P-glycoprotein (Pgp) detoxifies cells by exporting hundreds of chemically unrelated toxins but has been implicated in multidrug resistance in the treatment of cancers. Substrate promiscuity is a hallmark of Pgp activity, thus a structural description of polyspecific drug-binding is important for the rational design of anticancer drugs and MDR inhibitors. The x-ray structure of apo-Pgp at 3.8 Å reveals an internal cavity of ∼6,000 Å 3 with a 30 Å separation of the two nucleotide binding domains (NBD). Two additional Pgp structures with cyclic peptide inhibitors demonstrate distinct drug binding sites in the internal cavity capable of stereo-selectivity that is based on hydrophobic and aromatic interactions. Apo-and drug-bound Pgp structures have portals open to the cytoplasm and the inner leaflet of the lipid bilayer for drug entry. The inward-facing conformation represents an initial stage of the transport cycle that is competent for drug binding.
A novel avian influenza A(H7N9) virus causing human infection emerged in February 2013 in China. To elucidate the mechanism of interspecies transmission, we compared the signature amino acids of avian influenza A(H7N9) viruses from human and non-human hosts and analysed the reassortants of 146 influenza A(H7N9) viruses with full genome sequences. We propose a genetic tuning procedure with continuous amino acid substitutions and reassorting that mediates host adaptation and interspecies transmission. When the early influenza A(H7N9) virus, containing ancestor haemagglutinin (HA) and neuraminidase (NA) genes similar to A/Shanghai/05 virus, circulated in waterfowl and transmitted to terrestrial poultry, it acquired an NA stalk deletion at amino acid positions 69 to 73. Then, receptor binding preference was tuned to increase the affinity to human-like receptors through HA G186V and Q226L mutations in terrestrial poultry. Additional mammalian adaptations such as PB2 E627K were selected in humans. The continual reassortation between H7N9 and H9N2 viruses resulted in multiple genotypes for further host adaptation. When we analysed a potential association of mutations and reassortants with clinical outcome, only the PB2 E627K mutation slightly increased the case fatality rate. Genetic tuning may create opportunities for further adaptation of influenza A(H7N9) and its potential to cause a pandemic. www.eurosurveillance.org Methods Virus sampling and isolation Specimens as well as clinical and epidemiological information were collected from human cases. Environmental samples and avian samples were collected in the area where human cases identified. Virus isolation was conducted by Chinese National Influenza Center (CNIC) in a biosafety level 3 facility using nineday-old specific pathogen-free (SPF) embryonated chicken eggs and incubated at 37 °C for 48-72 hours. The allantoic fluid was harvested, aliquoted and stored at-80 ºC until use. RNA extraction and genome sequencing Viral RNA was extracted using QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). Gene segments were amplified using the Qiagen OneStep RT-PCR Kit. A total of 48 primer pairs were used to generate PCR amplicons between 378 and 1,123 bp in length for full genome sequencing. Primer sequences are available from the authors on request. Amplified PCR products were purified using ExoSAP-IT reagent (USB, Cleveland, US). Complete genome sequencing was performed with an ABI 3730XL automatic DNA analyser (Applied Biosystems, Foster City, US) using the ABI BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems; Foster City, US). HA: haemagglutinin; NA: neuraminidase. Red dots represent the common ancestor of the novel H7N9 virus. A/Shanghai/5/2013 and A/Shanghai/1/2013 are highlighted in pink and green, respectively. Schematic unrooted trees of HA and NA genes are shown in lower left boxes. The authors gratefully acknowledge the originating and submitting laboratories who contributed sequences used in the phylogenetic analysis to GISAID, and recognise in ...
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