Proteolytic processing of poliovirus polyprotein: elimination of 2Apro-mediated, alternative cleavage of polypeptide 3CD by in vitro mutagenesis.
The polypeptide 3CD of many poliovirus strains can be cleaved at two different amino acid pairs. The viral proteinase 3C and the viral polymerase 3D result from cleavage at a Gln-Gly pair by proteinase 3C, whereas cleavage at a Tyr-Gly pair by proteinase 2A yields the alternative products 3C' and 3D'. Specific mutations were introduced into the 3C'/3D' cleavage site in an infectious cDNA clone of poliovirus type 1 (Mahoney) by oligonucleotide-directed mutagenesis in order to investigate the role of 3C' and 3D' in viral proliferation and to obtain information about the cleavage specificity of 2Apro. Substitution of a threonine residue by an alanine residue at position -2 (P2) of this cleavage site abolished cleavage, whereas substitution of a tyrosine residue by a phenylalanine residue at amino acid position -1 (P1) of the cleavage site did not influence processing. Both mutated cDNA clones produced infectious viruses (T147A and Y148F) on transfection. The phenotypes of the mutant viruses were similar to that of the parental strain. We conclude that (i) 3C' and 3D' are not essential for virus replication, (ii) a Phe-Gly pair at the cleavage site can be cleaved by 2Apro, and (iii) a threonine residue in the P2 position of the cleavage site may be important in substrate recognition by 2Apro.
Lee
C K
CK
Department of Microbiology, School of Medicine, State University of New York, Stony Brook 11794.
Wimmer
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E
D000595:Amino Acid Sequence; D004252:DNA Mutational Analysis; D006367:HeLa Cells; D008969:Molecular Sequence Data; D017955:Poliovirus / Q000378:metabolism* / Q000502:physiology; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication
D011506:Proteins; D012367:RNA, Viral; D014764:Viral Proteins