A mutant poliovirus containing a novel proteolytic cleavage site in VP3 is altered in viral maturation.
A six-amino-acid insertion containing a Q-G amino acid pair was introduced into the carboxy terminus of the capsid protein VP3 (between residues 236 and 237). Transfection of monkey cells with full-length poliovirus cDNA containing the insertion described above yields a mutant virus (Sel-1C-02) in which cleavage occurs almost entirely at the inserted Q-G amino acid pair instead of at the wild-type VP3-VP1 cleavage site. Mutant Sel-1C-02 is delayed in the kinetics of virus production at 39 degrees C and exhibits a defect in VP0 cleavage into VP2 and VP4 at 39 degrees C. Sucrose gradient analysis of HeLa cell extracts prepared from cells infected by Sel-1C-02 at 39 degrees C shows an accumulation of fast-sedimenting replication-packaging complexes and a significant amount of uncleaved VP0 present in fractions containing mature virions. Our data provide in vivo evidence for the importance of determinants other than the conserved amino acid pair (Q-G) for recognition and cleavage of the P1 precursor by proteinase 3CD and show that an alteration in the carboxy terminus of VP3 or the amino terminus of VP1 affects the process of viral maturation.
Blair
W S
WS
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine 92717.
Hwang
S S
SS
Ypma-Wong
M F
MF
Semler
B L
BL
D000595:Amino Acid Sequence; D000818:Animals; D002213:Capsid / Q000235:genetics*; D036022:Capsid Proteins; D002499:Centrifugation, Density Gradient; D006801:Humans; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010449:Peptide Mapping; D017955:Poliovirus / Q000235:genetics* / Q000254:growth & development; D011499:Protein Processing, Post-Translational / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics
D036022:Capsid Proteins; D012367:RNA, Viral; C442540:VP1 protein, Poliovirus; C442542:VP3 protein, Poliovirus; D008715:Methionine