Studies of a putative amphipathic helix in the N-terminus of poliovirus protein 2C.
Poliovirus protein 2C contains near its N-terminus a putative amphipathic helix which is well conserved among picornaviruses. Three mutants were constructed within this region by site-directed mutagenesis. In the first mutant (pT7XL2-2C-N1) two glutamic acids were replaced with valines at the boundary of the charged and uncharged faces of the helix. The second mutant (pT7XL2-2C-N2) contains an isoleucine to lysine change in the hydrophobic half; in the third mutant (pT7XL2-2C-N3) two lysines were replaced with threonines in the hydrophilic half of the helix. Upon transfection of HeLa cells with RNA transcripts made from these plasmids only pT7XL2-2C-N1 yielded viable virus (W1-2C-N1) which had a small-plaque phenotype. A large-plaque revertant of this virus, W1-2C-N1R, was found to contain the original glutamic acid at one of the mutated sites (E19). There is no detectable minus-stranded RNA synthesis following transfection of HeLa cells with transcript RNAs of the other two plasmids, pT7XL2-2C-N2 and -N3. In vitro translation of these two mutant RNA transcripts in HeLa extracts revealed processing abnormalities in the P2/P3 region of the polyprotein. This leads to a nearly complete absence of 2C and 3AB, which might be the primary cause of defective viral RNA synthesis. The putative amphipathic helix was found to overlap a consensus binding site for double-stranded RNA.
Paul
A V
AV
Department of Microbiology, School of Medicine, State University of New York at Stony Brook 11794-5222.
Molla
A
A
Wimmer
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D004279:DNA, Viral; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics; D014867:Water / Q000737:chemistry
D002352:Carrier Proteins; D004279:DNA, Viral; D017361:Viral Nonstructural Proteins; D014867:Water; C076023:2C protein, viral