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PubMed1978

The crystal sheaths from bivalve hinge ligaments.

The aragonite crystals in the molluscan bivalve hinge ligament are surrounded by an organic sheath which is distinct from the remainder of the ligament matrix. Methods have been developed to isolate these sheathed crystals from the ligaments of Spisula solidissima and Mercenaria mercenaria employing a papain digestion of the matrix protein. The sheathed crystals from Spisula have a CaCO3/protein ratio of 11.1 and those from Mercenaria a ratio of 29.6. The sheathed crystals and the empty crystal sheaths have been examined by electron microscopy for structural integrity. The sheath proteins exhibit much smaller proportions of the amino acids glycine and methionine than the hinge ligaments. These are characteristic amino acids of high concentrations in the hinge ligaments of both species. The concentrations of acidic and basic amino acids are increased about two fold in the sheaths over those of the ligaments. Otherwise there is little similarity in the amino acid composition of the sheaths in the two species. However, SDS electrophoresis shows the sheaths of both to contain a major protein component with a molecular weight of about 25,000. The sheath protein from the Mercenaria ligament contains about 5% carbohydrate and that of Spisula sheaths less than 1% carbohydrate.

Amino Acids↗
PubMed1987

Purification of chicken testicular müllerian inhibiting substance by ion exchange and high-performance liquid chromatography.

A highly purified protein molecule was obtained from the secretory proteins of 8-week-old chicken testes using ion-exchange column chromatographic procedures, including DEAE Bio-Gel A, CM Bio-Gel A, wheat germ lectin columns, and high-performance liquid chromatographic (hplc) separation techniques. This protein molecule has a molecular weight of 74,000 Da (74K protein). The isolated 74K protein induces regression of chicken Müllerian ducts grown in vitro. The 74K protein does not cause regression of cultured embryonic intestine or Wolffian duct. When the total testicular secretory proteins are resolved in a two-dimensional gel electrophoresis, approximately 120 polypeptides are obtained. The purified 74K protein has a pI of 6.1. Analysis of amino acid composition indicates that the 74K protein is relatively acidic in nature with a ratio of acidic to basic amino acids of 1.93.

Animals↗
PubMed2002

Evidence for the involvement of the unique C-tail of S100A9 in the binding of arachidonic acid to the heterocomplex S100A8/A9.

Protein complexes formed by S100A8 and S100A9 represent the only AA-binding capacity in the human neutrophilic cytosol and are involved in the intracellular arachidonic acid metabolism. The formation of S100A8/A9 protein complexes and the binding of calcium to the complexes are prerequisites for the specific binding of polyunsaturated fatty acids. The present study was undertaken to characterize the fatty acid binding site within the protein complex. Deletions at both termini and point mutations of different basic amino acids especially within the extended C-terminal tail of human S100A9 were introduced. The S100A9 mutant proteins were then analyzed with respect to protein-protein interaction (GST pull down-assay and yeast two-hybrid system) and functional properties (arachidonic acid and calcium binding). The data give strong evidence that the unique C-tail of S100A9 containing the three consecutive histidine residues (His103-His105) represents the region to which the fatty acid carboxy-group is bound to the protein complex. The localization of the AA-binding site within the unique C-tail of S100A9 correlates with the fact that fatty acid binding has not yet been reported for other S100 proteins.

Amino Acid Sequence↗
PubMed1997

Distribution of a sub-class of bacterial ABC polar amino acid transporter and identification of an N-terminal region involved in solute specificity.

A new sub-class of binding protein-dependent transporter with specificity for a broad range of polar amino acids has been identified by sequence comparison, in Rhizobium leguminosarum, Rhodobacter capsulatus, Escherichia coli and Pseudomonas fluorescens. Southern blotting and PCR analysis has shown that transporters from this new sub-class are widely distributed in Gram-negative bacteria, including, in addition to the above, Citrobacter freundii, Erwinia carotovorum and Rhizobium meliloti. ABC transporters of polar amino acids can be divided into two groups: those with narrow solute specificity and the newly identified sub-class with broad solute specificity. The binding and inner membrane proteins from transporters with a broad solute specificity are larger by approximately 30% than those with a narrow solute specificity. Multiple alignment of the inner membrane proteins from all sequenced polar amino acid transporters indicates there is an N-terminal conserved region that may be involved in solute specificity. A conserved arginine or lysine at residue 30 of this region is changed to glutamate in arginine transporters. Residue 53 also has a strong correlation with the charge on the transported solute, with basic amino acid transporters replacing an aliphatic amino acid at this position with a negatively charged amino acid. The general amino acid permease from R. leguminosarum, which will transport aliphatic as well as basic and acidic amino acids, juxtaposes two prolines at residues 52 and 53 of the N-terminal conserved region.

ATP-Binding Cassette Transporters↗
PubMed1982

Isolation and partial characterization of a nuclear antigen (68/6.3) from the Namalwa cell line (a Burkitt lymphoma).

A nuclear antigen was purified from the 0.01 M Tris-HCl/pH8 extract of nuclei of the Burkitt tumor Namalwa cell line to electrophoretic homogeneity by DEAE cellulose chromatography, affinity chromatography, and preparative isoelectric focusing. The yield of antigens was 0.02% of the nuclear 0.01 M Tris-HCl/pH8 extract. On two-dimensional gel electrophoresis, the major antigen separated into two adjacent protein spots with molecular weights of 68,000 and an approximate pI of 6.3 (68/6.3 A and 68/6.3 B). A minor antigen had a molecular weight of 61,000 and pI of 6.0 (61/6.0). Fourteen 125I-labeled peptides were obtained from the tryptic digest of the major antigen (68/6.3 A and 68/6.3 B). The amino-acid composition analysis of the purified antigens indicated that the amino acids in the highest content were glycine (15%), glutamic acid (11.6%), and serine (9%); the ratio of acidic to basic amino acids was 1.95. In studies on nucleolytic activity, the purified antigen produced a single-stranded and then a double-stranded cleavage of PM 2 and pBR 322 DNA. This antigen is the first purified nuclear antigen that reacts with the HeLa-specific nucleolar antibodies.

Amino Acids↗
PubMed1982

Major high mobility group like proteins of Drosophila melanogaster embryonic nuclei.

Nuclei from Drosophila melanogaster embryos contain three major proteins which are extracted by 0.35 M NaCl and by 2% perchloric acid. One of these is histone H1, and we refer to the other two as A63 and A13 in accordance with their molecular weights determined by electrophoresis on sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels (63,000 and 13,000, respectively). The molecular weight of A13, based on its amino acid composition, is approximately 10,000. The amino acid analyses of A63 and A13 show that both of these proteins have high proportions of acidic and basic amino acid residues, a property characteristic of the high mobility group proteins isolated from vertebrate tissues. While A13 comigrates with histone H2A on NaDodSO4-polyacrylamide gels and with H2B on acid/urea gels, it can be readily resolved from the histones by Triton/acid/urea-Na DodSO4 two-dimensional electrophoresis.

Amino Acids↗
PubMed1996

Isolation and characterization of a copper containing protein from blue cell walls of Neurospora crassa.

Culturing Neurospora crassa in presence of toxic amounts of copper (0.63 mM) resulted in blue coloured mycelia and cell walls. Significant amounts (approximately 45%) of total mycelial copper were associated with cell wall isolates under conditions of copper toxicity. Hence, such blue cell walls were analysed to identify specific ligands involved in copper binding. While decuprification of the blue cell walls with 8-hydroxy quinoline (8 HQ) did not alter their copper binding abilities, similar treatment with EDTA (10 mM) decreased such abilities indicating that EDTA treatment lead to loss of copper binding ligands from cell walls. Treatment of blue cell walls with 8 HQ followed by EDTA resulted in the solubilization of a copper binding protein (relative MW approximately 14 kDa) which was associated with phosphate and carbohydrate moieties. On amino acid analysis, this protein was found to be devoid of free thiol groupings but enriched in acidic and basic amino acids, distinguishing it from classical intracellular metal binding proteins such as metallo-thioneins and phytochelatins that are inducively synthesized under conditions of metal toxicity. The biological significance of the isolated wall-bound copper binding protein, which appears to be a normal constituent of cell walls, is discussed in relation to cytoplasmic metal binding proteins and mechanism(s) adapted by fungi in countering metal toxicity.

Cell Wall↗
PubMed1989

[Dynamics of amino acid and protein metabolism in laying hens after the administration of 15N-labeled wheat protein. 10. 15N-labeling of crude protein and amino acids of the oviducts].

Over a period of 4 days 12 colostomized laying hens daily received 36 g coarse wheat meal containing 14.37 atom-% 15N excess (15N') together with a conventional ration. After the homogenisation of each oviduct N and 15N' were determined. After the precipitation with TCA the 15N' of the amino acids was analysed in both the precipitate and the supernatant. In addition, the free amino acids and the peptides were determined in the TCA soluble fraction. The atom-% 15N' in the total N and in the non-basic amino acid N showed a parallel decrease; it diminished from 1.75 atom-% 15N' to 0.64. Of the three basic amino acids, lysine shows the lowest labelling at all four measuring points. The quotas of non-basic amino acid 14N and 15N' in the total 14N and 15N' of the oviduct are the same and amount to 53%. In contrast to this, the quota of the 14N of the basic amino acids in the total 14N of the oviduct only amounts to 21.6% and that of 15N' only to 15.4%. The average atom-% 15N' of the free amino acids 12 h after the last 15N application is 1.54 and is considerably above that of the peptides with 1.15 atom-% 15N'. 36 h after the last 15N application the ascertained value of 1.25 is identical in both fractions. The labelling of the free amino acids decreases more quickly than that of the peptides the more time has passed after the last 15N application.

Amino Acids↗
PubMedDate not supplied

Survey of the hemagglutinin (HA) cleavage site sequence of H5 and H7 avian influenza viruses: amino acid sequence at the HA cleavage site as a marker of pathogenicity potential.

The deduced amino acid sequence at the hemagglutinin (HA) cleavage site of 76 avian influenza (AI) viruses, subtypes H5 and H7, was determined by reverse transcription-polymerase chain reaction and cycle sequencing techniques to assess pathogenicity. Eighteen of the 76 viruses were isolated in 1993 and 1994 from various sources in the United States. In addition, 34 H5 (4 highly pathogenic [HP] and 30 non-highly pathogenic [non-HP]) and 24 H7 (3 HP and 21 non-HP) repository viruses, isolated between 1927 and 1992, were sequenced and the sequences compared to those in recent isolates. All repository HP H5 and H7 viruses studied had multiple basic amino acids adjacent to the HA cleavage site and most had basic amino acids in excess of the proposed minimum motif B-X-B-R (B = basic amino acids arginine or lysine, X = nonbasic amino acid, R = arginine) that has been associated with high pathogenicity. Of the non-HP viruses studied, 35 of 38 for H5 and 30 of 31 for H7 conformed to the motif B-X-X-R and B-X-R, respectively. Two non-HP H5 viruses had the motif X-X-X-R at the cleavage site and a third had the motif B-X-X-K (K = basic amino acid lysine). One non-HP H7 (A/Pekin robin/CA/30412-5/94) had four basic amino acids (K-R-R-R) adjacent to the cleavage site. Although the Pekin robin isolate did not produce disease in chickens under the conditions of the study it did have the amino acid sequence compatible with that in HP AI viruses and, therefore, is considered potentially HP. This is the first account of an H7 virus that is non-HP in chickens but meets the molecular criterion to be classified as HP.

Amino Acid Sequence↗
PubMed2003

Dual subcellular distribution of cytochrome b5 in plant, cauliflower, cells.

Subfractionation studies showed that cytochrome b(5) (cyt b5), which has been considered to be a typical ER protein, was localized in both the endoplasmic reticulum membrane (ER) and the outer membrane of mitochondria in cauliflower (Brassica olracea) cells and was a component of antimycin A-insensitive NADH-cytochrome c reductase system in both membranes. When cDNA for cauliflower cyt b5 was introduced into mammalian (COS-7) and yeast cells as well as into onion cells, the expressed cytochrome was localized both in the ER and mitochondria in those cells. On the other hand, rat and yeast cyt b5s were specifically localized in the ER membranes even in the onion cells. Mutation experiments showed that cauliflower cyt b5 carries information that targets it to the ER and mitochondria within the carboxy-terminal 10 amino acids, as in the case of rat and yeast cyt b5s, and that replacement of basic amino acids in this region of cauliflower cyt b5 with neutral or acidic ones resulted in its distribution only in the ER. Together with the established findings of the importance of basic amino acids in mitochondrial targeting signals, these results suggest that charged amino acids in the carboxy-terminal portion of cyt b5 determine its location in the cell, and that the same mechanism of signal recognition and of protein transport to organelles works in mammalian, plant, and yeast cells.

Amino Acids, Basic↗
PubMed1995

The biological activity of hydrogen peroxide. VI. Mechanism of the enhancing effects of L-histidine: the role of the formation of a histidine-peroxide adduct and membrane transport.

Further details of the mechanism of the enhancing effects of L-histidine (L-His) on the clastogenic activities of hydrogen peroxide (H2O2) were investigated. The L-His-H2O2 adduct was prepared and its physicochemical properties and biological activities were compared with those of a mixture of L-His plus H2O2 and of H2O2 alone. When the stabilities of the three test samples against glucose were determined in terms of residual H2O2 content in solutions of various pH values over the course of 11 days, the adduct was found to be more stable than H2O2 alone and very similar in terms of stability to the mixture. The almost equivalent stability of the adduct and the mixture suggested formation of the adduct in the mixture even though the interaction between L-His and H2O2 in solution seems, from 13C-NMR analysis, to be rather weak. In cell-free DNA after lysis of cell membranes, the induction of single-strand breaks (SSB) by the adduct and by the mixture was less effective than by H2O2 alone. These results contrast with previous results obtained in intact cells (Oya et al., 1992) and demonstrate the indispensability of the cell membrane for the enhancing effects of L-His. In the presence of inhibitors of the active transport of L-His, namely, 10 different neutral amino acids, effective suppression of the clastogenic activity of the adduct and of the mixture was observed, whereas four acidic and basic amino acids had no effect. Thus, the participation of active transport in the enhancing effects of L-His was apparent. The formation of the adduct of L-His with H2O2 brings about the stabilization or reduces the reactivity of H2O2 and, as a result, the induction of SSB is prevented to some extent in cell-free DNA systems. By contrast, in a cellular system, the accumulation of the adduct in cells by active transport is potentiated by the enhancing effect of L-His, although the mediation of some factors that can generate hydroxyl radicals (*OH) from the adduct in cells must be postulated.

Biological Transport↗
PubMed1984

Epidermal taurine transport in marine mussels.

We examined the structural specificity and kinetic characteristics of epidermal taurine transport in the marine mussels Mytilus edulis and M. californianus. The gill was the primary site of taurine uptake in both species, responsible for 70% of the accumulation of radioactively labeled substrate. Taurine transport was inhibited by short-chain beta-amino acids, including beta-alanine and beta-aminobutyric acid (beta-ABA), and by gamma-ABA; alpha-neutral, acidic, and basic amino acids had no effect on this transport. The uptake process was clearly a saturable phenomenon and was adequately described by Michaelis-Menten-type kinetics in both intact animals and isolated preparations of gill. The maximal rates of taurine uptake, expressed per gram of gill tissue, were 8.4 and 3.2 mumol X g-1 X h-1 for M. edulis and M. californianus, respectively. In intact mussels the half-saturation constant for both species ranged from 5 to 8 microM. A mathematical model was developed that describes the epidermal transport of taurine in the gill of the actively pumping mussel. The model takes into account the geometry and pattern of water flow in the gill. Calculations based on this model indicate that because of the convective movement of water through the gill, the actual Michaelis constant of the taurine transporter must be much lower (i.e., 0.5-2.5 microM) than the half-saturation constants determined with intact animals and isolated gills. The model also predicts that the presence of the transporter in the gill can result in a significant reaccumulation of taurine lost from the gill by passive diffusion. We suggest that epidermal taurine transport is involved with maintenance of the high concentrations of this compound commonly associated with gill tissue in mussels.

Amino Acids↗
PubMed1992

Cloning and characterization of SRP1, a suppressor of temperature-sensitive RNA polymerase I mutations, in Saccharomyces cerevisiae.

The SRP1-1 mutation is an allele-specific dominant suppressor of temperature-sensitive mutations in the zinc-binding domain of the A190 subunit of Saccharomyces cerevisiae RNA polymerase I (Pol I). We found that it also suppresses temperature-sensitive mutations in the zinc-binding domain of the Pol I A135 subunit. This domain had been suggested to be in physical proximity to the A190 zinc-binding domain. We have cloned the SRP1 gene and determined its nucleotide sequence. The gene encodes a protein of 542 amino acids consisting of three domains: the central domain, which is composed of eight (degenerate) 42-amino-acid contiguous tandem repeats, and the surrounding N-terminal and C-terminal domains, both of which contain clusters of acidic and basic amino acids and are very hydrophilic. The mutational alteration (P219Q) responsible for the suppression was found to be in the central domain. Using antibody against the SRP1 protein, we have found that SRP1 is mainly localized at the periphery of the nucleus, apparently more concentrated in certain regions, as suggested by a punctate pattern in immunofluorescence microscopy. We suggest that SRP1 is a component of a larger macromolecular complex associated with the nuclear envelope and interacts with Pol I either directly or indirectly through other components in the structure containing SRP1.

Amino Acid Sequence↗
PubMed1991

Role of potentially charged transmembrane residues in targeting proteins for retention and degradation within the endoplasmic reticulum.

The selective breakdown of newly synthesized proteins retained within the endoplasmic reticulum (ER) is probably mediated by the specific recognition of structural features of protein substrates by components of a degradative system. Within the alpha chain of the multisubunit T-cell antigen receptor (TCR) complex, a transmembrane sequence containing two basic amino acid residues has been shown to act as a determinant for retention and rapid degradation in the ER. We now demonstrate that single basic or acidic amino acid residues can cause targeting for retention and degradation in the ER when placed within the transmembrane domain of an integral membrane protein normally destined for the cell surface. The effect of such potentially charged residues is dependent on their relative position within the transmembrane sequence and on the nature of the amino acid side chains. The phenotypic changes induced by potentially charged transmembrane residues occur without apparent alterations of the global folding or transmembrane topology of the mutant proteins. These observations test the hypothesis that potentially charged residues within transmembrane domains can provide the basis for a motif for ER degradation and explain the selective breakdown of some proteins retained within the ER.

Amino Acid Sequence↗
PubMed1991

Comparison of the amino acid uptake profile of reference and clinical isolates of Fusobacterium nucleatum subspecies.

Human isolates of Fusobacterium nucleatum subspecies appear to colonize different niches in the oral cavity, which may be reflected in their nutritional properties. Consequently the utilization of nitrogenous substrates, their sources of energy (supplied here as amino acids) were compared between the 3 subspecies using the reference strain and fresh clinical isolates of each subspecies. All strains incorporated mainly acidic and basic amino acids but significant differences occurred between subspecies. Both reference and clinical isolates of F. nucleatum subspecies polymorphum utilized all amino acids in the medium but the levels of glutamate, arginine and cysteine were noticeably higher in the reference strain. By contrast, F. nucleatum subspecies fusiforme used a very restricted range of amino acids, of which only glutamate, arginine, histidine and cysteine were taken up at greater than 0.5 mM. F. nucleatum subspecies nucleatum utilized fewer amino acids than F. nucleatum subspecies polymorphum but higher concentrations were taken up by the former. Clinical isolates of F. nucleatum subspecies nucleatum incorporated polar and nonpolar neutral amino acids poorly but their levels increased steadily as a clinical isolate was subcultured over a period of 4 months, and was eventually similar to the reference strain. The effect of adding the key catabolic substrate, glutamate (10 mM), on the amino acid uptake profile of F. nucleatum subspecies nucleatum resulted in the complete suppression of the dibasic amino acids arginine, ornithine and histidine. Strains of this subspecies could grow on glutamate as a major source of carbon and energy but, morphologically, the cells appeared somewhat distended and had a tendency to clump.

Amino Acids↗
PubMed1977

Effects of streptolysin O on transport of amino acids, nucleosides, and glucose analogs in mammalian cells.

The transport of several amino acids, nucleosides, and glucose analogs was studies in HeLa cells treated with sublethal concentrations of streptolysin O. A differential effect of toxin on the various solutes tested was observed. The uptake of the neutral amino acids alanine, alpha-aminoisobutyric acid, leucine, and phenylalanine was reduced by 60 about to 70%. Less inhibition of transport was observed with acidic and basic amino acids, and the uptake of nucleosides and glucose analogs was reduced by 20% or less. The decreased uptake of alpha-aminoisobutyric acid could be explained by the inability of toxin-treated cells to retain this amino acid. The altered transport of phenylalanine and lysine, however, appeared to be due to decreased initial rates of uptake rather than to the loss of these amino acids from intracellular pools in toxin-treated cells. After treatment with sublethal concentrations of streptolysin O, the cells recovered their ability to transport and accumulate alpha-aminoisobutyric acid in about 4 h. The data suggest that the alterations in membrane transport observed in toxin-treated cells are due to an effect of streptolysin O on specific transport systems, rather than to the production of holes or pores in the membrane.

Amino Acids↗
PubMed1982

Phosphoproteins in the parotid saliva from the subhuman primate Macaca fascicularis. Isolation and characterization of a proline-rich phosphoglycoprotein and the complete covalent structure of a proline-rich phosphopeptide.

Parotid saliva from the cynomolgus monkey (Macaca fascicularis) and from pooled human collections displayed the same groups of proteins when fractionated by anion exchange and gel filtration chromatography. We have isolated and characterized a proline-rich phosphoglycoprotein (MPRP) and a proline-rich phosphopeptide (M-statherin) from macaque parotid saliva. MPRP has an apparent molecular weight of 16,900 and displays an unusual chemical composition. It is enriched in proline, glycine, and acidic amino acids, but lacks cysteine, methionine, and tyrosine. MPRP contains 25% (w/w) carbohydrate with 7.0 mol of neutral hexoses, 5.3 mol of galactosamine, 5.9 mol of sialic acid, and 3 mol of phosphorus/mol of protein. M-statherin is a 42-residue phosphopeptide with a high proline, glutamic acid, and tyrosine content, but which lacks threonine, valine, cysteine, methionine, isoleucine, and histidine. The complete covalent structure of M-statherin (Mr = 5,368) is: NH2-Asp-Pse-Pse-Glu-Glu-Lys-Phe-Leu-Arg-Arg-Leu-Arg-Arg-Phe-Asp-Glu-Gly-Arg-Tyr- -Gly-Pro-Tyr-Gln-Pro-Phe-Ala-Pro-Gln-Pro-Leu-Tyr-Pro-Gln-Pro-Tyr-Gln-Pro-Tyr-Gln-Pro-Gln-Tyr-COOH This is the first complete amino acid sequence of a component in the salivary secretion of a subhuman primate. Phosphoserine occurs at residues 2 and 3. All 13 acidic and basic amino acids are located in the NH2-terminal half of the molecule. The carboxyl-terminal half of the molecule is hydrophobic where the tripeptide Tyr-Gln-Pro is repeated three times, the dipeptide Gln-Pro occurs twice, and the tripeptides Tyr-Gly-Pro, Phe-Ala-Pro, and Leu-Tyr-Pro occur once. Evaluation of secondary structure by the Chou-Fasman method predicts an alpha helix in the NH2-terminal half (residue 4-16) and a beta pleated sheet in the carboxyl-terminal half (residues 22-26; 38-42) of the molecule. Both MPRP and M-statherin inhibit spontaneous and seeded precipitation from solutions supersaturated with respect to calcium phosphate salts. This suggests that these macaque compounds may function by maintaining saliva supersaturated with respect to calcium phosphate salts, a necessary requirement for stabilization of hydroxyapatite in the surface layers of teeth.U

Amino Acid Sequence↗
PubMed2006

Pathogenic potential of North American H7N2 avian influenza virus: a mutagenesis study using reverse genetics.

An H7N2 subtype avian influenza virus (AIV) first appeared in the live bird marketing system (LBMS) in the Northeastern United States in 1994. Since then this lineage of virus has become the predominant subtype of AIV isolated from the LBMS and has been linked to several costly commercial poultry outbreaks. Concern for this low pathogenicity isolate mutating to the highly pathogenic form has remained high because of the increasing number of basic amino acids at the hemagglutinin (HA) cleavage site, which is known to be associated with increased pathogenicity of AIV. To address the risk of low pathogenic LBMS-lineage H7N2 virus mutating to the highly pathogenic form of the virus, we generated a series of mutant viruses that have changes in the sequence at the HA cleavage site by using plasmid-based reverse genetics. We confirmed that a conserved proline at -5 position from the HA cleavage site could be changed to a basic amino acid, producing a virus with five basic amino acids in a row at the cleavage site, but with no increase in virulence. Increased virulence was only observed when additional basic amino acids were inserted. We also observed that the virus preferred the arginine instead of lysine at the -4 position from the cleavage site to manifest increased virulence both in vitro and in vivo. Using helper virus-based reverse genetics, where only one transcription plasmid expressing a mutated HA vRNA is used, we identified specific HA cleavage site sequences that were preferentially incorporated into the low pathogenic wild-type virus. The resultant reassortant viruses were highly pathogenic in chickens. This study provides additional evidence that H7 avian influenza viruses require an insertional event to become highly pathogenic, as compared to H5 viruses that can become highly pathogenic strictly by mutation or by insertions.

Amino Acid Sequence↗