacts by means of a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation.     

A common function for polyoma virus large-T and papillomavirus E1 proteins?

Nucleotide sequencing has revealed a common genetic organization for three papillomaviruses: BPV-1 (bovine papillomavirus type 1), HPV-1 (human papillomavirus type 1a) and HPV-6 (human papillomavirus type 6b). Several open reading frames, corresponding to as yet uncharacterized proteins, were observed in these genomes in the region that is required for oncogenic transformation by BPV-1 and for plasmidial maintenance of its genome. The longest of these frames, E1, is also the most conserved between the three viruses; we have compared the amino acid sequence of its putative product ('E1 protein') with those of the large-T proteins of three polyoma viruses and report here significant homologies in their carboxy-terminal halves, extending for over 200 amino acids. Moreover, similar secondary structures were predicted in this region, especially in two blocks of homologous residues, which correspond in the large-T proteins of polyoma and simian virus 40 (SV40) viruses to sites involved in the ATPase and nucleotide-binding activities. These observations suggest that the papillomavirus E1 proteins might have a function in common with the polyoma virus large-T proteins (which are required for the initiation of viral DNA replication). As it was suggested recently that the E1 gene product is involved in maintaining the BPV-1 genome as a plasmid in transformed cells, we speculate that the structural features conserved in these otherwise very different viruses are general characteristics of eukaryotic proteins involved in the control of DNA replication.     

流感病毒胰酶不依赖性的研究

 在无胰酶条件下将流感病毒在MDCK细胞和Vero细胞上连续传代,研究流感病毒生长对胰酶的依赖性.选择出细胞培养流感病毒的适宜pH值,在无胰酶条件下将流感病毒毒株在Vero细胞和MDCK细胞上连续传代,然后将无效价的病毒收获液与原毒种混合后再进行传代.获得了6株在MDCK细胞上无需胰酶就能扩增的流感病毒毒株.     

Neutralizing antibodies derived from the B cells of 1918 influenza pandemic survivors

Investigation of the human antibody response to influenza virus infection has been largely limited to serology, with relatively little analysis at the molecular level. The 1918 H1N1 influenza virus pandemic was the most severe of the modern era. Recent work has recovered the gene sequences of this unusual strain, so that the 1918 pandemic virus could be reconstituted to display its unique virulence phenotypes. However, little is known about adaptive immunity to this virus. We took advantage of the 1918 virus sequencing and the resultant production of recombinant 1918 haemagglutinin (HA) protein antigen to characterize at the clonal level neutralizing antibodies induced by natural exposure of survivors to the 1918 pandemic virus. Here we show that of the 32 individuals tested that were born in or before 1915, each showed seroreactivity with the 1918 virus, nearly 90 years after the pandemic. Seven of the eight donor samples tested had circulating B cells that secreted antibodies that bound the 1918 HA. We isolated B cells from subjects and generated five monoclonal antibodies that showed potent neutralizing activity against 1918 virus from three separate donors. These antibodies also cross-reacted with the genetically similar HA of a 1930 swine H1N1 influenza strain, but did not cross-react with HAs of more contemporary human influenza viruses. The antibody genes had an unusually high degree of somatic mutation. The antibodies bound to the 1918 HA protein with high affinity, had exceptional virus-neutralizing potency and protected mice from lethal infection. Isolation of viruses that escaped inhibition suggested that the antibodies recognize classical antigenic sites on the HA surface. Thus, these studies demonstrate that survivors of the 1918 influenza pandemic possess highly functional, virus-neutralizing antibodies to this uniquely virulent virus, and that humans can sustain circulating B memory cells to viruses for many decades after exposure-well into the tenth decade of life.     

Structures of the multidrug exporter AcrB reveal a proximal multisite drug-binding pocket

AcrB and its homologues are the principal multidrug transporters in Gram-negative bacteria and are important in antibiotic drug tolerance. AcrB is a homotrimer that acts as a tripartite complex with the outer membrane channel TolC and the membrane fusion protein AcrA. Minocycline and doxorubicin have been shown to bind to the phenylalanine cluster region of the binding monomer. Here we report the crystal structures of AcrB bound to the high-molecular-mass drugs rifampicin and erythromycin. These drugs bind to the access monomer, and the binding sites are located in the proximal multisite binding pocket, which is separated from the phenylalanine cluster region (distal pocket) by the Phe-617 loop. Our structures indicate that there are two discrete multisite binding pockets along the intramolecular channel. High-molecular-mass drugs first bind to the proximal pocket in the access state and are then forced into the distal pocket in the binding state by a peristaltic mechanism involving subdomain movements that include a shift of the Phe-617 loop. By contrast, low-molecular-mass drugs, such as minocycline and doxorubicin, travel through the proximal pocket without specific binding and immediately bind to the distal pocket. The presence of two discrete, high-volume multisite binding pockets contributes to the remarkably broad substrate recognition of AcrB.     

高致病性禽流感分子鉴别诊断方法的建立

为了快速、敏感、特异地鉴别诊断高致病性禽流感,根据H 5,H 7亚型禽流感病毒HA基因序列的保守区域设计出并合成两对特异性引物,利用这两对引物对H 5,H 7亚型禽流感病毒的HA基因片断进行二联RT-PCR扩增,并对二联RT-PCR检测方法进行敏感性及特异性试验。结果表明:这两对引物能特异性地扩增出H 5,H 7亚型禽流感病毒HA基因片段,大小分别为490 bp和375 bp;该检测方法敏感性高,特异性强;初步建立起快速、敏感、特异地鉴别检测H 5,H 7亚型高致病性禽流感病毒的分子诊断方法。     

生物信息学及其在病毒研究中的应用

介绍生物信息学的原理与方法及其在病毒研究中的应用。保守序列承担着极其重要的功能，通过多重序列对齐搜寻保守序列，是生物信息学方法的基础；敏感位点是一种反映蛋白质或核酸功能的特定模式，因此，通过数量关系的优化推导敏感位点，可用于分离病毒蛋白质与核酸相互作用位点；核苷酸和氨基酸序列只有形成了三级或四级结构才能表现功能，通过同源建模预测蛋白质的高级结构，有助于疫苗的研制、抗病毒药物的筛选以及药物的分子设计。预测RNA的三级结构大多从RNA折叠入手．病毒蛋白质三级结构预测比较成功的是日本脑炎病毒包膜糖蛋白的三级结构。     

Structure of the human histamine H1 receptor complex with doxepin

The biogenic amine histamine is an important pharmacological mediator involved in pathophysiological processes such as allergies and inflammations. Histamine H(1) receptor (H(1)R) antagonists are very effective drugs alleviating the symptoms of allergic reactions. Here we show the crystal structure of the H(1)R complex with doxepin, a first-generation H(1)R antagonist. Doxepin sits deep in the ligand-binding pocket and directly interacts with Trp?428(6.48), a highly conserved key residue in G-protein-coupled-receptor activation. This well-conserved pocket with mostly hydrophobic nature contributes to the low selectivity of the first-generation compounds. The pocket is associated with an anion-binding region occupied by a phosphate ion. Docking of various second-generation H(1)R antagonists reveals that the unique carboxyl group present in this class of compounds interacts with Lys?191(5.39) and/or Lys?179(ECL2), both of which form part of the anion-binding region. This region is not conserved in other aminergic receptors, demonstrating how minor differences in receptors lead to pronounced selectivity differences with small molecules. Our study sheds light on the molecular basis of H(1)R antagonist specificity against H(1)R.     

Methylated lysine 79 of histone H3 targets 53BP1 to DNA double-strand breaks

The mechanisms by which eukaryotic cells sense DNA double-strand breaks (DSBs) in order to initiate checkpoint responses are poorly understood. 53BP1 is a conserved checkpoint protein with properties of a DNA DSB sensor. Here, we solved the structure of the domain of 53BP1 that recruits it to sites of DSBs. This domain consists of two tandem tudor folds with a deep pocket at their interface formed by residues conserved in the budding yeast Rad9 and fission yeast Rhp9/Crb2 orthologues. In vitro, the 53BP1 tandem tudor domain bound histone H3 methylated on Lys 79 using residues that form the walls of the pocket; these residues were also required for recruitment of 53BP1 to DSBs. Suppression of DOT1L, the enzyme that methylates Lys 79 of histone H3, also inhibited recruitment of 53BP1 to DSBs. Because methylation of histone H3 Lys 79 was unaltered in response to DNA damage, we propose that 53BP1 senses DSBs indirectly through changes in higher-order chromatin structure that expose the 53BP1 binding site.