In Vivo Synthesis of Crown Gall-specific Agrobacterium tumefaciens-directed Derivatives of Basic Amino Acids

Highly pathogenic (HP) avian influenza viruses (AIVs) are naturally restricted to H5 and H7 subtypes with a polybasic cleavage site (CS) in hemagglutinin (HA) and any AIV with an intravenous pathogenicity index (IVPI) ≥ 1.2. Although only a few non-H5/H7 viruses fulfill the criteria of HPAIV; it remains unclear why these viruses did not spread in domestic birds. In 2012, a unique H4N2 virus with a polybasic CS 322PEKRRTR/G329 was isolated from quails in California which, however, was avirulent in chickens. This is the only known non-H5/H7 virus with four basic amino acids in the HACS. Here, we investigated the virulence of this virus in chickens after expansion of the polybasic CS by substitution of T327R (322PEKRRRR/G329) or T327K (322PEKRRKR/G329) with or without reassortment with HPAIV H5N1 and H7N7. The impact of single mutations or reassortment on virus fitness in vitro and in vivo was studied. Efficient cell culture replication of T327R/K carrying H4N2 viruses increased by treatment with trypsin, particularly in MDCK cells, and reassortment with HPAIV H5N1. Replication, virus excretion and bird-to-bird transmission of H4N2 was remarkably compromised by the CS mutations, but restored after reassortment with HPAIV H5N1, although not with HPAIV H7N7. Viruses carrying the H4-HA with or without R327 or K327 mutations and the other seven gene segments from HPAIV H5N1 exhibited high virulence and efficient transmission in chickens. Together, increasing the number of basic amino acids in the H4N2 HACS was detrimental for viral fitness particularly in vivo but compensated by reassortment with HPAIV H5N1. This may explain the absence of non-H5/H7 HPAIV in poultry.

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The carboxy-terminal domain of IGF-binding protein-5 inhibits heparin binding to a site in the central domain

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0260229 ◽

2001 ◽

Vol 26 (3) ◽

pp. 229-239 ◽

Cited By ~ 14

Author(s):

H Song ◽

JH Shand ◽

J Beattie ◽

DJ Flint ◽

GJ Allan

Keyword(s):

Amino Acids ◽

Binding Protein ◽

Binding Activity ◽

Heparin Binding ◽

Central Domain ◽

Basic Amino Acids ◽

Terminal Domain ◽

Igf Binding ◽

Igf Binding Protein

The IGF-binding protein (IGFBP)-5 protein contains consensus heparin binding motifs in both its carboxy (C)-terminal and central domains, although only the C-terminal site has previously been shown to be functional. We have made two chimeric IGFBP proteins by switching domains between rat IGFBP-5 and -2, named BP552 and BP522 to reflect the domains present, and a truncated rat IGFBP-5 mutant (1-168), named BP550. The ability of these proteins and wild-type (wt) IGFBPs-5 and -2 to bind to either IGFs or heparin was determined using biosensor real-time analysis and heparin ligand blotting respectively. We report that the chimeric molecules have IGF binding affinities comparable to those of the native IGFBPs from which they were derived and, as expected, the binding of BP550 to IGFs was greatly compromised. More surprising was the finding that the ability of BP552 and BP550 to bind to heparin was equivalent to that of wtIGFBP-5, whereas wtIGFBP-2 and BP522 failed to bind. These results demonstrate that the active heparin binding site in BP552 and BP550 is contained within the central domain of IGFBP-5, and that this site is active only in the absence of the C-terminal domain. We subsequently mutated two basic amino acids (R136A:R137A) in the central consensus binding sites between residues 132-140. This resulted in the loss of heparin binding for BP550, confirming the importance of these two basic amino acids in the central domain heparin binding activity. In light of these findings, we suggest that C-terminally truncated fragments of IGFBP-5 generated in vivo by proteolysis could retain heparin/extracellular matrix binding properties.

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Investigation of exponential RNA amplification by the Qβ replicase complex

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314083971 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1602-C1602

Author(s):

Heidi Olesen ◽

Charlotte Knudsen ◽

Paulina Seweryn ◽

Ditlev Brodersen ◽

Zarina Kutlubaeva ◽

...

Keyword(s):

Amino Acids ◽

Nmr Spectroscopy ◽

Rna Virus ◽

Β Subunit ◽

Rna Amplification ◽

Nmr Studies ◽

Basic Amino Acids ◽

Rna Backbone

Positive-stranded RNA viruses are common among human pathogenic viruses, which often cooperate with host proteins to fulfill essential functions during infection. One function is replication of the viral genome. The Qβ phage is a positive-stranded RNA virus that infects E.coli. The Qβ replicase holo enzyme comprises the phage-encoded RNA-dependent RNA polymerase (β-subunit) and the host-encoded translation elongation factors, EF-Ts and EF-Tu as well as the ribosomal protein S1. The Qβ replicase has an extraordinary ability to exponentially amplify RNA in vivo and in vitro. A prerequisite for this is release of product and template RNA as single strands that can serve as new templates in subsequent rounds of replication. The role of S1 in the Qβ replicase is not clear. Recently, S1 was found to promote release of single-stranded product in Qβ replicase–mediated RNA synthesis. We have undertaken NMR spectroscopy and crystallization trials to improve our understanding of distinct S1 domains in solution as well as the ribosome- and replicase-binding properties of S1. Expression of distinct S1 domains for NMR spectroscopy has been optimized by use of autoinduction and results in high yields of [13C15N]-labelled protein fragments. These have proven very suitable for NMR studies and spectra revealed both ordered and disordered regions in the protein. Studies are ongoing. The structure of the Qβ core complex was recently determined at 2.5Å resolution. Thus, co-crystallization of the Qβ core in complex with S1 domains was undertaken and different crystal forms were obtained. These initial crystals diffracted to 3.2Å resolution and data processing as well as further optimization of the crystals is ongoing. S1 is thought to bind the β-subunit close to a region lined with basic amino acids, which potentially could facilitate interactions with the template RNA backbone and split it from the product strand. We demonstrate that neutralization of these basic amino acids indeed decrease or abolish infectivity of the Qβ phage. However, only one mutation, R503A affects the exponential replication in vitro. Crystallization of the Qβ holo enzyme bound to a truncated legitimate RNA template will be the next step for investigation of the mechanism of exponential RNA amplification by Qβ replicase.

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Intestinal transport of certain N-substituted amino acids

American Journal of Physiology-Legacy Content ◽

10.1152/ajplegacy.1962.203.4.637 ◽

1962 ◽

Vol 203 (4) ◽

pp. 637-640 ◽

Cited By ~ 63

Author(s):

Hiroshi Hagihira ◽

T. Hastings Wilson ◽

Edmund C. C. Lin

Keyword(s):

Amino Acids ◽

Transport System ◽

Intestinal Transport ◽

Transport Systems ◽

Concentration Gradients ◽

Methyl Derivatives ◽

Derivatives Of ◽

Amino Acid Carrier ◽

Everted Sacs

Sarcosine, N,N-dimethylglycine and betaine were transported against concentration gradients by everted sacs of hamster small intestine. These compounds shared a common transport system which differed from that which acted on most neutral l-amino acids. The N-methyl derivatives of glycine, while competing with each other, had no effect on the transport of l-valine. Furthermore, l-valine and l-methionine, which were powerful inhibitors of the transport of most other neutral l-amino acids, had little effect on the absorption of betaine. Two other N-substituted amino acids, l-proline and hydroxy-l-proline, possessed affinity for both transport systems, a greater affinity being shown for the betaine carrier than for the neutral amino acid carrier. It is postulated that the transport system for N-substituted amino acids is important for the absorption of l-proline and hydroxy-l-proline in vivo.

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