ABSTRACT
The molecular basis of antigenic drift was determined for the hemagglutinin (HA) of human influenza A/H3N2 virus. From 1968 to 2003, antigenic change was caused mainly by single amino acid substitutions, which occurred at only seven positions in HA immediately adjacent to the receptor binding site. Most of these substitutions were involved in antigenic change more than once. Equivalent positions were responsible for the recent antigenic changes of influenza B and A/H1N1 viruses. Substitution of a single amino acid at one of these positions substantially changed the virus-specific antibody response in infected ferrets. These findings have potentially far-reaching consequences for understanding the evolutionary mechanisms that govern influenza viruses.
Subject(s)
Antigens, Viral/immunology , Evolution, Molecular , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Influenza A Virus, H3N2 Subtype/immunology , Amino Acid Substitution/genetics , Amino Acid Substitution/immunology , Antigens, Viral/genetics , Binding Sites/genetics , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Humans , Influenza A Virus, H3N2 Subtype/genetics , MutationABSTRACT
The currently available reverse-genetics systems for Influenza A virus are all based on transcription of genomic RNA by RNA polymerase I, but the species specificity of this polymerase is a disadvantage. A reverse-genetics vector containing a T7 RNA polymerase promoter, hepatitis delta virus ribozyme sequence and T7 RNA polymerase terminator sequence has been developed. To achieve optimal expression in minigenome assays, it was determined that viral RNA should be inserted in this vector in the negative-sense orientation with two additional G residues downstream of the T7 RNA polymerase promoter. It was also shown that expression of the minigenome was more efficient when a T7 RNA polymerase with a nuclear-localization signal was used. By using this reverse-genetics system, recombinant influenza virus A/PR/8/34 was produced more efficiently than by using a similar polymerase I-based reverse-genetics system. Furthermore, influenza virus A/NL/219/03 could be rescued from 293T, MDCK and QT6 cells. Thus, a reverse-genetics system for the rescue of Influenza A virus has been developed, which will be useful for fundamental research and vaccine seed strain production in a variety of cell lines.
Subject(s)
DNA-Directed RNA Polymerases/metabolism , Genetic Vectors , Influenza A virus/genetics , RNA, Viral/genetics , Viral Proteins/metabolism , Animals , Cell Line , DNA-Directed RNA Polymerases/genetics , Dogs , Genes, Reporter , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Hepatitis Delta Virus/genetics , Humans , Nuclear Localization Signals/genetics , Promoter Regions, Genetic , Quail , RNA Polymerase I/metabolism , RNA, Catalytic/genetics , RNA, Viral/biosynthesis , Terminator Regions, Genetic , Transcription, Genetic , Transfection , Viral Proteins/geneticsABSTRACT
Ethylene dibromide (EDB) is a widespread environmental pollutant and mutagen/carcinogen. Certain Theta-class glutathione transferases (GSTs), enzymes that catalyze the reaction of reduced glutathione (GSH) with electrophiles, activate EDB to a mutagen. Previous studies have shown that human GST T1-1, but not rat GST T2-2, activates EDB. We have constructed an E. coli lacZ reversion mutagenicity assay system in which expression of recombinant GST supports activation of EDB to a mutagen. Hexa-histidine N-terminal tagging of GST T1-1 results in greatly enhanced expression of the recombinant enzyme and gives a lacZ strain that shows a mutagenic response to EDB at extremely low levels (approximately 1 ng EDB per plate). The hexa-histidine-tagged enzyme was purified in one step by Ni(2+)-affinity chromatography. We applied the lacZ mutagenicity assay to the rapid screening of a library of variant GST Theta enzymes. Sequence variants with altered catalytic activities were identified, purified, and characterized.
