scholarly journals FlexibilityIn Vitroof Amino Acid 226 in the Receptor-Binding Site of an H9 Subtype Influenza A Virus and Its EffectIn Vivoon Virus Replication, Tropism, and Transmission

2018 ◽  
Vol 93 (6) ◽  
Author(s):  
Adebimpe O. Obadan ◽  
Jefferson Santos ◽  
Lucas Ferreri ◽  
Andrew J. Thompson ◽  
Silvia Carnaccini ◽  
...  
Keyword(s):  
Public Health ◽  
Amino Acids ◽  
Influenza Virus ◽  
Amino Acid ◽  
Influenza A ◽  
Amino Acid Change ◽  
Single Amino Acid ◽  
Receptor Binding Site ◽  
Single Amino Acid Change

ABSTRACTInfluenza A viruses (IAVs) remain a significant public health threat, causing more than 300,000 hospitalizations in the United States during the 2015–2016 season alone. While only a few IAVs of avian origin have been associated with human infections, the ability of these viruses to cause zoonotic infections further increases the public health risk of influenza. Of these, H9N2 viruses in Asia are of particular importance as they have contributed internal gene segments to other emerging zoonotic IAVs. Notably, recent H9N2 viruses have acquired molecular markers that allow for a transition from avian-like to human-like terminal sialic acid (SA) receptor recognition via a single amino acid change at position 226 (H3 numbering), from glutamine (Q226) to leucine (L226), within the hemagglutinin (HA) receptor-binding site (RBS). We sought to determine the plasticity of amino acid 226 and the biological effects of alternative amino acids on variant viruses. We created a library of viruses with the potential of having any of the 20 amino acids at position 226 on a prototypic H9 HA subtype IAV. We isolated H9 viruses that carried naturally occurring amino acids, variants found in other subtypes, and variants not found in any subtype at position 226. Fitness studies in quails revealed that some natural amino acids conferred anin vivoreplication advantage. This study shows the flexibility of position 226 of the HA of H9 influenza viruses and the resulting effect of single amino acid changes on the phenotype of variantsin vivoandin vitro.IMPORTANCEA single amino acid change at position 226 in the hemagglutinin (HA) from glutamine (Q) to leucine (L) has been shown to play a key role in receptor specificity switching in various influenza virus HA subtypes, including H9. We tested the flexibility of amino acid usage and determined the effects of such changes. The results reveal that amino acids other than L226 and Q226 are well tolerated and that some amino acids allow for the recognition of both avian and human influenza virus receptors in the absence of other changes. Our results can inform better avian influenza virus surveillance efforts as well as contribute to rational vaccine design and improve structural molecular dynamics algorithms.

scholarly journals A single amino acid change in the cytoplasmic domain alters the polarized delivery of influenza virus hemagglutinin.

1991 ◽  
Vol 114 (3) ◽  
pp. 413-421 ◽  
Author(s):  
C B Brewer ◽  
M G Roth
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Amino Acid Change ◽  
Mdck Cells ◽  
Single Amino Acid ◽  
Apical Surface ◽  
Membrane Glycoproteins ◽  
Coated Pits ◽  
Influenza Virus Hemagglutinin ◽  
Single Amino Acid Change

In the polarized kidney cell line MDCK, the influenza virus hemagglutinin (HA) has been well characterized as a model for apically sorted membrane glycoproteins. Previous work from our laboratory has shown that a single amino acid change in the cytoplasmic sequence of HA converts it from a protein that is excluded from coated pits to one that is efficiently internalized. Using trypsin or antibodies to mark protein on the surface, we have shown in MDCK cells that HA containing this mutation is no longer transported to the apical surface but instead is delivered directly to the basolateral plasma membrane. We propose that a cytoplasmic feature similar to an endocytosis signal can cause exclusive basolateral delivery.

scholarly journals A Point Mutation in the Rhesus Rotavirus VP4 Protein Generated through a Rotavirus Reverse Genetics System Attenuates Biliary Atresia in the Murine Model

2017 ◽  
Vol 91 (15) ◽  
Author(s):  
Sujit K. Mohanty ◽  
Bryan Donnelly ◽  
Phylicia Dupree ◽  
Inna Lobeck ◽  
Sarah Mowery ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biliary Atresia ◽  
Amino Acid Change ◽  
Reverse Genetics ◽  
Single Amino Acid ◽  
Wild Type ◽  
Neonatal Mice ◽  
Single Amino Acid Change

ABSTRACT Rotavirus infection is one of the most common causes of diarrheal illness in humans. In neonatal mice, rhesus rotavirus (RRV) can induce biliary atresia (BA), a disease resulting in inflammatory obstruction of the extrahepatic biliary tract and intrahepatic bile ducts. We previously showed that the amino acid arginine (R) within the sequence SRL (amino acids 445 to 447) in the RRV VP4 protein is required for viral binding and entry into biliary epithelial cells. To determine if this single amino acid (R) influences the pathogenicity of the virus, we generated a recombinant virus with a single amino acid mutation at this site through a reverse genetics system. We demonstrated that the RRV mutant (RRVVP4-R446G) produced less symptomatology and replicated to lower titers both in vivo and in vitro than those seen with wild-type RRV, with reduced binding in cholangiocytes. Our results demonstrate that a single amino acid change in the RRV VP4 gene influences cholangiocyte tropism and reduces pathogenicity in mice. IMPORTANCE Rotavirus is the leading cause of diarrhea in humans. Rhesus rotavirus (RRV) can also lead to biliary atresia (a neonatal human disease) in mice. We developed a reverse genetics system to create a mutant of RRV (RRVVP4-R446G) with a single amino acid change in the VP4 protein compared to that of wild-type RRV. In vitro, the mutant virus had reduced binding and infectivity in cholangiocytes. In vivo, it produced fewer symptoms and lower mortality in neonatal mice, resulting in an attenuated form of biliary atresia.

scholarly journals Structural characterization of human aryl sulphotransferases

1999 ◽  
Vol 337 (2) ◽  
pp. 337-343 ◽  
Author(s):  
Lulu A. BRIX ◽  
Ronald G. DUGGLEBY ◽  
Andrea GAEDIGK ◽  
Michael E. McMANUS
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Change ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Wild Type ◽  
Substrate Specificities ◽  
Loop Region ◽  
Single Amino Acid Change

Human aryl sulphotransferase (HAST) 1, HAST3, HAST4 and HAST4v share greater than 90% sequence identity, but vary markedly in their ability to catalyse the sulphonation of dopamine and p-nitrophenol. In order to investigate the amino acid(s) involved in determining differing substrate specificities of HASTs, a range of chimaeric HAST proteins were constructed. Analysis of chimaeric substrate specificities showed that enzyme affinities are mainly determined within the N-terminal end of each HAST protein, which includes two regions of high sequence divergence, termed Regions A (amino acids 44–107) and B (amino acids 132–164). To investigate the substrate-binding sites of HASTs further, site-directed mutagenesis was performed on HAST1 to change 13 individual residues within these two regions to the HAST3 equivalent. A single amino acid change in HAST1 (A146E) was able to change the specificity for p-nitrophenol to that of HAST3. The substrate specificity of HAST1 towards dopamine could not be converted into that of HAST3 with a single amino acid change. However, compared with wild-type HAST1, a number of the mutations resulted in interference with substrate binding, as shown by elevated Ki values towards the co-substrate 3´-phosphoadenosine 5´-phosphosulphate, and in some cases loss of activity towards dopamine. These findings suggest that a co-ordinated change of multiple amino acids in HAST proteins is needed to alter the substrate specificities of these enzymes towards dopamine, whereas a single amino acid at position 146 determines p-nitrophenol affinity. A HAST1 mutant was constructed to express a protein with four amino acids deleted (P87–P90). These amino acids were hypothesized to correspond to a loop region in close proximity to the substrate-binding pocket. Interestingly, the protein showed substrate specificities more similar to wild-type HAST3 than HAST1 and indicates an important role of these amino acids in substrate binding.

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