A heterologous coiled coil can substitute for helix I of the Sindbis virus capsid protein.
Alphavirus core assembly proceeds along an assembly pathway involving a dimeric assembly intermediate. Several regions of the alphavirus capsid protein have been implicated in promoting and stabilizing this dimerization, including a putative heptad repeat sequence named helix I. This sequence, which spans residues 38 to 55 of the Sindbis virus capsid protein, was implicated in stabilizing dimeric contacts initiated through the C-terminal two-thirds of the capsid protein and nucleic acid. The studies presented here demonstrate that helix I can be functionally replaced by the corresponding sequence of a related alphavirus, western equine encephalitis virus, and also by an unrelated sequence from the yeast transcription activator, GCN4, that was previously shown to form a dimeric coiled coil. Replacing helix I with the entire leucine zipper domain of GCN4 (residues 250 to 281) produced a virus with the wild-type phenotype as determined by plaque assay and one-step growth analysis. However, replacement of helix I with a GCN4 sequence that favored trimer formation produced a virus that exhibited approximately 40-fold reduction in virus replication compared to the wild-type Sindbis virus. Changing residues within the Sindbis virus helix I sequence to favor trimer formation also produced a virus with reduced replication. Peptides corresponding to helix I inhibited core-like particle assembly in vitro. On the basis of these studies, it is proposed that helix I favors capsid protein-capsid protein interactions through the formation of dimeric coiled-coil interactions and may stabilize assembly intermediates in the alphavirus nucleocapsid core assembly pathway.
Perera
Rushika
R
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA.
Navaratnarajah
Chanakha
C
Kuhn
Richard J
RJ
D000595:Amino Acid Sequence; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004667:Encephalitis Virus, Western Equine / Q000235:genetics / Q000378:metabolism; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012845:Sindbis Virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000378:metabolism; D019065:Virus Assembly
D036022:Capsid Proteins; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases