Site-directed mutations in the Sindbis virus 6K protein reveal sites for fatty acylation and the underacylated protein affects virus release and virion structure.
A small hydrophobic polypeptide of 55 amino acids, noted as the 6K protein, is formed during processing of the polyprotein translated from the Sindbis virus subgenomic 26 S mRNA. In the accompanying paper we show that this 6K protein can be found in purified preparations of virions and that it is palmitoylated via thioester bonds with about four covalently bound fatty acids per molecule. To determine acylation sites on 6K and define a role for these fatty acids, we used site-directed mutagenesis to alter cysteine codons in the 6K gene of Sindbis virus cDNA. One of these mutants had a single cysteine replaced with a serine and the second had two adjacent cysteines replaced with an alanine-serine sequence. Transfection of the transcribed RNA from these two cDNAs produced infectious virus which contained 6K proteins that had decreased amounts of fatty acids. Intracellular formation and maturation of virus glycoproteins appeared to be unaffected by the mutations but the release of virus particles from mutant-infected cells was decreased about 70 to 90% from that observed with wild-type virus. Electron microscopy of virus-infected cells and of isolated virions showed that the 6K mutations led to large numbers of aberrant enveloped particles containing multiple nucleocapsids. These results indicate that the 6K protein and its state of acylation are important factors in Sindbis virus assembly and budding. Additional phenotypic changes are reported for virions released from cells infected with the mutationally altered viruses.
Gaedigk-Nitschko
K
K
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Ding
M X
MX
Levy
M A
MA
Schlesinger
M J
MJ
D000215:Acylation; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D002642:Chick Embryo; D005347:Fibroblasts / Q000378:metabolism; D005814:Genes, Viral; D007700:Kinetics; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation; D015183:Restriction Mapping; D012845:Sindbis Virus / Q000235:genetics* / Q000502:physiology / Q000648:ultrastructure; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D014771:Virion / Q000235:genetics* / Q000502:physiology / Q000648:ultrastructure; D014779:Virus Replication
D014764:Viral Proteins