Myristoylation is important at multiple stages in poliovirus assembly.
The N-terminal glycine of the VP4 capsid subunit of poliovirus is covalently modified with myristic acid (C14 saturated fatty acid). To investigate the function of VP4 myristoylation in poliovirus replication, amino acid substitutions were placed within the myristoylation consensus sequence at the alanine residue (4003A) adjacent to the N-terminal glycine by using site-directed mutagenesis methods. Mutants which replace the alanine residue with a small hydrophobic residue such as leucine, valine, or glycine displayed normal levels of myristoylation and normal growth kinetics. Replacement with the polar amino acid histidine (4003A.H) also resulted in a level of myristoylation comparable to that of the wild type. However, replacement of the alanine residue with aspartic acid (4003A.D) caused a dramatic reduction (about 40 to 60%) in myristoylation levels of the VP4 precursors (P1 and VP0). In contrast, no differences in modification levels were found in either VP0 and VP4 proteins isolated from mature mutant virions, indicating that myristoylation is required for assembly of the infectious virion. The myristoylation levels of the VP0 proteins found in capsid assembly intermediates indicate that there is a strong but not absolute preference for myristoyl-modified subunits during pentamer formation. Complete myristoylation was observed in mature virions but not in assembly intermediates, indicating that there is a selection for myristoyl-modified subunits during stable RNA encapsidation to form the mature virus particle. In addition, even though mutant infectious virions are fully modified, the severe reduction in specific infectivity of both 4003A.D and 4003A.H purified viruses indicates that the amino acid residue adjacent to the N-terminal glycine apparently has an additional role early during viral infection and that mutations at this position induce pleiotropic effects.
Moscufo
N
N
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Simons
J
J
Chow
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D004279:DNA, Viral; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009227:Myristic Acids / Q000378:metabolism*; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D017955:Poliovirus / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014779:Virus Replication
D004279:DNA, Viral; D009227:Myristic Acids