Mutational analysis of the genome-linked protein VPg of poliovirus.
Using a mutagenesis cartridge (R. J. Kuhn, H. Tada, M. F. Ypma-Wong, J. J. Dunn, B. L. Semler, and E. Wimmer, Proc. Natl. Acad. Sci. USA 85:519-523, 1988), we have generated single and multiple amino acid replacement mutants, as well as a single amino acid insertion mutant in the genome-linked protein VPg of poliovirus. Moreover, we constructed three different 5-amino-acid insertion mutants that map close to the C terminus of 3A, a viral polypeptide whose coding sequence is adjacent to VPg. Transfection of HeLa cells with RNA synthesized in vitro was used to test the effect of the mutation on viral proliferation. Mutations were either lethal or nonlethal. A temperature-sensitive phenotype was not observed. The arginine at position 17 of VPg could not be exchanged with any other amino acid without loss of viability, whereas the lysine at position 20, an amino acid conserved among all known polioviruses, coxsackieviruses, and echoviruses, was replaceable with several neutral amino acids and even with glutamic acid. Replacement of poliovirus VPg with echovirus 9 VPg yielded viable virus with impaired growth properties. Our results suggest considerable flexibility in the amino acid sequence of a functional VPg. All insertions in polypeptide 3A proved to be lethal. In vitro translation of mutated viral RNAs gave patterns of proteolytic processing that in some cases was aberrant, even though the mutation was nonlethal.
Kuhn
R J
RJ
Department of Microbiology, State University of New York, Stony Brook 11794.
Tada
H
H
Ypma-Wong
M F
MF
Semler
B L
BL
Wimmer
E
E
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics; D001483:Base Sequence; D004251:DNA Transposable Elements; D010450:Endopeptidases / Q000235:genetics; D008239:Lysine / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D017955:Poliovirus / Q000235:genetics* / Q000254:growth & development; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism; D013912:Threonine / Q000235:genetics; D014758:Viral Core Proteins / Q000235:genetics / Q000502:physiology*; D014764:Viral Proteins / Q000235:genetics / Q000502:physiology*; D014776:Virus Cultivation; D014779:Virus Replication
D004251:DNA Transposable Elements; D012367:RNA, Viral; D014758:Viral Core Proteins; D014764:Viral Proteins; D013912:Threonine; D001120:Arginine; D010450:Endopeptidases; D008239:Lysine