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 ...

We report the identification of three cyclic peptide ligands of K-Ras(G12D) using an integrated in vitro translation–mRNA display selection platform. These cyclic peptides show preferential binding to the GTP-bound state of K-Ras(G12D) over the GDP-bound state and block Ras-Raf interaction. A co-crystal structure of peptide KD2 with K-Ras(G12D)·GppNHp reveals that this peptide binds in the Switch II groove region with concomitant opening of the Switch II loop and a 40° rotation of the α2 helix, and that a threonine residue (Thr10) on KD2 has direct access to the mutant aspartate (Asp12) on K-Ras. Replacing this threonine with non-natural amino acids afforded peptides with improved potency at inhibiting the interaction between Raf1-RBD and K-Ras(G12D) but not wildtype K-Ras. The union of G12D over wildtype selectivity and GTP state/GDP state selectivity is particularly desirable, considering that oncogenic K-Ras(G12D) exists predominantly in the GTP state in cancer cells, and wildtype K-Ras signaling is important for the maintenance of healthy cells.

Engineering protein expression in vitro or in vivo is usually straightforward for single genes, but remains challenging for multiple genes because of the requirement of coordinated control. RNA and protein overexpression strategies often exploit T7 RNA polymerase and its natural TΦ Class I terminator. However, this terminator’s inefficiency and large size (100 bp) are problematic for multigene construction and expression. Here, we measure the effects of tandem copies of a small (18 bp) Class II T7 terminator from vesicular stomatitis virus on transcription in vitro and on translation in vitro and in vivo. We first test monomeric and dimeric gene constructs, then attempt extension to pentameric gene constructs. “BioBrick” versions of a pET vector and translation factor genes were constructed to facilitate cloning, and His-tags were incorporated to allow copurification of all protein products for relatively unbiased analysis and easy purification. Several results were surprising, including imbalanced expression of the pentameric constructs in vivo, illustrating the value of synthetic biology for investigating gene expression. However, these problems were solved rationally by changing the orders of the genes and by adding extra promoters to the upstream gene or by moving to a more predictable in vitro translation system. These successes were significant, given our initial unexpected results and that we are unaware of another example of coordinated overexpression of five proteins. Our modular, flexible, rational method should further empower synthetic biologists wishing to overexpress multiple proteins simultaneously.

a b s t r a c tThe changeabilities of individual modules of aminoacyl-tRNAs are poorly understood, despite the relevance for evolution, translational accuracy and incorporation of unnatural amino acids (AAs). Here, we dissect the effect of successive changes in four domains of Ala-tRNA Ala 3 on translation in a purified system. Incorporating five AAs, not one, was necessary to reveal major effects on yields of peptide products. Omitting tRNA modifications had little affect, but anticodon mutations were very inhibitory. Surprisingly, changing the terminal CCA to CdCA was sometimes inhibitory and non-cognate AAs were sometimes compensatory. Results have implications for translational fidelity and engineering.